Neil R. Bastian

Assembly and regulation of NADPH oxidase and nitric oxide synthase

Research over the past year has revealed several interesting advances in the biosynthesis of the superoxide anion and nitric oxide. Highlights include the demonstration that the G protein Rac 2 is required for NADPH oxidase activation, the finding that nitric oxide is a feedback inhibitor of nitric oxide synthase isoforms, and the discovery that the continuous catalytic activity of the immune/inflammatory nitric oxide synthase is due to strong calmodulin binding, which is independent of elevated calcium levels. Interferon-gamma primes neutrophils and macrophages for both O2- and nitric oxide synthesis. However, NADPH oxidase and immune/inflammatory nitric oxide synthase are differentially regulated such that their activities are not simultaneously induced.

Nω-monomethyl-l-arginine inhibits nitric oxide production in murine cardiac allografts but does not affect graft rejection

Endogenous nitric oxide biosynthesis in mice receiving allogeneic heterotopic heart transplants was monitored as a function of time post-transplant. Nitric oxide production was measured by daily urine nitrate levels and by formation of paramagnetic heme-nitrosyl complexes in the cardiac tissue. Exogenous sources of urine nitrate and EPR signal were minimized by maintaining the animals on a low nitrite/nitrate diet. Urine nitrate peaked on postoperative day 7. A heme-nitrosyl EPR signal also appeared in the cardiac tissue on postoperative day 7 and remained unchanged in size until rejection on postoperative day 9 at which time the peak height of the signal nearly tripled. Some of the animals in the study were treated with the nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine which caused marked inhibition of urinary nitrate excretion and prevented heme-nitrosyl complex formation in beating hearts. However, administration of the inhibitor did not increase graft survival time. Low intensity heme-nitrosyl signals were identified in inhibitor-treated allogeneic hearts after rejection. Syngeneic heart transplants did not induce urinary nitrate excretion nor EPR signal formation. These results show that cytokine induced high output nitric oxide synthesis from L-arginine is a prominent biochemical component of the cell-mediated immune response to cardiac allografts in mice. However, nitric oxide production was not essential for rejection of cardiac allografts mismatched at the major histocompatibility locus.

The amino acid sequence of Rhodobacter sphaeroides dimethyl sulfoxide reductase

The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the proteins composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.

The Discovery of the Biological Synthesis of Nitric Oxide

We have reviewed the experimental work reported through 1988 that provided the foundation for the new field of NO* biology. In addition, we have described experiments carried out between 1988 and 1990 that led directly to the purification of the first NOS isoform. A wide range of biomedical scientists have extended and refined the core information published in the period through 1988, and the study of NO*biology has entered many new fields and resulted in many new discoveries. It is clear that this work has had, and will continue to have, a major impact on our understanding of physiology and pathophysiology.

Nitric Oxide Effects on Murine Cardiac Allografts

Biosynthesis of nitric oxide (NO) by mammalian cells has recently been demonstrated (Hibbs et al., 1987a; Iyengar et al., 1987; Hibbs et al., 1988; Marietta et al., 1988). NO synthesis is induced during a cell-mediated immune response to infection or tumor development. NO is biologically produced by the enzyme immune/inflammatory NO synthase (iNOS), which utilizes molecular oxygen to oxidatively deiminate a guanidino nitrogen atom of L-arginine with formation of L-citrulline and NO as the products (Hibbs et al., 1988; Marietta et al., 1988, Tayeh et al., 1989). iNOS is competitively inhibited by N-substituted L-arginine derivatives, including NG -monomethyl-L-arginine (MLA) (Hibbs et al., 1987a, 1987b; Granger et al., 1990, 1991). Nitric oxide reacts with O2 in a termolecular reaction to produce nitrate (NO3 −) and nitrite (NO2 −- NO2 − entering the vascular system is further oxidized by hemoglobin to NO3 − (Granger et al., 1991) and is excreted in the urine. When exogenous sources of nitrate are eliminated from the diet, urine nitrate can be used as a measure of NO biosynthesis (Granger et al., 1991).

Identification of Nitric Oxide-Derived EPR Signals in Human Cancers

Progressively growing cancers in animals and humans often have significant infiltration by host macrophages (Svennevig et al., 1979;; Wood & Gillespie, 1975; Eccles & Alexander, 1974a). Conflicting data exist concerning the role of these tumor-infiltrating macrophages. In some studies, the degree of tumor infiltration by macrophages has correlated with a decrease in metastases or increased survival (Lauder et al. 1977; Eccles & Alexander, 1974b; Underwood, 1974; Wood & Gillespie, 1975; Mahoney & Heppner, 1987). Other reports have suggested that macrophage infiltration of tumors may adversely affect host immune function and promote tumor growth (Mantovani et al., 1992; McBride, 1986; Eccles & Alexander, 1974b; Gillespie & Russell, 1980; Mills et al., 1992).

Interactions Between Cytokine Induced Nitric Oxide and Intracellular Iron

Cooperation between macrophages and T-lymphocytes is necessary for the development of cell-mediated immune (CMI) responses (Hibbs et al., 1990). It has long been understood that the CMI response is important in host defense against intracellular microorganisms (Mackaness, 1971; Hibbs et al., 1980). What was not known until relatively recently, however, was the identity of an important biochemical defense, induced during the CMI response as well as during innate resistance. As is now well documented, the unidentified defense system turned out to be cytokine induced biosynthesis of nitric oxide (NO). This previously unknown biochemical activity participates in defense of the host against certain intracellular pathogens, and has a complex role in the host-tumor interaction.