Neale Foxwell

A Lethal Role For Lipid A In Salmonella Infections

Salmonella infections in naturally susceptible mice grow rapidly, with death occurring only after bacterial numbers in vivo have reached a high threshold level, commonly called the lethal load. Despite much speculation, no direct evidence has been available to substantiate a role for any candidate bacterial components in causing death. One of the most likely candidates for the lethal toxin in salmonellosis is endotoxin, specifically the lipid A domain of the lipopolysaccharide (LPS) molecule. Consequently, we have constructed a Salmonella mutant with a deletion–insertion in its waaN gene, which encodes the enzyme that catalyses one of the two secondary acylation reactions that complete lipid A biosynthesis. The mutant biosynthesizes a lipid A molecule lacking a single fatty acyl chain and is consequently less able to induce cytokine and inducible nitric oxide synthase (iNOS) responses both in vivo and in vitro. The mutant bacteria appear healthy, are not sensitive to increased growth temperature and synthesize a full‐length O‐antigen‐containing LPS molecule lacking only the expected secondary acyl chain. On intravenous inoculation into susceptible BALB/c mice, wild‐type salmonellae grew at the expected rate of approximately 10‐fold per day in livers and spleens and caused the death of the infected mice when lethal loads of approximately 108 were attained in these organs. Somewhat unexpectedly, waaN mutant bacteria grew at exactly the same rate as wild‐type bacteria in BALB/c mice but, when counts reached 108 per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 109 per organ were attained, when approximately 10% of the mice died. Most of the animals carrying these high bacterial loads survived, and the bacteria were slowly cleared from the organs. These experiments provide the first direct evidence that death in a mouse typhoid infection is directly dependent on the toxicity of lipid A and suggest that this may be mediated via pro‐inflammatory cytokine and/or iNOS responses.

Glucocorticoids do not affect the induction of a novel calcium-dependent nitric oxide synthase in rabbit chondrocytes

Incubation of rabbit articular chondrocytes with interleukin-1 beta caused time-dependent expression of NO synthase, determined as nitrite, after a lag period of 6h. The synthesis of nitrite was concentration-dependent and was inhibited by cycloheximide and NG-monomethyl-L-arginine, but not by dexamethasone or hydrocortisone. The synthesis of NO in the 100,000g supernatant of activated chondrocytes was inhibited by EGTA, but not by the calmodulin inhibitors W-13 or trifluoperazine. The synthesis of NO was half-maximal at approximately 20nM free Ca2+. Endotoxin also induced the expression of this NO synthase. Thus, rabbit articular chondrocytes express a novel inducible NO synthase which is Ca(2+)-dependent, and whose induction is not prevented by glucocorticoids.

Crystal Structure of SANOS, a Bacterial Nitric Oxide Synthase Oxygenase Protein from Staphylococcus aureus

Prokaryotic genes related to the oxygenase domain of mammalian nitric oxide synthases (NOSs) have recently been identified. Although they catalyze the same reaction as the eukaryotic NOS oxygenase domain, their biological function(s) are unknown. In order to explore rationally the biochemistry and evolution of the prokaryotic NOS family, we have determined the crystal structure of SANOS, from methicillin-resistant Staphylococcus aureus (MRSA), to 2.4 A. Haem and S-ethylisothiourea (SEITU) are bound at the SANOS active site, while the intersubunit site, occupied by the redox cofactor tetrahydrobiopterin (H(4)B) in mammalian NOSs, has NAD(+) bound in SANOS. In common with all bacterial NOSs, SANOS lacks the N-terminal extension responsible for stable dimerization in mammalian isoforms, but has alternative interactions to promote dimer formation.

Transcellular regulation of cell respiration by nitric oxide generated by activated macrophages

A macrophage cell line (J774), activated with interferon‐γ and endotoxin to express the inducible form of NO synthase (iNOS), immediately inhibited the cellular respiration of co‐incubated L‐929 fibroblasts or non‐activated J774 macrophages. The inhibition was potent, rapid and reversible when the NO was removed by adding oxyhaemoglobin or by inhibiting iNOS. Exogenously added NO also rapidly and reversibly inhibited cellular respiration over the same range of NO concentrations. This inhibition was competitive with oxygen and due to direct inhibition of cytochrome oxidase. Thus, NO generated by one cell can regulate the respiration of adjacent cells, supporting the hypothesis that NO may be a physiological and/or pathological regulator of cellular respiration, via its inhibition of cytochrome oxidase.

Signalling activity of beta-catenin targeted to different subcellular compartments.

Beta-catenin plays a dual role as an adhesion molecule in adherens junctions at the plasma membrane and as a key intermediate in the canonical Wnt signalling pathway. The cytosolic soluble pool of beta-catenin, involved in the transmission of the Wnt signal, is normally subjected to rapid protein degradation. On activation of the Wnt cascade, beta-catenin becomes stabilized and then translocates into the nucleus where it co-activates transcription factors of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family. The expression of plasma membrane-targeted forms of beta-catenin has been shown to also activate TCF/LEF-dependent transcription and different mechanisms have been put forward. In the present study, we have undertaken a systematic analysis of the signalling capability of non-degradable forms of beta-catenin targeted to different cellular compartments. beta-Catenin targeted to the plasma membrane activated transcription to a greater extent compared with non-targeted beta-catenin, and led to a marked stabilization of cytosolic soluble beta-catenin. These effects were independent of the competition with endogenous beta-catenin for binding to E-cadherin at the plasma membrane, since targeting non-degradable beta-catenin to other cellular compartments, i.e. the outer mitochondrial membrane and the endoplasmic reticulum membrane, also resulted in the accumulation of cytosolic wild-type beta-catenin and activation of beta-catenin-dependent signalling. In contrast, nuclear-targeted beta-catenin was without significant effect on cytosolic wild-type beta-catenin and did not activate transcription. Our results suggest that cytosolic accumulation of beta-catenin is a prerequisite for the activation of TCF/LEF-dependent transcription in the nucleus.

Expression of human nitric oxide synthase isozymes

Abstract (1) High levels of human eNOS and nNOS can be produced using the Sf 21/baculovirus expression system. (2) Recombinant iNOS, although produced at lower levels, provides significant amounts of enzyme that can be purified for further biochemical characterization. (3) Recombinant baculovirus-derived human NO synthase isozymes have biochemical properties that are highly similar to their native counterparts. (4) Large-scale production of recombinant NO synthase enzymes reduces the risk of batch-to-batch variation. (5) The production of recombinant human NO synthases obviates the need for a supply of tissue and also reduces the risks associated with handling human material.