It has generally been concluded that two divalent cations are required for enolase activity, even though the enzyme is a homodimer that specifically binds four metal ions in the presence of substrate. This paper reports a reinvestigation of the stoichiometry of enolase activation. Specific ion electrode measurements of Mg2+ binding in the presence and absence of substrate are compared with stopped-flow measurements of the velocity of 2-phosphoglycerate dehydration. It is concluded that the enzyme is inactive when only two metal-binding sites are filled and that four sites must be populated with Mg2+ for full activity. An ordered binding mechanism is proposed that quantitatively predicts the activation of enolase by the four Mg2+ ions from their measured dissociation constants and the Michaelis constant for the dehydration reaction. To explain the loss of enzymatic activity at still higher metal concentrations, the binding of additional, inhibitory Mg2+ ions is postulated.
Finding correct species relationships using phylogeny reconstruction based on molecular data is dependent on several empirical and technical factors. These include the choice of DNA sequence from which phylogeny is to be inferred, the establishment of character homology within a sequence alignment, and the phylogeny algorithm used. Nevertheless, sequencing and phylogeny tools provide a way of testing certain hypotheses regarding the relationship among the organisms for which phenotypic characters demonstrate conflicting evolutionary information. The protozoan family Sarcocystidae is one such group for which molecular data have been applied phylogenetically to resolve questionable relationships. However, analyses carried out to date, particularly based on small-subunit ribosomal DNA, have not resolved all of the relationships within this family. Analysis of more than one gene is necessary in order to obtain a robust species signal, and some DNA sequences may not be appropriate in terms of their phylogenetic information content. With this in mind, we tested the informativeness of our chosen molecule, the large-subunit ribosomal DNA (lsu rDNA), by using subdivisions of the sequence in phylogenetic analysis through PAUP, fastDNAml, and neighbor joining. The segments of sequence applied correspond to areas of higher nucleotide variation in a secondary-structure alignment involving 21 taxa. We found that subdivision of the entire lsu rDNA is inappropriate for phylogenetic analysis of the Sarcocystidae. There are limited informative nucleotide sites in the lsu rDNA for certain clades, such as the one encompassing the subfamily Toxoplasmatinae. Consequently, the removal of any segment of the alignment compromises the final tree topology. We also tested the effect of using two different alignment procedures (CLUSTAL W and the structure alignment using DCSE) and three different tree-building methods on the final tree topology. This work shows that congruence between different methods in the formation of clades may be a feature of robust topology; however, a sequence alignment based on primary structure may not be comparing homologous nucleotides even though the expected topology is obtained. Our results support previous findings showing the paraphyly of the current genera Sarcocystis and Hammondia and again bring to question the relationships of Sarcocystis muris, Isospora felis, and Neospora caninum. In addition, results based on phylogenetic analysis of the structure alignment suggest that Sarcocystis zamani and Sarcocystis singaporensis, which have reptilian definitive hosts, are monophyletic with Sarcocystis species using mammalian definitive hosts if the genus Frenkelia is synonymized with Sarcocystis.
Ventral anomalies of accessory pulmonary tissue have been classified as "bronchopulmonary foregut malformations." Between July 1, 1981, and May 31, 1985, 10 children with bronchopulmonary malformations have been cared for on the Pediatric Surgical Service at the University of Virginia. Six patients had bronchogenic cysts, one in an extrathoracic location and one associated with a pulmonary sequestration. Diagnosis was suspected in each case by plain chest radiographs and confirmed by computed tomography scans and ultrasound. Four patients had pulmonary sequestrations, two in association with diaphragmatic hernias. One patient had accessory pulmonary tissue, best classified as a tracheal lobe. Diagnosis in this patient was confirmed by bronchography. Nine patients underwent excision of the malformation without event. In one patient, a bronchogenic cyst was treated successfully by thoracoscopy. Review of the anatomy of these malformations leads to the conclusion that three embryologic events are cardinal in determining their ultimate form: (1) investment of the anomalous pulmonary tissue by the pulmonary artery; (2) the degree of involution of the original foregut communication; and (3) the stage of development leading to pleural investment.
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