Measurement of the immunoperoxidase staining of macrophages within liver granulomata of mice infected with Mycobacterium tuberculosis.

A quantitative image analysis technique developed for the measurement of the extent of macrophage activation and epithelioid cell differentiation was performed on mice infected experimentally with Mycobacterium tuberculosis. The granulomatous inflammatory response within the liver reached a peak at day 23 and declined by day 33. Animals of strain B10.BR (H-2k) showed an increased granuloma fraction as compared to Balb/k (H-2k) mice, thus confirming the influence of non-H2 genes in the control of granuloma formation in mice. Using a monoclonal antibody against CD11b/CD18 (Mac1;CR3), we observed two subpopulations of macrophages within the granulomata. The small, darkly staining cells at the periphery of granulomata appear to be newly recruited macrophages. Larger, paler staining cells toward the center of granulomata represent activated and mature epithelioid macrophages. Using a semiautomated image analyzer (Quantimet 970), we measured the relative numbers of these macrophage subpopulations. There were more activated macrophages (epithelioid cells) associated with the increased granuloma fraction in the B10.BR mice than in the Balb/k. However, similar numbers of newly recruited peripheral macrophages were found in both Balb/k and B10.BR strains. This technique has shown qualitative as well as quantitative differences in the granulomatous inflammatory response in this murine model of tuberculosis in strains of mice with quite different antibody repertoires to mycobacterial antigens.     

Phylogenetic analysis of the outer-membrane-protein genes of Chlamydiae, and its implication for vaccine development

Examination of 18 complete and 6 partial sequences of the major outer- membrane protein from 24 chlamydiae isolates was used to reconstruct their evolutionary relationships. From this analysis, assuming that the clades with 100% bootstrap support are correct, come the following conclusions: (1) The tree of these sequences is not congruent with the phylogeny of the hosts, and thus host switching would seem to have occurred, thereby limiting the extent to which there has been coevolution of parasite and host. (2) The tree is also noncongruent with clustering by type of cell infected, thereby limiting the extent to which there has been coevolution of parasite and the cell type that it infects. (3) The tree is also noncongruent with clustering by the organ infected (eyes or genitalia), thereby limiting the extent to which there has been coevolution of parasite and the organ that it infects. (4) The tree is also noncongruent with genital strains arising from lymphogranuloma venereum strains. (5) The tree is also noncongruent with the geographic site at which the isolates were obtained, thereby limiting the extent of divergence explained by geographic separation. (6) There are estimated to be 185 amino acid positions that are invariable (as opposed to unvaried) in the major outer-membrane protein. There are 10 unvaried positions in the variable domains, of which 9 appear to be invariable, giving some reason to hope that development of a vaccine might be possible. (7) The rate of change of this protein is too small to see increased divergence over the time span of isolation of these genes, giving hope to any vaccine having longevity. Bootstrapping supports those portions of the tree on which the first five conclusions above depend. The picture that these results provide is more one of pathogen versatility than one of coevolutionary constraints. In addition, we examined 10 60-KDa, outer-membrane protein- 2 genes, all but one of which were from these same strains. The tree was not, among the trachomatis strains, congruent with the major-outer- membrane protein tree, suggesting that gene exchange could be occurring among strains. Moreover, there is an apparent slowdown in divergence in this gene, among the trachomatis strains.      

The effect of aquatic plant abundance on shell crushing resistance in a freshwater snail

Most of the shell material in snails is composed of calcium carbonate but the organic shell matrix determines the properties of calcium carbonate crystals. It has been shown that the deposition of calcium carbonate is affected by the ingestion of organic compounds. We hypothesize that organic compounds not synthesized by the snails are important for shell strength and must be obtained from the diet. We tested this idea indirectly by evaluating whether the abundance of the organic matter that snails eat is related to the strength of their shells. We measured shell crushing resistance in the snail Mexipyrgus churinceanus and the abundance of the most common aquatic macrophyte, the water lily Nymphaea ampla, in ten bodies of water in the valley of Cuatro Ciénegas, Mexico. We used stable isotopes to test the assumption that these snails feed on water lily organic matter. We also measured other factors that can affect crushing resistance, such as the density of crushing predators, snail density, water pH, and the concentration of calcium and phosphorus in the water. The isotope analysis suggested that snails assimilate water lily organic matter that is metabolized by sediment bacteria. The variable that best explained the variation in crushing resistance found among sites was the local abundance of water lilies. We propose that the local amount of water lily organic matter provides organic compounds important in shell biomineralization, thus determining crushing resistance. Hence, we propose that a third trophic level could be important in the coevolution of snail defensive traits and predatory structures.     

Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.

An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.     

Phylogeography and Conservation Genetics of a Distinct Lineage of Sunfish in the Cuatro Ciénegas Valley of Mexico

The valley of Cuatro Ciénegas, an aquatic oasis located in the Mexican Chihuahuan Desert, exhibits the highest level of endemism in North America and is a Mexican National Protected Area. However, little is known about the evolutionary distinctiveness of several vertebrate species present in the Cuatro Ciénegas valley. We conducted a phylogeographic study using mitochondrial haplotypes from the centrarchid fish Lepomis megalotis to determine if the populations found within the valley were evolutionarily distinct from populations outside the valley. We also examined if there was evidence of unique haplotypes of this sunfish within the valley. Genetic divergence of L. megalotis suggests populations within the valley are evolutionarily unique when compared to L. megalotis outside the valley. Significant mitochondrial sequence divergence was also discovered between L. megalotis populations on either side of the Sierra de San Marcos that bisects the valley. Our results reinforce previous studies that suggest the organisms occupying aquatic habitats not only within Cuatro Ciénegas but also in each of the two lobes of the valley generally deserve independent consideration during management decisions.