Nitric oxide (NO) is important in many biological functions. It is generated from L-arginine by the enzyme NO synthase (NOS). The cytokine-inducible NOS (iNOS) is activated by several immunological stimuli, leading to the production of large quantities of NO which can be cytotoxic. To define the biological role of iNOS further, we generated iNOS mutant mice. These are viable, fertile and without evident histopathological abnormalities. However, in contrast to wild-type and heterozygous mice, which are highly resistant to the protozoa parasite Leishmania major infection, mutant mice are uniformly susceptible. The infected mutant mice developed a significantly stronger Th1 type of immune response than the wild-type or heterozygous mice. The mutant mice showed reduced nonspecific inflammatory response to carrageenin, and were resistant to lipopolysaccharide-induced mortality.

Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

We have ex ed the effects of pregnancy and sex hormones on calciumdependent and m-independent nitric oxide synthas (NOSs) in the guinea pig. Pregnancy (near term) caused a >4-fold iase in the activity of cacium-dependent NOS in the uterine artery and at least a doubling in the heart, kidney, skeletal muscle, esophagus, and cerebeflum. The increase in NOS activity in the cerebellum during pregnanc was nhibited by the estn-receptor antagonittam len. Treatment with a ol (but not p rone) also i ed calcium-dependent NOS activity in the tissues examined from both females and males. Testosterone a calcium-dependent NOS only in the cerebeflum. No sign nt change in calcium-independent NOS acv was observed either during pregency or after the admiration f any sex hormone. Both pregan and estradol treatment increased the amount of mRNAs for NOS Isogymes eNOS and nNOS in skeletal muscle, 8 that the ireases in NOS activity result from enzyme induction. Thus both eNOS and nNOS are subject to rution by esten, an action that could explain some of the changes that occur during pegn and some gender differences in physiology and pa polg.Nitric oxide (NO) synthases (NOSs) constitute a family of isozymes that catalyze the oxidation of L-arginine to NO and citrulline. First identified in the vascular endothelium (1, 2), NO synthesis has subsequently been shown to play important roles in the regulation of vascular and gastrointestinal tone, in cell-mediated cytotoxicity against bacteria and tumors, and in a variety of central and peripheral nervous system activities (for review, see ref.3). NOSs can be divided into two functional classes based on their sensitivity to calcium (3). The cytokine-or bacterial product-inducible isoenzyme iNOS binds calmodulin tightly at resting intracellular calcium concentrations. The constitutive forms, isozymes eNOS (originally described in endothelial cells) and nNOS (originally described in neuronal tissue), bind calmodulin in a reversible and calcium-dependent fashion. The mechanisms by which their synthesis is controlled are unknown.The cDNA species encoding the rat, mouse, and human nNOS, the human and bovine eNOS, and iNOS from several species and cell types have been cloned and sequenced (4-17). ref. 28). This protection is lost with the onset of menopause or after surgical castration. This observation could be linked to reports that NO might slow the development ofatherosclerosis by inhibiting the proliferation of smooth muscle cells while stimulating endothelial cell proliferation (29,30).We therefore decided to investigate the hypothesis that sex hormones could regulate constitutive NOSs. We tested this in female and male guinea pigs by determining the effect of pregnancy on NOS activity and by measuring both NOS activity and NOS mRNA before and after sex hormone therapy. MATERIALS AND METHODSHartley guinea pigs of similar chronological age were obtained from a commercial breeder (Charles River Breeding Laboratories): males and nonpregnant females (600-700 g) and pregnant females of >5...

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties. Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis by upregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53, poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease.