The interaction of catecholamines and adrenal corticosteroids in the induction of phosphopyruvate carboxylase in rat liver and adipose tissue

Catecholamines induced an increase in the activity of rat adipose tissue and liver phosphopyruvate carboxylases that was maintained for 48h. The response of adipose tissue phosphopyruvate carboxylase was blocked by actinomycin D, corticosteroids and propranolol, whereas corticosteroids and propranolol did not affect the liver enzyme. Cortisol phosphate, like actinomycin D, interfered only with the initiation of the increase in enzyme activity caused by noradrenaline, but not with the process of enzyme accumulation. In contrast, cycloheximide was effective in blocking enzyme induction throughout the course of the catecholamine effect. Adrenocorticotrophic hormone caused a short-term induction of adipose tissue phosphopyruvate carboxylase, which could be blocked by propranolol. Hepatic phosphopyruvate carboxylase, but not the adipose tissue enzyme, was induced by dibutyryladenosine 3':5'-cyclic monophosphate and by glucagon. Both nicotinic acid and nicotinamide decreased the normal induction of adipose tissue phosphopyruvate carboxylase caused by starvation, but only nicotinamide increased the activity of the liver enzyme.     

Nitrogen fertilization alters the functioning of arbuscular mycorrhizas at two semiarid grasslands

The effects of nitrogen (N) fertilization on arbuscular mycorrhizas were studied at two semiarid grasslands with different soil properties and N-enrichment history (Shortgrass Steppe in Colorado, and Sevilleta National Wildlife Refuge in New Mexico). These sites are part of the National Science Foundation's Long-Term Ecological Research Network. The experimental plots at Shortgrass Steppe were fertilized with ammonium nitrate (NH₄NO₃) from 1971 to 1975, and have not received additional N since then. The experimental plots at Sevilleta were also fertilized with NH₄NO₃, but were established in 1995, 2 years before the soils were used for this study. Greenhouse experiments were conducted to compare the growth response of local grasses to arbuscular mycorrhizal (AM) fungi from fertilized (FERT) and unfertilized (UNFERT) field soils, at each site. Two species per site were chosen, Bouteloua gracilis and Elymus elymoides from Shortgrass Steppe, and B. gracilis and B. eriopoda from Sevilleta. Plants were grown for 3 months at HIGH N and LOW N levels, with FERT or UNFERT soil inoculum and in a non-mycorrhizal condition. Fertilization with N altered the functioning of AM fungi at both sites. Grasses inoculated with AM fungi from UNFERT soils had the most tillers, greatest biomass and highest relative growth rates. There were no significant differences in the growth response of plants inoculated with AM fungi from FERT soils and the non-mycorrhizal controls. These results were consistent across sites and species except for the plants grown at LOW N in Sevilleta soils. These plants were deficient in N and phosphorus (P) and did not show growth enhancement in response to AM inoculation with either FERT or UNFERT soils. Percent root length colonized by AM fungi was not directly related to plant performance. However, enrichment with N consistently decreased root colonization by AM fungi in the grasses grown in soils from Shortgrass Steppe with high P availability (18.4 mg kg^–1), but not in the grasses grown in Sevilleta soils with low P availability (6.6 mg kg^–1). Our study supports the hypotheses that (1) fertilization with N alters the balance between costs and benefits in mycorrhizal symbioses and (2) AM fungal communities from N fertilized soils are less beneficial mutualists than those from unfertilized soils.     

Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.     

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55)

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.