W A Scott

Cyclic 3′,5′-AMP phosphodiesterase of Neurosporacrassa

Abstract Cyclic 3′,5′-AMP (cAMP) phosphodiesterase activity can be demonstrated in extracts of Neurospora crassa . The activity is particulate, has a pH optimum of 7.4, and consists of two forms that have different cAMP binding constants. Methylxanthines, inorganic phosphate, and EDTA are inhibitors of the diesterase as are ATP, ADP, and 8-bromo-cAMP. The enzymatic activity is stimulated by histamine and imidazole. These properties suggest that the Neurospora enzyme is more closely related to the mammalian than to bacterial cAMP phosphodiesterases.

Susceptibility of Entamoeba histolytica to oxygen intermediates.

To explore the susceptibility of the extracellular protozoan, Entamoeba histolytica, to toxic oxygen intermediates, trophozoites were exposed to fluxes of O2, H2O2, and OH. generated enzymatically by the glucose oxidase and xanthine oxidase reactions. HM-1 trophozoites were resistant to O2, but were readily killed by H2O2 alone. OH. and 1O2 were not required for effective amebicidal activity. The addition of peroxidase and halide enhanced trophozoite killing by H2O2. Sonicates of amebae contained virtually no catalase and little glutathione peroxidase activity which may contribute to susceptibility to H2O2. Coupled with our previous studies with Toxoplasma gondii and Leishmania spp. these observations indicate that there is a broad spectrum of susceptibility of intra- and extracellular pathogenic protozoa to killing by oxygen intermediates.

Leukotriene C4 Is Released from the Anaphylactic Heart: A Case for Its Direct Negative Inotropic Effect

Data from our laboratory indicate that the heart reacts as a target organ in systemic hypersensitivity reactions (Capurro and Levi, 1975; Graver et al., 1983). Cardiac dysfunction observed during anaphylaxis in the guinea pig (Capurro and Levi, 1975; Zavecz and Levi, 1977) resembles that reported in humans (Bernreiter, 1959; Both and Patterson, 1970; Criep and Woehler, 1971; Petsas and Kotier, 1973; Sullivan, 1982) and is caused by mediators released intracardially and reaching the heart from the lung (Zavecz and Levi, 1977). “Cardiac anaphylaxis” (Feigen and Prager, 1969) is characterized by tachycardia, arrhythmias, contractile failure, coronary constriction, and mediator release (Capurro and Levi, 1975; Levi and Allan, 1980; Levi et al., 1982). Tachycardia and arrhythmias are caused by the release of endogenous cardiac histamine, since they are reproduced by the intracardiac administration of histamine and abolished by antihistamines (Levi and Allan, 1980; Levi et al., 1982). On the other hand, anaphylactic coronary constriction is markedly reduced by cyclooxygenase inhibitors such as indomethacin or aspirin or by thromboxane syn-thetase inhibitors such as 1-(2-isopropylphenyl)imidazole (Allan and Levi, 1981). Furthermore, the intracardiac administration of U 46619, a stable thromboxane analogue, causes coronary constriction (Allan and Levi, 1980a). Thus, prostanoate compounds, particularly thromboxane, contribute to the fall in coronary flow rate that characterizes cardiac anaphylaxis (Levi et al., 1982). Other potent coronary-constricting agents, such as platelet-activating factor (PAF, AGEPC), are also likely to contribute to anaphylactic coronary constriction (Levi et al., 1984).

Defects of Glucose-6-phosphate and 6-Phosphogluconate Dehydrogenases in Neurospora and Their Pleiotropic Effects

Publisher Summary This chapter focuses on the defects of glucose-6-phosphate (G6P) and 6-phosphogluconate (6PG) dehydrogenases in neurospora and their pleiotropic effects. The various reaction sequences of an intact cell are not discrete entities in that activities of many metabolic pathways are regulated by the availability of intermediates or products derived from different areas of metabolism, particularly intermediary metabolism. Often different reaction sequences also utilize common metabolites and cofactors. These interactions provide the basis for elaborate control mechanisms and a method for coordinating the diverse functions of cellular physiology. Progress in dissecting the coordinate regulation of metabolism has been limited because such controls are complex. Mutants of Neurospora crassa can be divided into three major classes: nutritional, mitochondrial, and morphological. Strains that carry a defective G6P dehydrogenase or 6PG dehydrogenase have morphologically distinct phenotypes. These mutants, grouped together because of their common phenotypic traits, are characterized by abnormal mycelial growth patterns, aberrant hyphal morphologies, and greatly reduced growth rates.

Requirements of Δ9 and Δ12 fatty acid desaturation in Neurospora

Abstract Microsomes prepared from the wild-type strain and lipid auxotrophs of Neurospora were analyzed for Δ9-(stearoyl-CoA) and Δ12-(oleoyl-CoA) desaturase activities. The wild-type Δ9-desaturase was found to have a 20-fold higher specific activity and 2-fold lower activation energy than the Δ12-desaturase. In addition, Δ12-desaturase had higher Kmapp values for oleoyl-CoA and for NADH than the equivalent values for Δ9-desaturase. These properties were correlated with a rate-limiting role of Δ12-desaturase in the production of 18:2, the major fatty acid of Neurospora. The Δ12-desaturase also exhibited a higher tolerance to pH changes and to cyanide than did the Δ9-desaturase. Both activities could be measured in the same reaction mixture using stearoyl-CoA as the substrate, indicating a coupling of the two enzymes. Enrichment of cellular membranes of the wild-type Neurospora with 18:0 and 18:1, 18:2, 18:3 fatty acids led to the conclusion that the presence of excess substrate in the membrane induces activation of the appropriate desaturase. These experiments also suggested that the membrane fluidity, as determined by the degree of unsaturation of membrane fatty acids, may influence the activities of the desaturating enzymes. Perturbation of the polar head groups of the membrane phospholipids indicated that the correct composition of anionic phospholipids is an absolute requirement for the function of both desaturases. These studies show that the activities of the Δ9-desaturase and the Δ12-desaturase are regulated by a variety of factors and that the Δ12-desaturase is subjected to less stringent controls than the Δ9-desaturase.

Cyclic AMP levels in relation to membrane phospholipid variations in Neurospora crassa

The correlation between membrane phospholipid composition and total cyclic AMP levels was investigated by using Neurospora lipid auxotrophs under various supplementation conditions.The lipid composition of the supplemented cultures was determined, and the intracellular and extracellular cyclic AMP levels were measured at various stages of the culture growth. Kinetic parameters and the thermostability of adenylate cyclase and of cyclic AMP-dependent phosphodiesterase were measured under all supplementation conditions.In inositol deficient inl cultures the levels of intracellular cyclic AMP decreased exponentially towards the end of the log phase and thereafter. In chol-l; chol-2 cultures, grown in N-monomethylethanolamine and low choline supplementation, the level of intracellular cyclic AMP decreased as function of decreasing exogenous choline supplement. Rates of cyclic AMP extrusion in all cultures were comparable on dry weight basis, and thus not affected by the mycelial lipid composition. Adenylate cyclase activity and thermostability decreased under those supplementation conditions resulting in reduction of cyclic AMP. Cyclic AMP-dependent phosphodiesterase was insensitive to phospholipid changes.Accordingly, it is suggested that specific perturbations in cellular phospholipid composition affect the membranebound adenylate cyclase and hence the cyclic AMP synthesis in vivo.

Secretory functions of the mononuclear phagocyte.

The functional properties of mononuclear phagocytes include their well-known capacities for phagocytosis and pinocytosis. Work in recent years has provided greater understanding of the effector functions of these cells; in particular, their secretory nature. The versality of the macrophage has been a surprise; over 50 secretory products including mediators, cytocidal factors, and growth-promoting agents have been identified and triggers for some activities described.

Membrane Lipids and the Control of Endocytosis

Cellular membranes are oriented, two-dimensional systems composed of heterogeneous mixtures of lipids and proteins. The most commonly accepted structure of a cellular membrane, the fluid mosaic model of Singer & Nicholson (1972), is a lipid bilayer, with some protein molecules partially embedded and some transversing the lipid matrix. Both lipids and proteins undergo lateral motion within the plane of the lipid bilayer. Much evidence supports this dynamic picture of membranes. The concept of fluidity (Singer & Nicholson 1972) predicts that chemical perturbations of lipids can effect a redistribution of membrane components through the viscous two-dimensional system and thereby lead to new interactions of both lipids and proteins. Attenuation of membrane fluidity is thought to have an important role in controlling membrane-associated functions.