Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (>90% for isoforms I and III, >80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(7?)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca 2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. It has also been implicated in T he initial evidence for the production of nitrogen oxides in mammals came from experiments demonstrating nitrate production in germ-free rats. 1This triggered the search for mammalian cells capable of synthesizing nitrogen oxides and in 1985 led to the discovery that macrophages could be induced with lipopolysaccharide to produce significant amounts of both nitrite and nitrate. 2 Further work demonstrated that L-arginine was the substrate for this pathway and that L-citrulline was formed as a coproduct.3 -4 One year later nitric oxide (NO) was identified as the initial product that is subsequently oxidized to nitrite and nitrate. 5 In parallel, Furchgott and coworkers* 7 had discovered endothelium-derived relaxing factor (EDRF). It had been established that, similar to nitrovasodilators, the EDRF-mediated vasodilatation was associated with increased levels of cyclic GMP and activation of cyclic GMP kinase activity in smooth muscle cells 810 and that the EDRF could directly stimulate purified soluble guanyh/1 cyclase. 1112 In 1987 it was concluded that NO can account for the biologic activity of EDRF, 1315 and, analogous to the macrophage system, L-arginine was established as a substrate for EDRF/NO synthesis in endothelial cells. physiological research demonstrated that stimulation of neuronal cells and brain slices with agonists leads to the release of a labile mediator that stimulates guanylyl cyclase and has the properties of NO.1820 During the past 4 years, significant progress has been made elucidating the mechanism of NO synthesis, the NO synthases involved, and the functions of NO in different biologic systems. The present review attempts to summarize this progress with some emphasis on the cardiovascular system. Isozymes of NO SynthaseMany cells are capable of synthesizing NO. Three isozymes of NO synthase (EC 1.14.13.39) have been...

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The integrity of peripheral nerves relies on communication between axons and Schwann cells. The axonal signals that ensure myelin maintenance are distinct from those that direct myelination and are largely unknown. Here we show that ablation of the prion protein PrP(C) triggers a chronic demyelinating polyneuropathy (CDP) in four independently targeted mouse strains. Ablation of the neighboring Prnd locus, or inbreeding to four distinct mouse strains, did not modulate the CDP. CDP was triggered by depletion of PrP(C) specifically in neurons, but not in Schwann cells, and was suppressed by PrP(C) expression restricted to neurons but not to Schwann cells. CDP was prevented by PrP(C) variants that undergo proteolytic amino-proximal cleavage, but not by variants that are nonpermissive for cleavage, including secreted PrP(C) lacking its glycolipid membrane anchor. These results indicate that neuronal expression and regulated proteolysis of PrP(C) are essential for myelin maintenance.

Summary The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ+-derived cells abolished FDC, indicating that FDC originate from PDGFRβ+ cells. Lymphotoxin-α-overexpressing prion protein (PrP)+ kidneys developed PrP+ FDC after transplantation into PrP mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ+ stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR) kidney capsules, differentiated into Mfge8+CD21/35+ FcγRIIβ+PrP+ FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ+ FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation.

The occurrence of multiple strains of prions may reflect conformational variability of PrP(Sc), a disease-associated, aggregated variant of the cellular prion protein, PrP(C). Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.