Paracoccidioides brasiliensis Interferes on Dendritic Cells Maturation by Inhibiting PGE2 Production

Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil, whose etiologic agent is the thermodimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii. The mechanisms involved in the initial interaction of the fungus with cells of the innate immune response, as dendritic cells (DCs), deserve to be studied. Prostaglandins (PGs) are eicosanoids that play an important role in modulating functions of immune cells including DCs. Here we found that human immature DCs derived from the differentiation of monocytes cultured with GM-CSF and IL-4 release substantial concentrations of PGE₂, which, however, were significantly inhibited after challenge with P. brasiliensis. In vitro blocking of pattern recognition receptors (PRRs) by monoclonal antibodies showed the involvement of mannose receptor (MR) in PGE₂ inhibition by the fungus. In addition, phenotyping assays showed that after challenge with the fungus, DCs do not change their phenotype of immature cells to mature ones, as well as do not produce IL-12 p70 or adequate concentrations of TNF-α. Assays using exogenous PGE₂ confirmed an association between PGE₂ inhibition and failure of cells to phenotypically mature in response to P. brasiliensis. We conclude that a P. brasiliensis evasion mechanism exists associated to a dysregulation on DC maturation. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.     

Philopatry, kin clusters, and genetic relatedness in a population of woodrats (Neotoma macrotis)

Studies of highly kin-structured mammal societies have revealedthe importance of natal philopatry in determining the distributionof genetic variation within populations. In comparison, therelationship between philopatry and genetic diversity withinpopulations of moderately kin-structured societies has receivedrelatively little attention. Previous studies of Neotoma macrotishave suggested that females form distinct kin clusters. Eachkin cluster overlaps spatially with the home range(s) of oneor more males that are not related to each other or to the femaleswith which they are spatially associated. To examine interactionsbetween philopatry and genetic structure in this apparentlymoderately kin-structured species, we characterized spatialand genetic relationships among individually marked femalesin a population of N. macrotis from central coastal California.Our field studies revealed that, contrary to expectation, femalesin this population were not strongly philopatric and spatiallyclustered females were not characterized by high levels of geneticrelatedness. Nevertheless, genetic structure was evident withinthe study population; spatial and genetic distances among femaleswere significantly correlated, suggesting that dispersal patternsinfluenced genetic structure even in the absence of marked femalephilopatry. Because females with overlapping spatial distributionswere not typically closely related to one another, opportunitiesfor the evolution of kin-selected social behavior (e.g., cooperativecare of young) appear to be limited in this population.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca2+/Mg2+)-ATPase,and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.     

Identification of metabolites of phenylacetic acid in rat brain by plasma desorption mass spectrometry

Mass spectra of acyl hydroxamates may be obtained directly, without prior derivatization or gas chromatography, by the new technique of plasma desorption mass spectrometry. Authentic alkyl and aryl hydroxamates showed the expected protonated molecular ions when isobutane was used as the carrier gas. Advantage was taken of this novel technique to identify phenylacetyl-CoA and phenylbutyric acid in brain extracts obtained from young rats after a loading dose of phenylacetic acid. The formation of these metabolic products lends strong support to our suggestion that brain dysfunction induced by phenylacetic acid in experimental phenylketonuria may be due to decreased availability of CoA and acetyl-CoA in the rapidly developing brain.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

Cloning and Functional Characterization of Two BTB Genes in the Predatory Mite Metaseiulus occidentalis

Proteins containing the BTB (Bric-à-brac, tramtrack, and Broad Complex) domain typically share low sequence similarities and are involved in a wide range of cellular functions. We previously identified two putative and closely related BTB genes, BTB1 and BTB2, in the genome of the predatory mite Metaseiulus occidentalis. In the current study, full-length BTB1 and BTB2 cDNAs were cloned and sequenced. BTB1 and BTB2 encode proteins of 380 and 401 amino acids, respectively. BTB1 and BTB2 proteins each contain an N-terminal BTB domain and no other identifiable domains. Thus, they belong to a large category of BTB-domain proteins that are widely distributed in eukaryotes, yet with largely unknown function(s). BTB1 and BTB2 gene knockdowns in M. occidentalis females using RNAi reduced their fecundity by approximately 40% and 73%, respectively, whereas knockdown had no impact on their survival or the development of their offspring. These findings suggest these two proteins may be involved in processes related to egg production in this predatory mite, expanding the list of functions attributed to these diverse proteins.