A subclone of the human colon adenocarci-

Inbred strains of mice differ considerably in their innate resistance to leishmanial infection. BALB/c mice are highly susceptible to cutaneous leishmaniasis caused by Leishmania major, whereas CBA mice are resistant. We now show that this resistance correlates with the ability of macrophages to synthesize nitric oxide (NO) following activation with interferon-gamma or tumor necrosis factor alpha. Furthermore, the larger amounts of NO generated by resistant macrophages are related to higher levels of NO synthase activity, a difference which is not attributable to the number or the affinity of the receptors for interferon-gamma on these cells. The level of NO synthesis by activated macrophages was also correlated to the resistance in a number of other inbred mouse strains tested; macrophages from the resistant B10.S, C57BL and C3H mice produced significantly higher levels of NO than the macrophages from the susceptible BALB.b and DBA/2 mice.

Incubation of human articular chondrocytes with interleukin 113 results in the time-dependent expression of nitric oxide (NO) synthase. We report here the isolation of a cDNA clone which encodes a protein of 1153 amino acids with a molecular mass of 131,213 Da and a calculated isoelectric point of 7.9. CHO cells transfected with a plasmid harboring this cDNA clone expressed NO synthase activity that was inhibited by some L-arginine analogues. The deduced amino acid sequence of the human chondrocyte inducible NO synthase shows 51% identity and 68% similarity with the endothelial NO synthase and 54% identity and 70% similarity with the neuronal NO synthase. The similarity (88%) between the human chondrocyte NO synthase cDNA sequence and that reported for the murine macrophage suggests that the inducible class of enzyme is conserved between different cell types and across species.The synthesis of nitric oxide (NO) from L-arginine is now recognized as an important pathway for regulating the function of a wide variety of cells and tissues. NO exerts many of these effects through activation of the soluble guanylate cyclase (1). In the vessel wall, NO is synthesized by the vascular endothelium, to regulate smooth muscle tone and thus blood pressure (2-7). NO synthase is also present in the central nervous system, where NO is a neurotransmitter/ neuromodulator mediating the action of glutamate on N-methyl-D-aspartate receptors (8-11) and mediating/ modulating transmission in nerves previously recognized as nonadrenergic and noncholinergic (12). NO can also act as an autocrine regulator of some cells, including platelets, where it modulates their activation (13).NO generated by activated macrophages is also an important effector molecule in host defense, through a mechanism involving its interaction with iron-sulfur-centered enzymes (14) and/or superoxide anions (15). In this role, NO has been shown to possess antitumor (16) and antimicrobial activity against various parasites in vitro (17) and in vivo (18).NO is synthesized from L-arginine by the action of NO synthase(s), generating citrulline as a co-product. These enzymes are all NADPH-, FAD-, FMN-, and tetrahydrobiopterin-dependent (19). Both the neuronal and inducible NO synthases are P450-type heme proteins (20). In addition to control at the enzyme level, NO synthesis can also be regulated through the expression of different enzymes in various cell types. In endothelial cells (21,22) and neuronal (9, 10) and other (13, 23, 24) tissues, NO synthase activity is constitutively expressed and has a requirement for Ca2+ and calmodulin (22). In contrast, NO synthase is synthesized de novo in macrophages, hepatocytes, Kupffer cells, vascular smooth muscle, and vascular endothelium following activation with endotoxin and/or cytokines (1,19). The inducibleThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.NO sy...

Activation of murine microglial and macrophage cell lines with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) resulted in the induction of the inducible form of nitric oxide synthase (NOS) and the release of micromolar amounts of NO into the surrounding medium. The synthesis of NO was associated with increased cellular membrane damage as assessed by trypan blue dye exclusion and the leakage of lactate dehydrogenase into the cell culture medium. However, the synthesis and release of cytokines was largely unaffected. NO-mediated cell damage was also accompanied by a marked decrease in the intracellular levels of reduced glutathione and ATP. In addition, significant inhibition of mitochondrial respiratory chain enzyme activities was seen following cellular activation. However, citrate synthase activity (a mitochondrial matrix enzyme) was not detectable in the extracellular supernatants, suggesting preservation of the integrity of the mitochondrial inner membrane following activation. These effects were largely prevented by the addition of the NOS inhibitor, N-guanidino monomethyl L-arginine during the activation period. Our observations demonstrate that induction of NOS activity in microglia results in damage to the plasma membrane leading to a loss of glutathione, complex-specific inhibition of the mitochondrial electron transport chain and depletion of cellular ATP. Our data suggest that pharmacological modulation of NOS activity in activated microglia in vivo may prevent cellular damage to bystander cells such as neurons, astrocytes and oligodendrocytes, as well as to microglia themselves.

The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.