Micellization and interactions with phospholipid vesicles of the lipopeptide iturin A, as monitored by time-resolved fluorescence of a D-tyrosyl residue.
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Anti-Bacterial Agents↗
Biomedical subjects
M Ptak
Publications and source records attributed to M Ptak.
Modelling and refinement of the conformation of mycosubtilin in solution from two-dimensional NMR data.
The conformation in solution of mycosubtilin, an antifungal lipopeptide [cyclo(L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser-beta-amino acid)]has been probed by two-dimensional nuclear magnetic resonance and restrained energy minimization. Several structures have been proposed belonging to the same family with minor local variations related to different orientations of amide planes. The molecular topology was found to be completely different from that of iturin A, an analogue which exhibits quite different biological properties. The cyclic peptide of mycosubtilin is shown to be rather rigid in the region of L-proline and stabilized by C7 structures; in contrast, the neighbourhood of D-tyrosine-2 was found to be more flexible. The validity of our models is discussed first in terms of distance violation and second on the basis of reconstructed NOE spectroscopy maps. The different limitations towards higher-resolution structures are discussed.
Antifungal Agents↗
Effect of the O-methylation of tyrosine on the pore-forming properties of iturins.
A comparison has been made between the pore-forming properties of the antibiotic lipopeptide iturin A and a derivative methylated on the tyrosine residue which shows a restricted biological activity. It is shown that this derivative increases the ion permeability of planar lipid membranes as does iturin A. Nevertheless, the global conductance of the doped membrane is very much lower at the same lipopeptide/phospholipid ratio and the ion selectivity is inverted (PK/PCl = 6 instead of 0.6 with iturin A). The characteristics of the induced conducting pores are also rather different. This suggests an important role of the D-Tyr2 residue, present in all the compounds of the iturin family, both in the biological and in the pore-forming properties of iturin A.
Anti-Bacterial Agents↗
Instability of bovine brain clathrin-coated vesicles on Sephacryl S-1000 gel chromatography.
Clathrin-coated vesicles have been isolated from bovine brain. To allow their further use in biophysical studies, the homogeneity of the preparations has been fully characterized after chromatography on Sephacryl S-1000, which is employed in many studies. It is demonstrated here that clathrin-coated vesicles are not stable on the gels and that their instability is increased in preparations using gels that are not presaturated with phospholipids. In addition, some fractionation occurs during chromatography. It is proposed that the slower migrating fractions contain mainly empty clathrin coats. Changes that occurred during the chromatography step are the result of reversible and irreversible events and are probably related to the assembly/disassembly cycle of clathrin observed in vitro.
Animals↗
Reconstruction of NOESY maps. A requirement for a reliable conformational analysis of biomolecules using 2D NMR.
The modelling of the conformation of a biomolecule in solution is based mainly on the internuclear distances deduced from measurements of nuclear Overhauser effects (nOe) in NOESY correlation maps. The distances are then used as restraints in the energy minimization procedure, which leads to one or several optimized conformations. A general and safe technique for validating these structures with respect to the experimental data is here proposed: from the internuclear distances, the relaxation matrix can be computed under the assumption of a unique rotational correlation time. By stepwise integration of these relaxation equations, the NOESY maps can be accurately reconstructed for any mixing time. Because multi-spin effects are correctly taken into account, any difference between the experimental and theoretical maps can be easily interpreted in terms of conformation, and possible inconsistencies due to conformational averaging can be pointed out. The technique is illustrated for a bacterial lipopeptide, mycosubtilin, the spectrum of which is completely assigned.
Biopolymers↗
Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix.
The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.
DNA↗
Isotype-like suppression of T cell-mediated immunity in vivo. I. Delayed-type hypersensitivity specificity of T cell suppression induced by antigen-binding T cell factors that initiate contact sensitivity.
A new form of immunoregulation is described that is based on the recent suggestion that the effector phase of delayed-type hypersensitivity (DTH) responses consists of a cascade of steps that are dependent on the sequential action of two types of antigen-specific Ly-1+ effector cells. According to this formulation, which is based on analysis of contact sensitivity (CS) in mice, DTH consists of at least two T cell-dependent steps that must occur in sequence. The first of these steps occurs within 2 hr of challenge and depends on DTH-initiating, antigen-binding, antigen-specific T cell factors that sensitize the tissues for an obligatory initial vasoactive step, which allows the antigen/major histocompatibility complex (MHC)-restricted, Ly-1+ effector T cells of classic 24 to 48 hr DTH responses to enter the tissues and produce chemoattractant lymphokines. We have now found that nonspecific suppression of CS responses can be induced by i.v. injection of these antigen-binding, CS-initiating T cell factors. Injection of the antigen-binding T cell factor induces Ly-2+, I-J-, cyclophosphamide sensitive, seemingly nonspecific suppressor T cells to inhibit initiation of CS responses. These suppressor cells do not affect the late-acting lymphokine-producing T cells, but probably act by preventing production of antigen-specific factors of the type that are required to initiate DTH responses. Furthermore, injection of CS-initiating antigen-binding T cell factors also induces suppression of sheep red blood cell (SRBC)-specific DTH, but does not affect classic anti-SRBC B cell responses, which are dependent on antigen/MHC-restricted Ly-1+ helper T cells; skin allograft rejection responses are also not affected. Thus, the suppression is DTH-specific. In addition, suppression induced by antigen-binding T cell factors is Igh and not MHC/H-2 restricted. These findings and data in the companion manuscript showing that these suppressor T cells act by production of soluble suppressor factors that bind to antigen-specific T cell factors of different antigenic specificities, cause us to suggest that the antigen-binding T cell factors are T cell isotype-like. Therefore, an isotype-like suppression is induced by these factors. This isotype-like suppression affects factor-producing cells of various antigenic specificities, may be mediated by T cell isotype-binding factors that are Igh restricted and block initiation of DTH responses, but does not affect conventional, antigen/MHC-restricted T cells, which may therefore have antigen receptors of a different isotype.
Animals↗
Isotype-like suppression of T cell-mediated immunity in vivo. II. Suppression of the early component of contact sensitivity by a Ly-2+ T cell-derived suppressor factor that binds to contact sensitivity-initiating, antigen-specific, Ly-1+ T cell-derived factors that are of different antigen specificities.
Recognition that delayed-type hypersensitivity (DTH) reactions, such as contact sensitivity (CS) in mice, are initiated by Ly-1+ T cell-derived, antigen-specific factors has led to identification of a new kind of suppressor T cell that regulates this initiation phase of CS. Regulation by these suppressor T cells is T cell isotype-like in that initiation of DTH of various antigenic specificities is suppressed, whereas, Ly-1+ T cells mediating the antigen/major histocompatibility complex-restricted, classic delayed phase of CS responses are not affected, nor are other T cell activities. This study shows that these isotype-specific suppressor T cells probably act by release of soluble, isotype-specific, suppressor factors. These isotype-specific T cell factors bind to and can be eluted from columns linked with antigen-specific Ly-1+ T cell factors that initiate CS, and are of different antigen specificities. These T cell regulating, anti-isotypic suppressor factors are derived from Lyt-2+ I-J- T cells and suppress CS-initiating T cells, but do not affect the delayed-acting T cells of CS. This is in contrast with antigen-specific T cell suppressor factors that affect the late-acting and not the early-acting T cells of CS. It is suggested that the antigen-binding, CS-initiating, T cell factors, and their regulatory, anti-isotypic T cell factors are, respectively, T cell analogues of immunoglobulin(Ig)E antibody, and IgE-binding factors, that regulate IgE antibody production by IgE+ B cells.
Animals↗
Conformational study of bacterial lipopeptides: refinement of the structure of iturin A in solution by two-dimensional 1H-NMR and energy calculations.
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Anti-Bacterial Agents↗
Deficiency of contrasuppressor lymphocytes in alloxan-diabetic mice.
Ly 1 immune cells of alloxan diabetic mice are inferior to cells of normoglycemic PCl sensitized animals in transferring adoptive responses into recipients. A new regulatory activity, contrasuppression, that prevents suppressor cells from influencing activity of immune cells, has been recently described. Two types of Ly 1 contrasuppressor (Tcs) cells modulate contact sensitivity reactions in mice. A non-specific Tcs cell, which by itself has no immune activity, helps Ly 1 immune effector cells in adoptive transfer to bypass, in the recipient, the suppressor cell barrier. The antigen-specific Tcs cell induced by immune complexes makes Ly 1 effector cells resistant to specific suppression. Both Tcs cell, in contrast to Ly 1 effector cells, adhere to Vicia villosa lectin and can easily be separated (VV+ and VV-, respectively). Our experiments show that diabetic mice are deficient in nonspecific Tcs cells. The most important finding was that when immune Ly 1 cells of diabetic mice, which otherwise transfer little immunity, were injected together with Ly 1 VV+ cells of normoglycemic animals (these cells have no ability to transfer immunity whatsoever), the adoptive transfer was significantly augmented. We also demonstrate that diabetic mice are unable, upon appropriate immunization, to produce antigen-specific Tcs cells. Since a hypoinsulinemic environment abolishes the function of promiscuous Tcs cells and prevents the development of antigen-specific Tcs cells, this may suggest that contrasuppressor cells have insulin receptors which make them particularly sensitive to insulin deficiency.
Animals↗
Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting.
The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.
Circular Dichroism↗
Preferential induction of antigen-specific contrasuppressor T lymphocytes by trinitrophenyl (TNP)-substituted Langerhans cells.
We have tested the ability of trinitrophenyl (TNP)-labelled antigen-presenting cells (Langerhans cells (LC) and peritoneal macrophages (M phi)) administered intravenously to induce cells that mediate and regulate contact sensitivity. TNP-M phi fail to induce contrasuppressor cells (Tcs) but activate efferent T suppressor (Ts) cells. However, the activity of immune cells can be recovered after removal of Ly 2 Ts cells. In sharp contrast, TNP-substituted purified LC produced a significant contact sensitivity reaction, which is roughly equivalent to that achieved by skin sensitization with picryl chloride. Immune cells from these animals were relatively resistant to suppression by antigen-specific Ts cells, and we have found that their resistance is due to the presence of Ly 1, Vicia villosa lectin adherent, I-J+ cells. The Tcs cell induced by TNP-LC is indistinguishable by surface markers and function from Tcs cell found in mice injected with antigen-antibody complexes. Both these Tcs cells are capable of protecting immune cells from the effects of suppression by antigen-specific Ts cells if added in the proper sequence. Although they have identical surface phenotypes, it is not known at present whether or not Tcs cells induced by two different antigen presentations are identical. The possible reasons why LC are such potent inducers of contrasuppression are discussed.
Animals↗
Revised structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis.
The structure of mycosubtilin, a peptidolipid antibiotic from Bacillus subtilis, was revised by FAB mass spectrometry, 2D NMR spectrometry and also by Edman degradation of the peptide resulting from the N-bromosuccinimide reaction. Four homologous beta-amino acid components were identified by capillary gas chromatography. The cyclopeptide mycosubtilin consists of seven alpha-amino acids in an LDDLLDL sequence closed by a beta-amino acid linkage similar to that found in other antibiotics of the iturin group.
Anti-Bacterial Agents↗
Structure of bacillomycin F, a new peptidolipid antibiotic of the iturin group.
The structure of a new antibiotic of the iturin group, bacillomycin F, has been demonstrated. It is a mixture of homologous peptidolipids, essentially C51H80N12O14 and C52N82N12O14. The lipid moiety consists of minor isoC15, anteisoC15 beta-amino acids and major isoC16, isoC17 and anteisoC17 beta-amino acids. The peptide sequence was determined by studying the peptides obtained after mild HCl hydrolysis and by Edman degradation of bacillomycin F treated with N-bromosuccinimide. The sequence was confirmed by two-dimensional NMR spectrometry and fast-atom-bombardment mass spectrometry gave the molecular masses of the homologous compounds. Bacillomycin F is a cyclic peptidolipid; its complete structure is given in the paper.
Amino Acid Sequence↗
Bacterial lipopeptides induce ion-conducting pores in planar bilayers.
Bacterial lipopeptides, known for their antibiotic activities, have been tested for their ability to interact with lipid membranes. These lipopeptides, Iturin A, Bacillomycin L and D and Peptidolipin NA present analogous structural characteristics: a heptapeptidic cycle is linked to a hydrocarbon chain. We present evidence that these lipopeptides modify the conductance of planar bilayers by forming ion-conducting pores.
Anti-Bacterial Agents↗
Pore-forming properties of iturin A, a lipopeptide antibiotic.
The addition of iturin A, a lipopeptide antibiotic extracted from Bacillus subtilis, to a bimolecular lipid membrane (BLM) increases dramatically its electrical conductance. For very low concentration of iturin A, discrete conductance steps are observed which are assigned to the formation of conducting pores. The characteristics of these pores depend on the lipid content of the BLM and they change with time. Cholesterol considerably increases the lifetimes of open states. The pores are slightly anion versus cation selective. These first observations unable us to briefly discuss the pore-forming properties of lipopeptides.
Anti-Bacterial Agents↗
Calculations of the conformations of Iturin A in relation with NMR studies.
Iturin A is an antifungal antibiotic which was isolated from a strain of Bacillus subtilis, and contains a lipophilic beta amino acid closing an heptapeptide cycle with polar L and D residues. Iturin A belongs to a lipopeptide family of which the LDDLLDL sequence is kept constant. NMR spectroscopy and semi-empirical energy calculations are combined to design the conformations of Iturin A in pyridine solution. J coupling constants and nOes (nuclear Overhauser enhancements) are used as guiding line for energy calculations. This preliminary study shows that Iturin A in pyridine appears as rather rigid, especially in the L Pro 5-D Asn 6 region, probably involved in a beta turn. The polar side chains can form different networks of intramolecular hydrogen bonds. The Tyr side chain, relatively mobile, could be involved in interactions with an hydrophobic environment as the beta amino acid side chain found away from the peptide cycle.
Amino Acid Sequence↗
Suppression and contrasuppression in the induction of contact sensitivity by the administration of cellbound antigen-antibody complexes.
The tolerogenic signal produced by the i.v. injection of haptenated peritoneal exudate cells can be converted to an immunogenic signal by treating the cells with antibody to the hapten before administration. We examined this phenomenon and found that immunity induced by antigen-antibody complexes, as opposed to skin sensitization, is resistant to suppressor T cell influences. This resistance to suppression is due to the activation of an I-J+, Ly-1 T cell population which adheres to the Vicia villosa lectin, all characteristics of contrasuppressor T cells. Because haptenated cells can induce immunity if injected subcutaneously or into cyclophosphamide-pretreated recipients (thereby avoiding the induction of suppressor cells), we suggest that the activation of contrasuppressor cells by antigen-antibody complexes overrides suppressive influences in the host, allowing immunity to become dominant. The possible roles of suppression and contrasuppression in channeling the effector arm of the immune response (e.g., contact sensitivity vs humoral immunity) are discussed.
Animals↗