Salmonella enterica serovar typhimurium trxA mutants are protective against virulent challenge and induce less inflammation than the live-attenuated vaccine strain SL3261.

In Salmonella enterica serovar Typhimurium, trxA encodes thioredoxin 1, a small, soluble protein with disulfide reductase activity, which catalyzes thiol disulfide redox reactions in a variety of substrate proteins. Thioredoxins are involved as antioxidants in defense against oxidative stresses, such as exposure to hydrogen peroxide and hydroxyl radicals. We have made a defined, complete deletion of trxA in the mouse-virulent S. Typhimurium strain SL1344 (SL1344 trxA), replacing the gene with a kanamycin resistance gene cassette. SL1344 trxA was attenuated for virulence in BALB/c mice by the oral and intravenous routes and when used in immunization experiments provided protection against challenge with the virulent parent strain. SL1344 trxA induced less inflammation in murine spleens and livers than SL3261, the aroA mutant, live attenuated vaccine strain. The reduced splenomegaly observed following infection with SL1344 trxA was partially attributed to a reduction in the number of both CD4(+) and CD8(+) T cells and B lymphocytes in the spleen and reduced infiltration by CD11b(+) cells into the spleen compared with spleens from mice infected with SL3261. This less severe pathological response indicates that a trxA mutation might be used to reduce reactogenicity of live attenuated vaccine strains. We tested this by deleting trxA in SL3261. SL3261 trxA was also less inflammatory than SL3261 but was slightly less effective as a vaccine strain than either the SL3261 parent strain or SL1344 trxA.

Structure of the ribosomal interacting GTPase YjeQ from the enterobacterial species Salmonella typhimurium.

The YjeQ class of P-loop GTPases assist in ribosome biogenesis and also bind to the 30S subunit of mature ribosomes. YjeQ ribosomal binding is GTP-dependent and thought to specifically direct protein synthesis, although the nature of the upstream signal causing this event in vivo is as yet unknown. The attenuating effect of YjeQ mutants on bacterial growth in Escherichia coli makes it a potential target for novel antimicrobial agents. In order to further explore the structure and function of YjeQ, the isolation, crystallization and structure determination of YjeQ from the enterobacterial species Salmonella typhimurium (StYjeQ) is reported. Whilst the overall StYjeQ fold is similar to those of the previously reported Thematoga maritima and Bacillus subtilis orthologues, particularly the GTPase domain, there are larger differences in the three OB folds. Although the zinc-finger secondary structure is conserved, significant sequence differences alter the nature of the external surface in each case and may reflect varying signalling pathways. Therefore, it may be easier to develop YjeQ-specific inhibitors that target the N- and C-terminal regions, disrupting the metabolic connectivity rather than the GTPase activity. The availability of coordinates for StYjeQ will provide a significantly improved basis for threading Gram-negative orthologue sequences and in silico compound-screening studies, with the potential for the development of species-selective drugs.

Structural characterization of Salmonella typhimurium YeaZ, an M22 O-sialoglycoprotein endopeptidase homolog.

The Salmonella typhimurium "yeaZ" gene (StyeaZ) encodes an essential protein of unknown function (StYeaZ), which has previously been annotated as a putative homolog of the Pasteurella haemolytica M22 O-sialoglycoprotein endopeptidase Gcp. YeaZ has also recently been reported as the first example of an RPF from a gram-negative bacterial species. To further characterize the properties of StYeaZ and the widely occurring MK-M22 family, we describe the purification, biochemical analysis, crystallization, and structure determination of StYeaZ. The crystal structure of StYeaZ reveals a classic two-lobed actin-like fold with structural features consistent with nucleotide binding. However, microcalorimetry experiments indicated that StYeaZ neither binds polyphosphates nor a wide range of nucleotides. Additionally, biochemical assays show that YeaZ is not an active O-sialoglycoprotein endopeptidase, consistent with the lack of the critical zinc binding motif. We present a detailed comparison of YeaZ with available structural homologs, the first reported structural analysis of an MK-M22 family member. The analysis indicates that StYeaZ has an unusual orientation of the A and B lobes which may require substantial relative movement or interaction with a partner protein in order to bind ligands. Comparison of the fold of YeaZ with that of a known RPF domain from a gram-positive species shows significant structural differences and therefore potentially distinctive RPF mechanisms for these two bacterial classes.

Early responses to Salmonella typhimurium infection in mice occur at focal lesions in infected organs.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid, with key roles for cytokines and possibly also inducible nitric oxide synthase (iNOS), in mediating host responses to infection. In this paper we demonstrate that iNOS mRNA, protein and enzyme activity is induced within spleens and livers of infected mice as early as 5 h post-infection. Immunohistochemistry and in situ hybridization indicated that iNOS expression occurs predominantly in macrophages in localized, discrete foci in the infected organs. iNOS activity in spleen and liver was not detectable in uninfected control mice. The presence of mRNA encoding pro-inflammatory cytokines (TNFalpha, IL-1beta and IFNgamma) in infected organs was measured using RT-PCR, all three being present from 2 h post-infection onwards, but not before. These data show that there is a very early host response to S. typhimurium infection in mice, limited to foci within the infected organs.

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases.

Survey of the allelic frequency of a NOS2A promoter microsatellite in human populations: assessment of the NOS2A gene and predisposition to infectious disease.

Allelic frequencies of a (CCTTT)(n) pentanucleotide repeat in the NOS2A promoter region were determined in a total of 1393 unrelated individuals from five specific population groups in four continents: Africa, Europe, Asia, and the Caribbean. There were highly significant differences in allele frequencies between the ethnically diverse populations. The repeat variation may have implications for the selective pressure of malaria or other infectious diseases that may operate at the NOS2 locus.

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding.

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.

The CCTTT polymorphism in the NOS2A gene is associated with dementia with Lewy bodies.

We report the analysis of the allele distribution of a (CCTTT)n pentanucleotide repeat within the promoter region of the NOS2A gene in DNA samples from patients with autopsy confirmed Alzheimer's disease (AD) and dementia with Lewy bodies (DLB) type. A significant difference was observed in the allelic distribution between the control group and the DLB group (chi2 = 15.175, df = 5; p<0.01), with an increased occurrence of the eight and nine repeat alleles, and a marked under representation of the 11 repeat allele. Genotype frequencies in the DLB group also differed significantly from controls (p<0.012). These results suggest that variations in the NOS2A gene may predispose to the development of DLB.

Molecular characterization and in situ localization of a full-length cyclic nucleotide-gated channel in rat brain.

Ion channels gated directly by cyclic nucleotides are required for the transduction of sensory signals in photoreceptor cells and olfactory cells. Cyclic nucleotide-gated (CNG) channels may also be expressed in the central nervous system because partial transcripts that share homology with CNG channels have been found therein. We have now isolated and cloned a full-length CNG channel cDNA from adult rat brain. The sequence is identical to that of the alpha-subunit originally found in the olfactory epithelium (CNCalpha3). In situ hybridization, using probes specific for the CNCalpha3 mRNA, suggest that this channel is expressed widely in the rat brain, albeit mostly at relatively low levels. Certain neuronal populations, however, such as deep cerebellar nuclei, olfactory bulb mitral cells and cerebellar Purkinje neurons, appeared specially enriched. The study demonstrates for the first time that a full-length CNG channel mRNA is expressed in the brain, supporting the possibility that CNG channels are involved in central neural communication and plasticity. The sequence reported in this paper has been deposited in the GenBank data base (accession no. AF126808).

Genotyping and functional analysis of a polymorphic (CCTTT)(n) repeat of NOS2A in diabetic retinopathy.

Accumulating evidence shows that the severity and rapidity of onset of diabetic retinopathy are influenced by genetic factors. Expression of the nitric oxide synthases is altered in the retinal vasculature in the early stages of diabetic retinopathy. We analyzed the allele distribution of a polymorphic pentanucleotide repeat within the 5' upstream promoter region of the NOS2A gene in samples of diabetic patients. In diabetic patients from Northern Ireland, the 14-repeat allele of the NOS2A marker was significantly associated with the absence of diabetic retinopathy. Carriers of this repeat had 0.21-fold the relative risk of developing diabetic retinopathy than noncarriers of this allele. They also had significantly fewer renal and cardiovascular complications. The ability of differing numbers of (CCTTT)(n) pentanucleotide repeats to induce transcription of the NOS2A gene was analyzed using a luciferase reporter gene assay in transfected colonic carcinoma cells. Interleukin 1beta (IL-1beta) induction was most effective in constructs carrying the 14-repeat allele. When cells were incubated in 25 mM glucose to mimic the diabetic state, IL-1beta induction was inhibited in all cases, but to a significantly lesser extent with the 14-repeat allele. These unique properties of the 14-repeat allele may confer selective advantages in diabetic individuals, which may delay or prevent microvascular complications of diabetes.

Identification of two human dimethylarginine dimethylaminohydrolases with distinct tissue distributions and homology with microbial arginine deiminases.

Methylarginines inhibit nitric oxide synthases (NOS). Cellular concentrations of methylarginines are determined in part by the activity of dimethylarginine dimethylaminohydrolase (DDAH; EC 3.5.3. 18). We have cloned human DDAH and identified and expressed a second novel DDAH isoform (DDAH I and II respectively). DDAH I predominates in tissues that express neuronal NOS. DDAH II predominates in tissues expressing endothelial NOS. These results strengthen the hypothesis that methylarginine concentration is actively regulated and identify molecular targets for the tissue and cell-specific regulation of methylarginine concentration.

Cytokine-induced venodilatation in humans in vivo: eNOS masquerading as iNOS.

OBJECTIVE: Venodilatation is a feature of endotoxaemia and sepsis. We have tested directly the hypothesis that three cytokines (IL-1 beta, TNF alpha and IL-6) generated during endotoxaemia affect venous tone in humans in vivo by increasing NO generation and explored whether the NO comes from the iNOS or eNOS isoform. DESIGN AND INTERVENTION: Cytokines were given into a superficial vein in very low doses sufficient only to produce changes in the study vessel. The effects of cytokines on the response to noradrenaline were examined. RESULTS: IL-1 beta increased basal NO-induced dilatation in the study vein, and this was sufficient to attenuate the constrictor response to exogenous noradrenaline or sympathetic stimulation. The effects were maximal at 6 h and both NG-monomethyl-L-arginine and aminoguanidine caused significant reversal of the IL-1 beta effects. However, no induction of iNOS mRNA was detected in the tissue samples. Instead, mRNA encoding eNOS and GTP cyclohydrolase-1 was detected in all vessels. CONCLUSION: The simplest explanation of these results is that IL-1 beta induces expression of GTP cyclohydrolase-1 which leads to increased generation of BH4 and activation of eNOS. This study identifies IL-1 beta as a key cytokine causing physiologically significant venodilatation in humans by increasing NO generation and suggests that this can occur even in the absence of iNOS expression.

Temporal expression of inducible nitric oxide synthase in mouse and human placenta.

The aim of this study is to investigate the changes in expression and activity of inducible nitric oxide synthase (iNOS) in the developing murine embryo and mouse and human placenta. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and in-situ hybridization (ISH) we identified iNOS mRNA in mouse placenta at 9.5, 12, 14, 16, 18 and 20 days post coitum. Northern blot analysis demonstrated that the quantity of murine iNOS transcript was expressed at a stable level between days 12-20 although the level of calcium-independent NOS activity declined with advancing gestation. RT-PCR detected iNOS-specific mRNA in murine embryonic stem cells, but not in embryos at later stages (4-cell or blastocyst). ISH failed to show iNOS-specific mRNA in either murine placenta or the underlying myometrium on day 7, but did so in the trophoblast by day 9.5. Later in gestation, extensive labelling was observed in both spongiotrophoblast and trophoblast giant cells. iNOS mRNA was also detected both in immature human placentae (16-18 weeks) and at term, predominantly in syncytiotrophoblasts and placental artery smooth muscle. In conclusion, iNOS is constitutively expressed in mouse and human placenta at a time and in a location that suggests a role in placentation.

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 10(8) 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 10(8) per organ, mice infected with mutant bacteria survived. Bacterial growth continued until unprecedentedly high counts of 10(9) 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.

Induction and role of NO synthase in hypotensive hepatic failure.

Nitric oxide (NO) plays an important role in the physiological and pathophysiological control of the vascular system. NO is synthesized by isoforms of the enzyme NO synthase (NOS). Hepatic failure is complicated by hypotension, low systemic vascular resistance, and resistance to vasoconstrictor drugs. The potential role of NO in these abnormalities was investigated by using in vitro pharmacological interventions on hepatic arteries obtained from both donor and recipient patients at the time of liver transplantation. The presence of NOS mRNA was investigated by reverse transcription polymerase chain reaction (RT-PCR) with primers designed from human endothelial NOS (eNOS) and inducible NOS (iNOS) cDNA sequences. Arteries from patients with hepatic failure had an impaired constrictor response to phenylephrine compared with those of donor arteries. The constrictor effect of phenylephrine was potentiated by NG-monomethyl-L-arginine, an inhibitor of NOS, which had no effect in donor control arteries. RT-PCR identified human eNOS mRNA in donor and recipient arteries and human iNOS mRNA in recipient arteries only. Induction of NOS in the vasculature with subsequent NO-induced vasodilatation may therefore contribute to the hemodynamic abnormalities observed in hepatic failure and potentially in other pathologies associated with endotoxemia. Whether selective inhibitors of iNOS will improve hemodynamic control or clinical outcome in these conditions requires further study.

Delineation of the arginine- and tetrahydrobiopterin-binding sites of neuronal nitric oxide synthase.

Nitric oxide synthase (EC 1.14.13.39) catalyses the conversion of arginine, NADPH and oxygen to nitric oxide and citrulline, using haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin), calmodulin, FAD and FMN as cofactors. The enzyme consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region that contains the arginine and tetrahydrobiopterin sites and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. By using domain boundaries defined by limited proteolysis of full-length enzyme, recombinant haem-binding regions of rat brain neuronal nitric oxide synthase were expressed and purified. Two proteins were made in high yield: one, corresponding to residues 221-724, contained bound haem and tetrahydrobiopterin and was able to bind Nomega-nitro-l-arginine (nitroarginine) or arginine; the other, containing residues 350-724, contained bound haem but was unable to bind tetrahydrobiopterin, nitroarginine or arginine. These results showed that rat brain neuronal nitric oxide synthase contains a critical determinant for arginine/tetrahydrobiopterin binding between residues 221 and 350. Limited proteolysis with chymotrypsin of the former protein resulted in a new species with an N-terminal residue 275 that retained the ability to bind nitroarginine, further defining the critical region for arginine binding as being between 275 and 350. Comparison of the sequences of nitric oxide synthase and the tetrahydrobiopterin-requiring amino acid hydroxylases revealed a similarity in the region between residues 470 and 600, suggesting that this might represent the core region of the pterin-binding site. The stoichiometries of binding of substrate and cofactors to the recombinant domains were not more than 0.5 mol/mol of monomer, suggesting that there might be a single high-affinity site per dimer.