Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.     

Natural variation in a testosterone-mediated trade-off between mating effort and parental effort

Male birds frequently face a trade-off between acquiring mates and caring for offspring. Hormone manipulation studies indicate that testosterone often mediates this trade-off, increasing mating effort while decreasing parental effort. Little is known, however, about individual covariation between testosterone and relevant behavior on which selection might act. Using wild, male dark-eyed juncos (Junco hyemalis), we measured individual variation in testosterone levels before and after standardized injections of gonadotropin-releasing hormone (GnRH). The GnRH challenges have been shown to produce short-term testosterone increases that are similar to those produced naturally in response to social stimuli, repeatable in magnitude, and greater in males with more attractive ornaments. We correlated these testosterone increases with behavioral measures of mating and parental effort (aggressive response to a simulated territorial intrusion and nestling feeding, respectively). Males that showed higher postchallenge testosterone displayed more territorial behavior, and males that produced higher testosterone increases above initial levels displayed reduced parental behavior. Initial testosterone levels were positively but nonsignificantly correlated with aggression but did not predict parental behavior. These relationships suggest that natural variation in testosterone, specifically the production of short-term increases, may underlie individual variation in the mating effort/parental effort trade-off. We discuss the implications of these results for the evolution of hormonally mediated trade-offs.     

THE CONTRIBUTION OF MATERNAL EFFECTS TO SELECTION RESPONSE: AN EMPIRICAL TEST OF COMPETING MODELS

Maternal effects can dramatically influence the evolutionary process, in some cases facilitating and in others hindering adaptive evolution. Maternal effects have been incorporated into quantitative genetic models using two theoretical frameworks: the variance‐components approach, which partitions variance into direct and maternal components, and the trait‐based approach, which assumes that maternal effects are mediated by specific maternal traits. Here, we demonstrate parallels between these models and test their ability to predict evolutionary change. First, we show that the two approaches predict equivalent responses to selection in the absence of maternal effects mediated by traits that are themselves maternally influenced. We also introduce a correction factor that may be applied when such cascading maternal effects are present. Second, we use several maternal effect models, as well as the standard breeder's equation, to predict evolution in response to artificial selection on flowering time in American bellflower, Campanulastrum americanum. Models that included maternal effects made much more accurate predictions of selection response than the breeder's equation. Maternal effect models differed somewhat in their fit, with a version of the trait‐based model providing the best fit. We recommend fitting such trait‐based models when possible and appropriate to make the most accurate evolutionary predictions.     

Hormone-mediated suites as adaptations and evolutionary constraints

Hormones mediate the expression of suites of correlated traits and hence may act both to facilitate and constrain adaptive evolution. Selection on one trait within a hormone-mediated suite may, for example, lead to a change in the strength of the hormone signal, causing either beneficial or detrimental changes in correlated traits. Theory and empirical methods for studying correlated trait evolution have been developed by the field of evolutionary quantitative genetics, and here we suggest that their application to the study of hormone-mediated suites may prove fruitful. We present hypotheses for how selection shapes the evolution of hormone-mediated suites and argue that correlational selection, which arises when traits interact in their effects on fitness, may act to alter or conserve the composition of hormone-mediated suites. Next, we advocate using quantitative genetic methods to assess natural covariation among hormone-mediated traits and to measure the strength of natural selection acting on them. Finally, we present illustrative examples from our own work on the evolution of testosterone-mediated suites in male and female dark-eyed juncos. We conclude that future work on hormone-mediated suites, if motivated by quantitative genetic theory, may provide important insights into their dual roles as adaptations and evolutionary constraints.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.