Site-directed mutagenesis of glutathione S-transferase YaYa: functional studies of histidine, cysteine, and tryptophan mutants.

The rat cytosolic glutathione S-transferase Ya subunit contains three histidine residues (at positions 8, 143, and 159), two cysteine residues (at positions 18 and 112), and a single tryptophan residue (at position 21). Histidine, cysteine, and tryptophan have been proposed to be present either near or at the active site of other glutathione S-transferase subunits. The functional role of these amino acids at each of the positions was evaluated by site-directed mutagenesis in which valine or asparagine, alanine, and phenylalanine were substituted for histidine, cysteine, and tryptophan, respectively. Mutant enzymes H8V, H143V, H159N, C112A, and W21F retained either full or better catalytic efficiencies (k(cat)/Km) toward 1-chloro-2,4-dinitrobenzene and glutathione. Lower but significant k(cat)/Km values were observed for H159V and C18A toward 1-chloro-2,4-dinitrobenzene. Some mutants displayed different thermal stabilities and intrinsic fluorescence intensities, but all retained the ability to bind heme. These results indicate that histidine, cysteine, and tryptophan in the glutathione S-transferase Ya subunit are not essential for catalysis nor are they involved in the binding of heme to the YaYa homodimer.     

Models,mechanisms and pathways of succession

The study of succession has been hampered by the lack of a general theory. This is illustrated by confusion over basic concepts and inadequacy of certain models. This review clarifies the basic ideas of pathway, mechanism, and model in succession. Second, in order to prevent inappropriate narrowness in successional studies, we analyze the mechanistic adequacy of the most widely cited models of succession, those of Connell and Slatyer. This analysis shows that models involving a single pathway or a dominant mechanism cannot be treated as alternative, testable hypotheses. Our review shows much more mechanistic richness than allowed by these widely cited models of succession. Classification of the mechanisms of specific replacement, called for by existing models, is problematic and less valuable than the search for the actual mechanisms of particular seres. For example, the “tolerance” mechanism of succession has at least two contrasting meanings and is unlikely to be disentangled from the “inhibition” mechanism in field experiments. However, the understanding of particular species replacements through experiment and knowledge of the conditions of a particular sere and species life histories is a reasonable and desirable goal. Finally, we suggest the need for a broad mechanistic concept of succession. Thus, based on classical causes of succession that have survived recent scrutiny, we erect a framework of successional mechanisms. This framework aims at comprehensiveness, and specific mechanisms are nested within more general causes. As a result of its breadth and hierarchical structure, the framework performs two important functions: First, it provides a context for studies at specific sites and, second, is a scheme for formulating general and testable hypotheses. The review of specific successional mechanisms and the general mechanistic framework can together guide future work on succession, and may foment the development of a broad theory.     

Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.     

Managing the social environments of flamingos for reproductive success

Recent research has shown that large flamingo flocks are more likely to experience reproductive success than small flocks and that there is a positive relationship between behavioral stimulation from group displays and reproductive success. This study compares the group displays and reproductive success in a captive flock of Caribbean flamingos over 6 years at the National Zoological Park. Birds were added to the flock in two different years. In each case, the flock produced fertile eggs during the breeding season following the addition of new birds. Furthermore, the addition of new birds in 1988, and then again in 1991, significantly increased group display activity over the previous year when the flock size was lower. That the addition of birds stimulated group display activity suggests an alternative management strategy for promoting reproduction in captivity: separate birds in a flock and then reunite them prior to the breeding season. © 1994 Wiley-Liss, Inc.