IL-1 induction-capacity of defined lipopolysaccharide partial structures

Natural and synthetic lipid A as well as natural and synthetic oligosaccharide partial structures of LPS were examined in dose-response experiments to define the minimal structure necessary for IL-1 induction and release in cultures of human mononuclear cells. Wild type LPS (S. abortus equi) and rough mutant LPS was active in minimal-doses of 1 to 100 pg/ml, whereas synthetic heptaacyl and hexaacyl lipid A (Salmonella minnesota and Escherichia coli lipid A, respectively) induced IL-1 in minimal-doses of 100 to 1,000 pg/ml and 10 to 1,000 pg/ml, respectively. Nanogram amounts (0.1 to 10 ng/ml) of synthetic monodephospho partial structures of E. coli lipid A were necessary for IL-1 induction. Synthetic pentaacyl partial structures induced IL-1 very weakly. Synthetic tetraacyl and bisacyl partial structures lacking non-hydroxylated fatty acids were not active. Compared to LPS million-fold higher doses of natural and synthetic 3-deoxy-D-manno-octulosonic acid containing core oligosaccharides were necessary for IL-1 induction. Dose-response investigations with LPS and natural or synthetic partial structures established the following hierarchy in IL-1 induction-capacity: LPS greater than lipid A much greater than lipid A partial structures greater than core oligosaccharides greater than oligoacyl lipid A. Lipid A was shown here to be the portion of LPS mainly responsible for induction of IL-1 activity. The high potency of lipid A in inducing IL-1 release and the failure of the precursor Ia of lipid A to induce IL-1 production and release was also observed measuring intracellular IL-1 activity after freeze-thawing the cells. Levels of IL-1 beta mRNA in extracts of mononuclear cells correlated with biologic activity. In co-incubation experiments, precursor Ia of lipid A produced dose-dependent inhibition of production and release of IL-1 activity induced by lipid A or LPS, but not by Staphylococcus epidermidis or PHA. Incubation of cells with precursor Ia for 1h, followed by a medium change and further incubation of stimulus without precursor Ia of lipid A also resulted in inhibition. We conclude that lipid A is the main portion of LPS responsible for induction of IL-1, and that specific activation- and/or binding-mechanisms are involved in stimulation of cells with LPS and/or lipid A.     

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.     

S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.     

Isolation and structural analysis of two lipid A precursors from a KDO deficient mutant of Salmonella typhimurium differing in their hexadecanoic acid content

The extraction, purification and structural characterization of two lipid A precursors (Ia and Ib) differing only in one hexadecanoic acid are described. Both precursors were synthesized at elevated temperatures by a new mutant of Salmonella typhimurium (mutant Ts5) which is conditionally defective in synthesis of the 3-deoxy-d-manno-octulosonic acid region of lipopolysaccharides.Both precursors were purified by repeated phenol/chloroform/petroleum ether (PCP) extractions followed by thin layer chromatography. Teh precursor preparation was free of lipopolysaccharides and phospholipids and contained less than 0.1% protein. Structural analysis which included chemical degradation procedures as well as positive ion laser desorption (LDMS) mass spectroscopy of dephosphorylated lipid A precursors showed together that precursor Ia represents a diphosphorylated glucosamine disaccharide containing two ester, two amide-linked residues of 3-hydroxytetradecanoic acid and lacks the ester-linked dodecanoic, tetradecanoic and hexadecanoic acid as well as 3-deoxy-d-manno-octulosonic acid. Precursor Ib has the same basic structure as precursor Ia, but contains in addition one mol of hexadecanoic acid per mol disaccharide which is linked to the 3-hydroxy group of the amide-bound 3-hydroxy-tetradecanoic acid of the reducing, terminal glucosamine residue.The structure of precursor Ib supports the conclusion that hexadecanoic acid incorporation occurs at an early stage in lipid A biosynthesis prior to the attachment of 3-deoxy-d-manno-octulosonic acid and/or other polar substituents.Abbreviations LDMS laser desorption mass spectrometry - KDO 3-Deoxy-d-manno-octulosonic acid - Ts5 Salmonella typhimurium mutant Ts5 - PCP phenol/chloroform/petroleum ether - H₂F₂ hydrogen fluoride This work is dedicated to Prof. Dr. Drews, Freiburg, on the occasion of his 60th birthday     

A new class of antihypertensive neutral lipid: 1-alkyl-2-acetyl-sn-glycerols, a precursor of platelet activating factor

A new type of neutral lipid is described that possesses hypotensive activity in genetic hypertensive (SHR) and normotensive (WKY) rats. 1-Alkyl-2-acetyl-sn-glycerols and 1-alkyl-2-propionyl-sn-glycerols are both equally effective in eliciting the hypotensive response. Requirement for the 1-alkyl and 2-acetyl or 2-propionyl structure of the active isomer was documented by the negative responses obtained with closely related neutral lipid analogs (1-alkyl-2-acyl-, 1-alkyl-3-acetyl-, 1-acyl-2-acetyl-, 1-alkyl-2,3-diacetyl-, and 1-alkyl-glycerols). Although less potent than PAF (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), the 1-alkyl-2-acetyl-sn-glycerols produce a response of significantly longer duration and may have fewer immediate side effects than PAF. The mechanism for the biological activity is unknown; however, we have demonstrated previously that the enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycerols to PAF occurs via a specific cholinephosphotransferase and therefore the observed blood pressure response might be due to the conversion of the neutral lipid precursor to PAF in vivo.     

Suppressive effect of lipid A partial structures on lipopolysaccharide or lipid A-induced release of interleukin 1 by human monocytes

Experiments were designed to investigate the significance of lipid A partial structures, precursor Ia (compound 406), and lipid X (compound 401) to serve as antagonists of interleukin 1 (IL-1) release from human mononuclear cells and monocytes induced by lipopolysaccharide (LPS, endotoxin) of Salmonella abortus equi or synthetic Escherichia coli lipid A (compound 506). A definite inhibition mediated by lipid A partial structures on IL-1 release induced by LPS or lipid A was found in repeated experiments. The inhibitory effect was exerted not only on IL-1 release, but also on IL-1 peptide synthesis at the intracellular level. The results also show that lipid A partial structures have suppressive effects even when added 1-4 h after LPS or lipid A. We conclude from these results that lipid A partial structures (precursor Ia and lipid X) have potent immunomodulatory effects on LPS- and lipid A-induced IL-1 release and may become useful reagents to study the mechanism of interaction of LPS and lipid A with cells of the immune system.     

Suppressive effect of lipid A partial structures on lipopolysaccharide or lipid A-induced release of interleukin 1 by human monocytes

Abstract Experiments were designed to investigate the significance of lipid A partial structures, precursor Ia (compound 406), and lipid X (compound 401) to serve as antagonists of interleukin 1 (IL-1) release from human mononuclear cells and monocytes induced by lipopolysaccharide (LPS, endotoxin) of Salmonella aborus equi or synthetic Escherichia coli lipid A (compound 506). A definite inhibition mediated by lipid A partial structures on IL-1 release induced by LPS or lipid A was found in repeated experiments. The inhibitory effect was exterted not only on IL-1 release, but also on IL-1 peptide synthesis at the intracellular level. The results also show that lipid A partial structures have suppressive effects even when added 1–4 after LPS or lipid A. We conclude from these results that lipis A partial structures (precursor Ia and lipid X) have potent immunomodulatory effects on LPS- and lipid A-induced IL-1 release and may become useful reagents to study the mechanism of interaction of LPS and lipid A with cells of the immune system.     

The interaction of cationic antimicrobial peptides with vesicles containing synthetic glycolipids as models of the outer membrane of gram-negative bacteria.

Two simple lipid A analogues methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside (GL1) and methyl 2,3-di-O-tetradecanoyl-alpha-D-glucopyranoside 4-O-phosphate (GL2) were synthesized and used for preparing mixed phosphocholine vesicles as models of the outer membrane of gram-negative bacteria. The interaction of these model membranes with magainin 2, a representative of the alpha-helical membrane active peptides, and apidaecin Ib and drosocin, two insect Pro-rich peptides which do not act at the level of the cellular membrane, were studied by CD and dye-releasing experiments. The CD spectra of apidaecin Ib and drosocin in the presence of GL1- or GL2-containing vesicles were consistent with largely unordered structures, whereas, according to the CD spectra, magainin 2 adopted an amphipathic alpha-helical conformation, particularly in the presence of negatively charged bilayers. The ability of the peptides to fold into amphipathic conformations was strictly correlated to their ability to bind and to permeabilize phospholipid as well as glycolipid membranes. Apidaecin Ib and drosocin, which are unable to adopt an amphipathic structure, showed negligible dye-leakage activity even in the presence of GL2-containing vesicles. It is reasonable to suppose that, as for the killing mechanism, the two classes of antimicrobial peptides follow different patterns to cross the bacterial outer membrane.     

Platelet glycoprotein Ib beta/IX mediates glycoprotein Ib alpha localization to membrane lipid domain critical for von Willebrand factor interaction at high shear

The localization of the platelet glycoprotein GP Ib-IX complex (GP Ibα, GP Ibβ, and GP IX) to membrane lipid domain, also known as glycosphingolipid-enriched membranes (GEMs or raft) lipid domain, is essential for the GP Ib-IX complex mediated platelet adhesion to von Willebrand factor (vWf) and subsequent platelet activation. To date, the mechanism for the complex association with the GEMs remains unclear. Although the palmitate modifications of GP Ibβ and GP IX were thought to be critical for the complex presence in the GEMs, we found that the removal of the putative palmitoylation sites of GP Ibβ and GP IX had no effects on the localization of the GP Ib-IX complex to the GEMs. Instead, the disruption of GP Ibα disulfide linkage with GP Ibβ markedly decreased the amount of the GEM-associated GP Ibα without altering the GEM association of GP Ibβ and GP IX. Furthermore, partial dissociation with the GEMs greatly inhibited GP Ibα interaction with vWf at high shear instead of in static condition or under low shear stress. Thus, for the first time, we demonstrated that GP Ibβ/GP IX mediates the disulfide-linked GP Ibα localization to the GEMs, which is critical for vWf interaction at high shear.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Availability and antiperoxidative effects of beta-carotene from Dunaliella bardawil in alcohol-drinking rats

The present study demonstrated the high bioavailability and antiperoxidative capacity of the natural beta-carotene isomer mixture of Dunaliella bardawil compared with synthetic beta-carotene under alcohol-induced oxidative stress. Weanling rats were adapted to ethanol by increasing ethanol levels in their drinking water to 30% at 5% intervals per week; other rats received water with no added ethanol. One water-drinking group and one alcohol-drinking group with no dietary carotene were used as controls. Two water-drinking groups were supplemented with 1 g/kg diet beta-carotene either from Dunaliella or a synthetic source, and due to reduced food intake, two ethanol-fed groups received 2 g beta-carotene per kilogram of diet from each source. Following 3 months of ethanol consumption, both carotene sources were found to prevent ethanol-induced lipid peroxidation as expressed by the hepatic conjugated oxidized dienes level. However, in the algal-fed rats, hepatic carotene and vitamin A levels were higher. In addition to a lower performance of the group fed ethanol and synthetic beta-carotene, there were three deaths in this group.     

Aggregates are the biologically active units of endotoxin

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.     

Residues involved in the pore‐forming activity of the Clostridium perfringens iota toxin

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334–359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β‐strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β‐pore‐forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β‐strands affected pore formation, single‐channel conductance and ion selectivity (S339E‐S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ‐clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single‐channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ‐clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single‐channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E‐S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.     

Highly purified lipid X is devoid of immunostimulatory activity. Isolation and characterization of immunostimulating contaminants in a batch of synthetic lipid X

Lipid X, an early precursor in the biosynthesis of lipid A has been reported to directly induce cytokine release in macrophages but also to inhibit endotoxin-induced tumor necrosis factor (TNF) induction. In this report we provide evidence that these conflicting results could be due to contaminants present in different batches of lipid X used. Thus, in an apparently pure batch of crystalline lipid X as obtained by a published procedure (Macher, I. (1987) Carbohydr. Res. 262, 79-84) small amounts of N,O-acylated disaccharide-1-phosphates could be identified. Their isolation was achieved by gel filtration on Sephadex LH-20 and further analysis of fractions showing elevated limulus amebocyte lysate values by thin layer chromatography and reverse-phase high performance liquid chromatography (HPLC) in combination with bioassays. Identification of immunostimulatory by-products was possible by testing HPLC-fractions for TNF-induction in bone marrow-derived mouse macrophages. Applying these procedures a disaccharide-1-phosphate, containing four 3(R)-hydroxymyristic acids at positions 2, 3, 2', 3', was identified as the main immunostimulatory side product. Two isomeric hydrolysis products of this compound with only three 3(R)-hydroxymyristic acid moieties attached to the disaccharide-1-phosphate were also identified. Surprisingly, these compounds behave quite differently in the TNF induction test. The disaccharide-1-phosphate, acylated at positions 2, 2', 3', is a very potent inducer of TNF-release whereas the corresponding isomer containing the 3(R)-hydroxymyristic acids in positions 2, 3, 2', does not induce TNF release, but strongly inhibits TNF release as induced by the former compound. Thus, contamination of "pure" lipid X with immunostimulatory or immunoinhibitory impurities may explain the divergent pharmacological profiles which were attributed to synthetic lipid X.     

Effects of backbone structures and stereospecificities of lipid A-subunit analogues on their biological activities

Among chemically synthesized analogues corresponding to the nonreducing sugar part of lipid A, we have found an analogue (GLA-27) which exhibits Limulus, mitogenic, polyclonal B cell activation (PBA), interferon-inducing, and tumor necrosis factor (TNF)-inducing activities but not pyrogenic activity. The structure of GLA-27 comprises 4-O-phosphono-D-glucosamine with tetradecanoyl and 3-tetradecanoyloxytetradecanoyl (C14-O-(C14] groups as the 3-O- and 2-N-acyl substituents, respectively. Derivatives of GLA-27 with different backbone structures, such as the 1-deoxy, 3-epimeric, 3-amino, and 1-deoxy-3-epimeric derivatives of glucosamine, were chemically synthesized, and their mediator-inducing activities such as interferon- and TNF-inducing activities were investigated in comparison with their B cell activation activities including mitogenic and PBA activities. Among these derivatives, a derivative with a 1-deoxyglucosamine backbone (GLA-40) exhibited stronger B cell activation activities than those of GLA-27 while the mediator-inducing activities of GLA-40 were weaker than those of GLA-27. In addition to these derivatives, stereoisomers of GLA-27 which possess the (R) and (S) forms of C14-O-(C14) as the 2-N-acyl substituent were also synthesized and their biological activities compared. The (S) isomer exhibited much stronger mediator-inducing activities than the (R) isomer. On the other hand, B cell activation activities of the (R) isomer were strong and those of the (S) isomer weak. These results clearly demonstrate that mediator-inducing activities and B cell activation activities can be selectively expressed by modifying the structures of lipid A analogues.     

Identification of a site in the alpha chain of platelet glycoprotein Ib that participates in von Willebrand factor binding

The binding of von Willebrand factor (vWF) to the platelet receptor glycoprotein (GP) Ib-IX complex is a key event in hemostasis and may participate in the development of thrombotic vascular occlusion. We present here evidence that residues Ser251-Tyr279 in the GP Ib alpha-chain participate in this function. Initial studies suggested that the modality of vWF interaction with GP Ib depended on the conditions used for induction of binding, either in the presence of ristocetin, or botrocetin, or with asialo-vWF. In fact, only the 45-kDa amino-terminal fragment of GP Ib alpha inhibited the vWF-GP Ib interaction under all conditions tested, while the 84-kDa macroglycopeptide was significantly effective only in the presence of ristocetin. Moreover, the 45-kDa fragment with reduced disulfide bonds still inhibited ristocetin-induced binding but had no effect, at the concentrations tested, on botrocetin-mediated or direct asialo-vWF binding. In order to localize in more detail the functional site, the entire sequence of the 45-kDa fragment was reproduced in 27 overlapping synthetic peptides that were then used in inhibition of binding assays. This led to the identification of a linear GP Ib alpha sequence (residues Ser251-Tyr279) that effectively inhibited platelet interaction with vWF mediated by ristocetin and, at higher concentration, also by botrocetin. A shorter peptide overlapping with the longer one (residues Gly271-Glu285) was the second most active inhibitory species. This region of the molecule contains several residues with a high surface probability index, as expected for a site involved in ligand binding. Thus, while native conformation of GP Ib alpha appears to be important for optimal interaction with vWF, the results obtained with short synthetic peptides may help in defining the amino acid residues participating in this essential function.     

Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Enhancement of O2- generation and tumoricidal activity of murine macrophages by a monosaccharide precursor of Escherichia coli lipid A

The effects of a monosaccharide precursor of Escherichia coli lipid A (lipid X) on murine macrophages were studied. Lipid X is a diacylglucosamine 1-phosphate bearing beta-hydroxymyristoyl groups at positions 2 and 3. Lipid X, as well as lipopolysaccharide and lipid A, enhanced O2- generation in mouse peritoneal macrophages and a macrophage-like cell line, J774.1, and further induced the tumor-cytotoxic activity of peritoneal macrophages. Elimination of a 1-phosphate or 3-O-beta-hydroxymyristoyl groups are essential for the elevated O2- generation and induction of tumoricidal activity due to lipid X.     

Synthesis of peptidoglycan and teichoic acid in Bacillus subtilis: role of the electrochemical proton gradient.

The effects of several ionophores and uncouplers on glycerol and N-acetylglucosamine incorporation by Bacillus subtilis 61360, a glycerol auxotroph, were tested at different pH values. In particular, the effect of valinomycin on the synthesis of teichoic acid and peptidoglycan was examined in more detail in both growing cells and in vitro biosynthetic systems. Valinomycin inhibited synthesis of wall teichoic acid and peptidoglycan in whole cells but not in the comparable in vitro systems. It did not inhibit formation of free lipid or lipoteichoic acid. The results were consistent with a role for the electrochemical proton gradient in maintaining full activity of cell wall synthetic enzymes in intact cells. Such an energy source would be required for a model in which rotation or reorientation of synthetic enzyme complexes is envisaged for the translocation of wall precursor molecules across the cytoplasmic membrane (Harrington and Baddiley, J. Bacteriol. 155:776-792, 1983).