Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis

Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across six pair of diverse cell lines and validated the database annotation process. Results suggest that splicing in humans is more complex than simple exon-skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.     

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.     

Percoll centrifugation eliminates mold contaminants from cell cultures

This work was supported by a grant and research associateship to N.A. from the Medical Research Council of Canada and the National Cancer Institute of Canada.     

Lineage sorting in multihost parasites: Eidmanniella albescens and Fregatiella aurifasciata on seabirds from the Galapagos Islands

Parasites comprise a significant percentage of the biodiversity of the planet and are useful systems to test evolutionary and ecological hypotheses. In this study, we analyze the effect of host species identity and the immediate local species assemblage within mixed species colonies of nesting seabirds on patterns of genetic clustering within two species of multihost ectoparasitic lice. We use three genetic markers (one mitochondrial, COI, and two nuclear, EF1‐α and wingless) and maximum likelihood phylogenetic trees to test whether (1) parasites show lineage sorting based on their host species; and (2) switching of lineages to the alternate host species depends on the immediate local species assemblage of individual hosts within a colony. Specifically, we examine the genetic structure of two louse species: Eidmanniella albescens, infecting both Nazca (Sula granti) and blue‐footed boobies (Sula nebouxii), and Fregatiella aurifasciata, infecting both great (Fregata minor) and magnificent frigatebirds (Fregata magnificens). We found that host species identity was the only factor explaining the patterns of genetic structure in both parasites. In both cases, there is evident genetic differentiation depending on the host species. Thus, a revision of the taxonomy of these louse species is needed. One possible explanation of this pattern is extremely low louse migration rates between host species, perhaps influenced by fine‐scale spatial separation of host species within mixed colonies, and low parasite infrapopulation numbers.     

Fluorescent and bimolecular-fluorescent protein tagging of genes at their native loci in Neurospora crassa using specialized double-joint PCR plasmids

The double-joint polymerase chain reaction (DJ-PCR) is a technique that can be used to construct vectors for targeted genome integration without laborious subcloning steps. Here we report the availability of plasmids that facilitate DJ-PCR-based construction of Neurospora crassa tagging vectors. These plasmids allow the creation of green or red fluorescent protein (GFP or RFP) tagging vectors for protein localization studies, as well as split-yellow fluorescent protein (YFP) tagging vectors for bimolecular fluorescence complementation (BiFC) analyses. We have demonstrated the utility of each plasmid with the tagging of known meiotic silencing proteins. Microscopic analysis of the tagged strains indicates that SMS-2 and QIP form macromolecular complexes in the perinuclear region during meiosis.     

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.     

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.     

Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.