Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon y, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shockIn biological systems, nitric oxide (NO) is produced via the oxidation of L-arginine by enzymes known as nitric oxide synthases (NOSs). Three NOS isozymes have been described (for recent reviews see refs. 1 and 2). These include constitutively expressed neuronal (3, 4) and endothelial (5, 6) enzymes and a cytokine-induced enzyme found in macrophages (7), hepatocytes (8), and a variety of other cells (9, 10). Although the biological functions of these enzymes are not completely understood, neuronal NOS is thought to play a role in neurotransmission (11), endothelial NOS is involved in regulation ofvascular tone (12, 13), and inducible NOS (iNOS) is involved in immune defense (14,15). The constitutively synthesized neuronal and endothelial enzymes produce small amounts of NO in response to increases in intracellular calcium levels. In contrast, iNOS is synthesized de novo in response to a variety of inflammatory stimuli and produces large amounts of NO over prolonged periods of time (16). NO produced by iNOS has been shown to be beneficial through its antitumor (17, 18) and antimicrobial (15) activities, but it is also thought to cause tissue damage and contribute to pathology in a variety of inflammatory conditions including rheumatoid arthritis (19,20), inflammatory bowel disease (21), and septic shock (22,23).Septic shock is usually the result of a systemic Gram-negative bacterial infection and is characterized by hypotension and the failure of a number of organ systems, especially the liver, kidney, and heart (24). The bacterial membrane component, lipopolysaccharide (LPS), induces the production of host inflammatory mediators such as tumor necrosis factor a, interferon y (IFN-,y), and interleukin 1,B, which in turn cause an increase in the expression of iNOS. The large amount of NO produced by iNOS has been hypothesized to contribute to LPS-induced hypotension and mortality.To be...

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2" over Mg2".

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A combination of cytokines induced the expression of nitric oxide synthase (NOS) in a human colorectal adenocarcinoma cell line, DLD-1. We have purified the enzyme and examined some of its biochemical properties. An antiserum to an inducible NOS from murine macrophages cross-reacted with the DLD-1 NOS. The purified human and murine enzymes displayed a similar lack of dependence on exogenous calcium and calmodulin for activity, which contrasts with the requirement for calcium and calmodulin of purified brain and endothelial isoforms of NOS. We have also isolated a cDNA for a cytokine-induced NOS from DLD-1 cells. Sequence analysis of this cDNA and NOS cDNAs from human liver, smooth muscle, and macrophages suggests that, at the genetic level, there is a single isoform of human-inducible NOS.