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Compounds with antiendotoxin properties have been extensively studied for their potential as therapeutic agents for sepsis attributable to gram-negative bacteria. However, with the increasing incidence of gram-positive sepsis, there is interest in identifying compounds with a broad spectrum of action against both gram-positive and gram-negative bacteria. A series of synthetic alpha-helical cationic peptides related to bee melittin and silk moth cecropin have previously been shown to bind lipopolysaccharide (LPS) with high affinity, inhibit LPS-induced tumor necrosis factor alpha (TNF-alpha) production in vitro and in vivo, and kill gram-negative bacteria. In this study, we analyzed whether these peptides were active against gram-positive bacteria; whether they could bind to lipoteichoic acid (LTA), the major proinflammatory structure on gram-positive bacteria; and whether they could block the ability of LTA to promote the release of cytokines by the RAW 264.7 murine macrophage cell line. We found that the cationic peptides demonstrated moderate growth-inhibitory activity toward gram-positive bacteria. In addition, the peptides bound LTA with high affinity. This correlated with the ability of the peptides to block LTA-induced production of TNF and interleukin-6 by RAW 264.7 cells but did not correlate with their ability to kill the bacteria. The peptides also effectively inhibited LTA-induced TNF production in a whole human blood assay. The peptides were also able to partly block the ability of heat-killed Staphylococcus aureus, as well as soluble products of live S. aureus, to stimulate cytokine production by macrophages. Our results indicate that these cationic peptides may be useful to prevent sepsis and inflammation caused by both gram-negative and gram-positive bacteria.

Voltage‐clamp techniques were used to record spontaneously occurring inhibitory post‐synaptic currents (i.p.s.c.s) from Müller cells in the brain stem of the lamprey. In normal bathing solution, the i.p.s.c.s in most cells had unimodally distributed amplitudes with a mean corresponding to a peak conductance of 107 nS and a coefficient of variation of about 15%. About 20% of the cells displayed, in addition, events of approximately twice the modal amplitude. The falling phase of the i.p.s.c.s was exponential with a mean time constant of about 32 msec. This is the same as the relaxation time constant of glycine‐activated channels in these cells under comparable conditions (Gold & Martin, 1983b). When tetrodotoxin (TTX) was added to the bathing solution the spontaneous i.p.s.c.s disappeared, suggesting that they were due to release of transmitter by action potentials in presynaptic terminals. Spontaneous activity was also abolished by removing Ca2+ from the bathing solution. When extracellular Ca2+ was increased, or 4‐aminopyridine (4‐AP) was added to the bathing solution, the mean amplitude of the i.p.s.c.s increased and the amplitude distribution showed two or more distinct peaks. Analysis of the amplitude distributions suggested that the peaks represented single and multiple quantal events and that the release process obeyed binomial statistics. In TTX‐blocked preparations, spontaneous i.p.s.c.s could be induced by raising extracellular K+. These had the same time constant of decay as in normal solution and a unimodal amplitude distribution, with the mean corresponding to a peak conductance of 45 nS. In solutions with raised extracellular K+ and reduced extracellular Cl‐, the mean i.p.s.c. amplitude corresponded to a peak conductance of 67 nS. These variations in conductance corresponded closely to variations in conductance with extracellular K+ and Cl‐ of single glycine‐activated channels. It is concluded that the i.p.s.c.s are produced by activation of conductance channels identical to those activated by glycine, and that in normal bathing solution i.p.s.c.s produced by individual presynaptic action potentials are the result of the release of one or, at most, two quanta of transmitter. Each quantum activates approximately 1500 elementary channels.

Voltage‐clamp techniques were used to measure fluctuations in membrane current produced by the application of glycine to Müller cells in the brain stem of the lamprey. The power density spectrum of the glycine‐induced current ‘noise’ was consistent with the hypothesis that glycine activated a single population of conductance channels with open times determined by first‐order kinetics. In normal bathing solution the channel conductance was 73 +/‐ 12 pS (mean +/‐ S.D.) and the channel open time 34 +/‐ 6 msec at 5 degrees C. The reversal potential for the response was 66 +/‐ 5 mV. Neither channel conductance nor mean open time was voltage‐dependent. Replacement of Cl‐ in the bathing solution by isethionate and sulphate reversibly abolished the response to glycine. Increasing intracellular Cl‐, either by using Cl‐ ‐filled micropipettes or by raising extracellular K+, decreased channel conductance. This unexpected decrease was a direct effect of intracellular Cl‐ and was not related to coincident changes in reversal potential. Channel open time was unaffected by intracellular Cl‐ concentration. Reducing extracellular Cl‐ concentration from 126.5 to 31 mM reduced channel conductance at all levels of intracellular Cl‐ without affecting open time. Increasing the temperature of the preparation resulted in increase in channel conductance and a decrease in mean open time. Q10S for the effects were of the order of 1.3 and ‐2.3 respectively in the range 4‐14 degrees C.

Abstract:  The effect of charge stratification by direct, in-cylinder injection of a small quantity of propane-air mixture in a single-cylinder, propane-fuelled, spart-ignition engine, has been determined in terms of in-cylinder pressure, flame visualisation and exhaust emissions. The operating conditions ranged from low load to wide open throttle at 1000 and 1500 rpm, and with quiescent and swirling in-cylinder flows. The effects of injection-driven flow and turbulence on combustion have been considered independently of mixture strength by assessing the consequences of injecting a local mixture of an equivalence ratio equal to that of the port-induced charge. The results provide further evidence to support the concept of direct in-cylinder mixture injection. At 1000 rpm and low load, faster development of the flame kernel and subsequent enflamed area were observed, together with a 60% increase in peak cylinder combustion pressure at an overall equivalence ratio of 0.7 (A/F of 22.5). The relative effect of local-charge stratification increased with reduction in equivalence ratio at this engine speed and without increase in emissions; at 1500 rpm, stable combustion was achieved even at an equivalence ratio of 0.61 (A/F of 25.8), which was below the flammability limit of the homogeneous port-only propane/ air mixture. Unthrottled engine operation produced similar results so that at an equivalence ratio of 0.55 (A/F of 28.5), for example, the Coefficient of variation of the indicated mean effective pressure was reduced from 0.4 to less than 0.1 by mixture local injection. With a shrouded inlet valve generating mean gas velocities of 6 m/s at the time of ignition in the vicinity of the spark plug, control of rich mixture injection both with and against the bulk in-cylinder flow, resulted in stable combustion with Coefficient of variation of the indicated mean effective pressure of less than 0.1 at an equivalence ratio of 0.55 (A/F of 28.5).

Crk proteins are Src homology (SH) 2/SH3-containing adapter proteins that can mediate the formation of signaling complexes. We show that engaging the B cell antigen receptor (BCR) on the RAMOS B cell line caused both Crk-L and Crk II to associate with several tyrosine-phosphorylated proteins. We identified two of these phosphoproteins as Cas and Cbl and showed that both bound to the Crk SH2 domain after BCR engagement. BCR ligation also increased the amount of Crk proteins in the particulate fraction of the cells and induced the formation of Crk.Cas and Crk.Cbl complexes in the particulate fraction. We propose that tyrosine phosphorylation of membrane-associated Cas and Cbl creates binding sites for the Crk SH2 domain and recruits Crk complexes to cellular membranes. Thus, Crk proteins may participate in BCR signaling by using their SH2 domains to direct the interactions and subcellular localization of proteins that bind to their SH3 domains. In RAMOS cells, we found that the SH3 domains of Crk-L and Crk II bound C3G. Since C3G activates Rap, a negative regulator of the Ras pathway, Crk proteins may participate in regulation of Ras signaling by the BCR.

Crosslinking membrane immunoglobulin (mIg), the B-cell antigen receptor, stimulates tyrosine phosphorylation of a number of proteins. Since many receptors are phosphorylated after ligand binding, we asked if components of the mIg receptor complexes were tyrosine-phosphorylated after mIg crosslinking. Both mIgM and mIgD are noncovalently associated with at least two other proteins. mIgM is associated with the MB-1 protein, which is disulfide-linked to a protein designated Ig-beta. mIgD is not associated with MB-1 but is with IgD-alpha, which is also disulfide-linked to Ig-beta. Using immunoprecipitation with a specific anti-MB-1 antiserum followed by anti-phosphotyrosine immunoblotting, we found that crosslinking mIgM stimulated tyrosine phosphorylation of MB-1, Ig-beta, and a previously unidentified 54-kDa polypeptide associated with MB-1. In mature splenic B cells that express both mIgM and mIgD, mIgM crosslinking stimulated tyrosine phosphorylation of the 32-kDa MB-1 protein, whereas mIgD crosslinking stimulated tyrosine phosphorylation of MB-1-related proteins of 33 and 34 kDa. The 32-kDa MB-1 protein was only associated with mIgM, whereas the 33- and 34-kDa MB-1-related proteins were specifically associated with mIgD and are most likely IgD-alpha. Thus, crosslinking either mIgM or mIgD stimulated tyrosine phosphorylation only of the MB-1-related proteins associated with that receptor.

Soluble non-cross-linked peptidoglycan polymers are released by gram-positive bacteria when beta-lactam antibiotics are administered to humans. In this report, we show that this type of peptidoglycan can stimulate monocyte-macrophage functions that cause inflammation. Non-cross-linked peptidoglycan polymers from penicillin-treated Streptococcus faecium were purified and shown to stimulate the production of interleukin 1 by human monocytes and of colony-stimulating factors by a murine macrophage cell line. In addition, the release of plasminogen activator by human monocytes was inhibited by the soluble peptidoglycan. These in vitro results suggest that prolonged treatment with beta-lactam antibiotics, by causing the production of soluble peptidoglycan, may result in interleukin 1-mediated inflammatory reactions, excessive production of monocytes and granulocytes, and increased fibrin deposition.

Cross-linking of membrane immunoglobulin (mIg), the B lymphocyte antigen receptor, with anti-receptor antibodies stimulates tyrosine phosphorylation of a number of proteins, including one of 42 kDa. Proteins with a similar molecular mass are tyrosine-phosphorylated in response to receptor stimulation in other cell types and have been identified as serine/threonine kinases, termed mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs). The MAP kinases constitute a family of related kinases, at least three of which have molecular masses of 40-45 kDa. In this paper we show that mIg cross-linking stimulated the myelin basic protein phosphotransferase activity characteristic of MAP kinase in both mature and immature murine B cell lines. This enzyme activity co-purified on three different columns with a 42 kDa protein that was tyrosine-phosphorylated (pp42) in response to mIg cross-linking and which reacted with a panel of anti-(MAP kinase) antibodies. Although immunoblotting with the anti-(MAP kinase) antibodies showed that these B cell lines expressed both 42 kDa and 44 kDa forms of MAP kinase, only the 42 kDa form was activated and tyrosine-phosphorylated to a significant extent. Activation of protein kinase C (PKC) with phorbol esters also resulted in selective tyrosine phosphorylation and activation of the 42 kDa MAP kinase. This suggested that mIg-induced MAP kinase activation could be due to stimulation of PKC by mIg. However, mIg-stimulated MAP kinase activation and pp42 tyrosine phosphorylation was only partially blocked by a PKC inhibitor, the staurosporine analogue Compound 3. In contrast, Compound 3 completely blocked the ability of phorbol esters to stimulate MAP kinase activity and induce tyrosine phosphorylation of pp42. Thus mIg may activate MAP kinase by both PKC-dependent and -independent mechanisms.

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.

B cell antigen receptor (BCR) cross-linking activates both Src family and Syk tyrosine kinases, resulting in increased cellular protein-tyrosine phosphorylation and activation of several downstream signaling enzymes. To define the role of Syk in these events, we expressed the BCR in the AtT20 mouse pituitary cell line. These nonlymphoid cells endogenously expressed the Src family kinase Fyn but not Syk. Anti-IgM stimulation of these cells failed to induce most of the signaling events that occur in B cells. BCR-expressing AtT20 transfectants were generated that also expressed Syk. Syk expression reconstituted several signaling events upon anti-IgM stimulation, including Syk phosphorylation and association with the BCR, tyrosine phosphorylation of numerous proteins including Shc, and activation of mitogen-activated protein kinase. In contrast, Syk expression did not reconstitute anti-IgM-induced inositol phosphate production. A catalytically inactive Syk mutant could associate with the BCR and become tyrosine phosphorylated but could not reconstitute downstream signaling events. Expression of the Src family kinase Lck instead of Syk also did not reconstitute signaling. Thus, wild type Syk was required to reconstitute several BCR-induced signaling events but was not sufficient to couple the BCR to the phosphoinositide signaling pathway.

The B-cell antigen receptors consist of membrane immunoglobulins (mIgs) noncovalently associated with two accessory proteins, MB-1 and Ig-beta. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-beta were sufficient to promote cell surface expression of mIgM capable of signal transduction. Expression of MB-1 and Ig-beta, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the signaling reactions characteristic of mIgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-beta and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other signaling events associated with mIgM.

Lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, stimulates immune responses by activating macrophages, B lymphocytes, and other cells of the immune system. The mechanisms by which LPS activates these cells are poorly characterized. Since protein tyrosine phosphorylation appears to be a major intracellular signaling event that mediates cellular responses, we examined whether LPS alters tyrosine phosphorylation in macrophages. We found that Escherichia coli K235 LPS increased tyrosine phosphorylation of several proteins in the RAW 264.7 murine macrophage cell line and in resident peritoneal macrophages from C3H/HeSNJ mice. Changes in tyrosine phosphorylation were detectable by 4-5 min, reached a maximum by 15 min, and declined after 30-60 min. Protein tyrosine phosphorylation increased following stimulation with LPS at 100 pg/ml and was maximal with 10 ng/ml. Similar changes in tyrosine phosphorylation were induced by Salmonella minnesota R595 LPS and by the biologically active domain of LPS, lipid A, but not by the inactive lipid A derivative N2-monoacylglucosamine 1-phosphate. Phorbol 12-myristate 13-acetate also stimulated protein tyrosine phosphorylation, but some of the modulated proteins were different than those phosphorylated by LPS. Treatment of RAW 264.7 cells with a tyrosine kinase inhibitor, herbimycin A, inhibited both LPS-stimulated tyrosine phosphorylation and LPS-stimulated release of arachidonic acid metabolites. Thus, increased protein tyrosine phosphorylation is a rapid LPS-activated signaling event that may mediate release of arachidonic acid metabolites in RAW 264.7 cells.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.

Gab1 is a member of the docking/scaffolding protein family which includes IRS-1, IRS-2, c-Cbl, p130(cas), and p62(dok). These proteins contain a variety of protein-protein interaction motifs including multiple tyrosine residues that when phosphorylated can act as binding sites for Src homology 2 (SH2) domain-containing signaling proteins. We show in the RAMOS human B cell line that Gab1 is tyrosine-phosphorylated in response to B cell antigen receptor (BCR) engagement. Moreover, tyrosine phosphorylation of Gab1 correlated with the binding of several SH2-containing signaling proteins to Gab1 including Shc, Grb2, phosphatidylinositol 3-kinase, and the SHP-2 tyrosine phosphatase. Far Western analysis showed that the SH2 domains of Shc, SHP-2, and the p85 subunit of phosphatidylinositol 3-kinase could bind directly to tyrosine-phosphorylated Gab1 isolated from activated RAMOS cells. In contrast, the Grb2 SH2 domain did not bind directly to Gab1 but instead to the Shc and SHP-2 associated with Gab1. We also show that Gab1 is present in the membrane-enriched particulate fraction of RAMOS cells and that Gab1/signaling protein complexes are found in this fraction after BCR engagement. Thus, tyrosine-phosphorylated Gab1 may recruit cytosolic signaling proteins to cellular membranes where they can act on membrane-bound targets. This may be a critical step in the activation of multiple BCR signaling pathways.

The B cell antigen receptor (BCR) activates Ras, a GTPase that promotes cell proliferation by activating the Raf-1/MEK/ERK signaling module and other signaling enzymes. In its active GTP-bound form, the Rap1 GTPase may act as a negative regulator of Ras-mediated signaling by sequestering Ras effectors (e.g., Raf-1) and preventing their activation. In this report, we show that BCR engagement activates Rap1 and that this is dependent on production of diacylglycerol (DAG) by phospholipase C-gamma. Activation of Rap1 by the BCR was greatly reduced in phospholipase C-gamma-deficient B cells, whereas both a synthetic DAG and phorbol dibutyrate could activate Rap1 in B cells. We had previously shown that C3G, an activator of Rap1, associates with the Crk adaptor proteins in B cells and that BCR engagement causes Crk to bind to the Cas and Cbl docking proteins. However, the DAG-dependent pathway by which the BCR activates Rap1 apparently does not involve Crk signaling complexes since phorbol dibutyrate could activate Rap1 without inducing the formation of these complexes. Thus, the BCR activates Rap1 via a novel DAG-dependent pathway.

The infrared and laser–Raman spectra of liquid dimethyliodoarsine, (CH3)2AsI, have been recorded. Assignments of the observed frequencies have been made to the 24 normal modes of vibration of the molecule. A normal coordinate calculation has been carried out in support of the assignment.

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