Evolutionary changes in sites and timing of actin gene expression in embryos of the direct- and indirect-developing sea urchins, Heliocidaris erythrogramma and H. tuberculata

 We describe an evolutionary comparison of expression of the actin gene families of two congeneric sea urchins. Heliocidaris tuberculata develops indirectly via a planktonic feeding pluteus that forms a juvenile rudiment after a long period of larval development. H. erythrogramma is a direct developer that initiates formation of a juvenile rudiment immediately following gastrulation. The developmental expression of each actin isoform of both species was determined by in situ hybridization. The observed expression patterns are compared with known expression patterns in a related indirect-developing sea urchin, Strongylocentrotus purpuratus. Comparisons reveal unexpected patterns of conserved and divergent expression. Cytoplasmic actin, CyIII, is expressed in the aboral ectoderm cells of the indirect developers, but is an unexpressed pseudogene in H. erythrogramma, which lacks aboral ectoderm. This change is correlated with developmental mode. Two CyII actins are expressed in S. purpuratus, and one in H. erythrogramma, but no CyII is expressed in H. tuberculata despite its great developmental similarity to S. purpuratus. CyI expression differs slightly between Heliocidaris and Strongylocentrotus with more ectodermal expression in Heliocidaris. Evolutionary changes in actin gene expression reflect both evolution of developmental mode as well as a surprising flexibility in gene expression within a developmental mode. Received: 27 July 1997 / Accepted: 30 December 1997     

Determination of phenylalanine hydroxylase activity by liquid chromatography/electrochemistry

An assay method is presented for the determination of phenylalanine hydroxylase activity in biological samples. The procedure is rapid and requires little sample. Multiple components of the enzyme system are determined and therefore serve as internal checks of the assay system. Liquid chromatography/electrochemistry is employed to follow the oxidation of the tetrahydropterin cofactor to the dihydropterin and to follow the formation of tyrosine. The KM and Vmax values of both phenylalanine and 6-methyl-5,6,7,8-tetrahydropterin were determined for mouse liver phenylalanine hydroxylase. Determination of the stoichiometry of the reaction showed that 1 mol of dihydropterin and 1 mol of tyrosine are formed per mole of tetrahydropterin that is oxidized. The reaction rate was linear for several minutes and over a wide range of enzyme (protein) concentrations.     

Determination of aniline and metabolites produced in vitro by liquid chromatography/electrochemistry

A method utilizing reverse-phase liquid chromatography/electrochemistry (LC/EC) was developed for the simultaneous determination of aniline and its hydroxylated derivatives, p-aminophenol, o-aminophenol, m-aminophenol, and N-phenylhydroxylamine. To achieve separation of these compounds, a mobile phase of 3.0% dimethylformamide and 97.0% 0.05 M piperazine acetate, pH 5.4, containing 0.05 M KNO3 was developed. A procedure is also presented for the determination of p-nitrophenol, nitrobenzene, and nitrosobenzene, possible aniline metabolites in higher N-oxidation states, using reductive amperometric detection. The hydroxylated compounds, including the hydroxylamine, and nitrosobenzene are easily detected as metabolites of aniline in mouse liver slice or microsomal preparations. No prior extraction, preconcentration, or derivatization steps are needed for the determinations, which can be accomplished by a direct injection of the incubation mixture. The Km value for the hepatic aniline 4-hydroxylase activity in male Cox-Swiss mice microsomal preparations has been determined to be 0.52 mM; the Vmax value is 2.90 +/- 0.64 nmol min-1 mg microsomal protein-1. Detection limits for all compounds of interest are in the picomole range.     

Origin, targeting, and function of the apicomplexan plastid.

The discovery of a plastid in Plasmodium, Toxoplasma and related protozoan parasites provides a satisfying resolution to several long-standing mysteries: the mechanism of action for various surprisingly effective antibiotics; the subcellular location of an enigmatic 35 kb episomal DNA; and the nature of an unusual intracellular structure containing multiple membranes. The apicomplexan plastid highlights the importance of lateral genetic transfer in evolution and provides an accessible system for the investigation of protein targeting to secondary endosymbiotic organelles. Combining molecular genetic identification of targeting signals with whole genome analysis promises to yield a complete picture of organellar metabolic pathways and new targets for drug design.     

New high performance liquid chromatographic analysis of brain catecholamines

A new high performance liquid chromatography analysis has been developed for catecholamines in brain tissue. The method retains alumina separation of the catecholamines. Quantitative read-out is by direct liquid chromatography, replacing the tedious trihydroxyindole chemistry and fluorescence measurements. The analysis is rapid and accurate and agrees well with existing literature data. The equipment is inexpensive and the technique can be utilized for routine analyses after 1–2 weeks of practice. The method is directly applicable to whole small animal brains and, depending on the NE and DA levels, to dissected sub-portions.     

Rapid evolution in a conserved gene family. Evolution of the actin gene family in the sea urchin genus Heliocidaris and related genera

Camarodont sea urchins possess a rapidly evolving actin gene family whose members are expressed in distinct cell lineages in a developmentally regulated fashion. Evolutionary changes in the actin gene family of echinoids include alterations in number of family members, site of expression, and gene linkage, and a dichotomy between rapidly and slowly evolving isoform-specific 3' untranslated regions. We present sequence comparisons and an analysis of the actin gene family in two congeneric sea urchins that develop in radically different modes, Heliocidaris erythrogramma and H. tuberculata. The sequences of several actin genes from the related species Lytechinus variegatus are also presented. We compare the features of the Heliocidaris and Lytechinus actin genes to those of the the actin gene families of other closely related sea urchins and discuss the nature of the evolutionary changes among sea urchin actins and their relationship to developmental mode.      

Genomics meets transgenics in search of the elusive Cryptosporidium drug target

Cryptosporidium is an important pathogen of humans, and a challenging model for the laboratory. The parasite genome sequence, accessible through a comprehensive database, now provides exciting opportunities for urgently needed advances. Comparative genomics, combined with the genetic system in the related parasite Toxoplasma gondii, outlines a detailed Cryptosporidium parvum metabolic map and facilitates cell biological analyses. New targets for Cryptosporidium drug and vaccine development can be identified and validated based on this approach.