Genetic requirement of p47phox for superoxide production by murine microglia.

An NADPH oxidase is thought to function in microglial cells of the central nervous system. These conclusions are based on pharmacological and immunochemical evidence, although these approaches are indirect and raise issues of specificity. For example, diphenyleneiodonium inhibits a variety of flavoenzymes, including xanthine oxidase, NADH dehydrogenase, and NADPH oxidase. Here, we provide genetic evidence that p47phox, an essential component of the phagocyte NADPH oxidase, is required for superoxide anion release from microglia. Microglia derived from newborn wild-type mice, but not from newborn p47phox-deficient (knockout; -/-) mice, produced superoxide after stimulation by opsonized zymosan or phorbol myristate acetate. Endogenous p47phox was detected only in wild-type microglia, consistent with selective superoxide production in these cells. Superoxide release was restored in p47phox-deficient microglia that were retrovirally transduced with human p47phox cDNA. Similar kinetics of superoxide generation were observed, consistent with the same enzyme functioning in wild-type and restored microglia. Immuno-detection of p47phox in transduced cells confirmed that restoration of superoxide release correlated with production of recombinant protein. These data provide genetic proof that p47phox is necessary for superoxide release by microglial cells and indicate that a system related to the phagocyte oxidase is active in these cells.     

Characterization of Amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tailbud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. The ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs. This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification.     

Evolutionary stability of mutualism: interspecific population regulation as an evolutionarily stable strategy

Interspecific mutualisms are often vulnerable to instability because low benefit : cost ratios can rapidly lead to extinction or to the conversion of mutualism to parasite-host or predator-prey interactions. We hypothesize that the evolutionary stability of mutualism can depend on how benefits and costs to one mutualist vary with the population density of its partner, and that stability can be maintained if a mutualist can influence demographic rates and regulate the population density of its partner. We test this hypothesis in a model of mutualism with key features of senita cactus (Pachycereus schottii)-senita moth (Upiga virescens) interactions, in which benefits of pollination and costs of larval seed consumption to plant fitness depend on pollinator density. We show that plants can maximize their fitness by allocating resources to the production of excess flowers at the expense of fruit. Fruit abortion resulting from excess flower production reduces pre-adult survival of the pollinating seed-consumer, and maintains its density beneath a threshold that would destabilize the mutualism. Such a strategy of excess flower production and fruit abortion is convergent and evolutionarily stable against invasion by cheater plants that produce few flowers and abort few to no fruit. This novel mechanism of achieving evolutionarily stable mutualism, namely interspecific population regulation, is qualitatively different from other mechanisms invoking partner choice or selective rewards, and may be a general process that helps to preserve mutualistic interactions in nature.     

The<Emphasis Type="Italic"> Trox-2</Emphasis> Hox/ParaHox gene of<Emphasis Type="Italic"> Trichoplax</Emphasis> (Placozoa) marks an epithelial boundary

Hox and ParaHox genes are implicated in axial patterning of cnidarians and bilaterians, and are thought to have originated by tandem duplication of a single ProtoHox gene followed by duplication of the resultant gene cluster. It is unclear what the ancestral role of Hox/ParaHox genes was before the divergence of Cnidaria and Bilateria, or what roles the postulated ProtoHox gene(s) played. Here we describe the full coding region, spatial expression and function of Trox-2, the single Hox/ParaHox-type gene identified in Trichoplax adhaerens (phylum Placozoa) and either a candidate ProtoHox or a ParaHox gene. Trox-2 is expressed in a ring around the periphery of Trichoplax, in small cells located between the outer margins of the upper and lower epithelial cell layers. Inhibition of Trox-2 function, either by uptake of morpholino antisense oligonucleotides or by RNA interference, causes complete cessation of growth and binary fission. We speculate that Trox-2 functions within a hitherto unrecognized population of possibly multipotential peripheral stem cells that contribute to differentiated cells at the epithelial boundary of Trichoplax.Edited by D. Tautz     

Early immunodiagnosis of fasciolosis in ruminants using recombinant Fasciola hepatica cathepsin L-like protease

A diagnostic ELISA with recombinant Fasciola hepatica cathepsin L-like protease as antigen was developed to detect antibodies against F. hepatica in sheep and cattle. The recombinant cathepsin L-like protease was generated by functional expression of the cDNA from adult stage F. hepatica flukes in Saccharomyces cerevisiae. Specificity and sensitivity of the cathepsin L enzyme-linked immunosorbent assay (ELISA) was assessed using sera from sheep and calves experimentally or naturally mono-infected with F. hepatica and six-seven other parasites. The sensitivity of the cathepsin L ELISA for sheep and cattle sera was 99.1 and 100%, respectively. In the experimental setting with established mono-infections, the specificity of the cathepsin L ELISA was 98.5% for cattle sera and 96.5% for sheep sera. In experimentally infected cattle and sheep, the first detection of F. hepatica-specific antibodies appeared first between 5 and 7 weeks post-infection, but depended on the infectious dose of F. hepatica. In ELISA the detection preceded first detection of the infection based on egg counts and remained detectable till at least 23 weeks after a primary F. hepatica infection. Detection of Fasciola gigantica infections was similar to detection of F. hepatica. The first detection occurred at week 5 and signals persisted for at least 20 weeks. All sera from naturally F. hepatica infected sheep were seropositive in the cathepsin L-like ELISA. The relevance of this ELISA format was also evaluated using sera from naturally infected cattle in the Netherlands, Ecuador and Vietnam and compared with results from egg-counts. For the latter two endemic areas with mixed parasitic infections the 'apparent' sensitivity of the cathepsin L ELISA was calculated for all serum samples together to be 90.2%. The 'apparent' specificity under these conditions was calculated to be 75.3%. In cattle, the cathepsin L ELISA was superior to the concurrently evaluated peptide ELISA format using a single epitope as the antigen both in controlled natural infections as well as in infections in endemic areas. The present ELISA-format contributes a relatively sensitive and reliable tool for the early serodiagnosis of bovine and ovine fasciolosis.     

Model studies on a synthetically facile series of N-substituted phenyl-N'-pyridin-3-yl ureas leading to 1-(3-pyridylcarbamoyl) indolines that are potent and selective 5-HT(2C/2B) receptor antagonists

A model series of 5-HT_2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N′-pyridin-3-yl ureas were found to have a range of 5-HT_2C receptor affinities and selectivities over the closely related 5-HT_2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridyl-carbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT_2C/2B antagonists.     

Mechanism of triclosan inhibition of bacterial fatty acid synthesis

Triclosan is a broad-spectrum antibacterial agent that inhibits bacterial fatty acid synthesis at the enoyl-acyl carrier protein reductase (FabI) step. Resistance to triclosan in Escherichia coli is acquired through a missense mutation in the fabI gene that leads to the expression of FabI[G93V]. The specific activity and substrate affinities of FabI[G93V] are similar to FabI. Two different binding assays establish that triclosan dramatically increases the affinity of FabI for NAD+. In contrast, triclosan does not increase the binding of NAD+ to FabI[G93V]. The x-ray crystal structure of the FabI-NAD+-triclosan complex confirms that hydrogen bonds and hydrophobic interactions between triclosan and both the protein and the NAD+ cofactor contribute to the formation of a stable ternary complex, with the drug binding at the enoyl substrate site. These data show that the formation of a noncovalent "bi-substrate" complex accounts for the effectiveness of triclosan as a FabI inhibitor and illustrates that mutations in the FabI active site that interfere with the formation of a stable FabI-NAD+-triclosan ternary complex acquire resistance to the drug.     

The Fine Structure of the Testis of a Lancelet (=Amphioxus), Branchiostoma floridae (Phylum Chordata: Subphylum Cephalochordata= Acrania)

The germinal and non-germinal cells of the ripe lancelet testis are described by transmission electron microscopy. The visceral peritoneum of the testis is composed of myoepithelial cells, and the haemal layer consists of regions of narrow sinuses and conspicuously thicker blood vessels filled with blood plasma and bounded by basal laminae. Within the germinal epithelium and the testicular lumen, the non-germinal cells, which are not abundant, contain conspicuous lysosomes and mitochondria with tubular cristae, indicating that they may be involved in steroid synthesis. In the ripe testis, the non-germinal cells do not appear to be organized into a blood-testis barrier. Ail types of spermatogenic cells may be flagellated and are joined in small groups by intercellular bridges. During differentiation of the spermatids, the Golgi complex is associated with formation of the acrosomal vesicle near the posterior pole of the cell. A remarkable feature is the dual origin of the subacrosomal material: one component originates at the posterior end of the spermatid, and the other at the anterior end. Subsequently, the two components merge into one after the acrosomal vesicle has migrated to its definitive anterior position in the mature spermatozoon.     

The evolution of multicellularity and early animal genomes

Several independent molecular datasets, including complete mtDNA sequence, indicate that Choanozoa are most closely related to multicellular animals. There is still confusion concerning basal animal phylogeny, although recent data indicate that Placozoa are not degenerate cnidarians and hence (along with sponges) occupy a pivotal position. The transition in evolution from diploblast to bilaterian animals is becoming better understood, with gene expression data arguing that cnidarians have forerunners of the anteroposterior and dorsoventral body axes, and even a putative homologue of mesoderm. The homeobox and kinase gene families have been further analysed in basal animals, although more data are required to enable detailed comparison with Bilateria.