Cytochemical localization of Na+-K+-ATPase in rat type II pneumocytes

The distribution of sodium-potassium-activated adenosinetriphosphatase (Na+-K+-ATPase) in the alveolar portion of rat lungs was examined by indirect immunofluorescence with the use of a mouse monoclonal anti-rat Na+-K+-ATPase and by ultrastructural cytochemistry using p-nitrophenylphosphate as substrate. The reaction was inhibitable by 10 mM ouabain or by the omission of K+ from the reaction mixture. Cysteine or levamisole was used to inhibit alkaline phosphatase activity. By immunofluorescence, staining was confined to cuboidal cells in alveolar spaces. These were tentatively identified as type II pneumocytes. By ultrastructural cytochemistry reaction product was present on the cytoplasmic side of the basolateral membranes of type II pneumocytes. No reaction product was observed in type I pneumocytes or in endothelium. These results indicate that type II pneumocytes contain more Na+-K+-ATPase, an enzyme important in vectorial electrolyte transport, than type I pneumocytes or endothelial cells. More sensitive methods, however, are required to determine the amounts and distribution of this enzyme in type I pneumocytes and pulmonary vascular endothelial cells.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

Tropical Vine Growth and the Effects on Forest Succession: A Review of the Ecology and Management of Tropical Climbing Plants

The climbing habit has evolved independently in many plant taxa, offering vines the ability to compete with non-climbing vegetation for resources such as light, nutrients, and water. This review examines the structural and functional characteristics that allow climbing plants to (1) achieve widespread dispersal, (2) transport large amounts of water throughout vessels, (3) maintain high photosynthesis levels through a large leaf area to biomass ratio, (4) achieve rapid vertical and horizontal expansion by fast growth rates and various climbing mechanisms and (5) survive and recover from disturbances. Due to the competitive effects of vines on trees, management of vine growth is used to preserve tropical timber plantations, combat invasive weeds, and promote rainforest recovery. In order to sustainably manage the vines into the future, it is necessary to understand the mechanisms by which they can alter tropical forest succession and the impacts of various management techniques.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Abstract. Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.