Regulation of V(D)J recombination by transcriptional promoters

Enhancer elements potentiate the rearrangement of antigen receptor loci via changes in the accessibility of gene segment clusters to V(D)J recombinase. Here, we show that enhancer activity per se is insufficient to target T-cell receptor beta miniloci for DbetaJbeta recombination. Instead, a promoter situated 5' to Dbeta1 (PDbeta) was required for efficient rearrangement of chromosomal substrates. A critical function for promoters in regulating gene segment accessibility was further supported by the ability of heterologous promoters to direct rearrangement of enhancer-containing substrates. Importantly, activation of a synthetic tetracycline-inducible promoter (Ptet) positioned upstream from the Dbeta gene segment was sufficient to target recombination of miniloci lacking a distal enhancer element. The latter result suggests that DNA loops, generated by interactions between flanking promoter and enhancer elements, are not required for efficient recognition of chromosomal gene segments by V(D)J recombinase. Unexpectedly, the Ptet substrate exhibited normal levels of rearrangement despite its retention of a hypermethylated DNA status within the DbetaJbeta cluster. Together, our findings support a model in which promoter activation, rather than intrinsic properties of enhancers, is the primary determinant for regulating recombinational accessibility within antigen receptor loci.     

Functional effects of enhancing or silencing adenosine A2b receptors in cardiac fibroblasts

Cardiac fibroblasts (CF) express adenosine (ADO) receptors, and pharmacological evidence suggests the possible involvement of the A2 (A2a and A2b) receptor (A2aR and A2bR) subtypes in inhibiting cell functions involved in fibrosis. The main objective of this study was to define the contributions of A2a and/or A2b receptors in modulating ADO-induced decreases in CF functions. For this purpose, CF were either treated pharmacologically or had the A2aR or A2bR levels modified through the use of recombinant adenovirus or siRNA. The assessment of mRNA expression in adult rat CF yielded evidence for A1R, A2bR, A2a), and A3R. Endogenously or exogenously enhanced ADO significantly inhibits CF proliferation, collagen, and protein synthesis. A2R and A2aR agonists, although capable of inhibiting CF protein and collagen synthesis, were unable to define the contributions derived from A2aR or A2bR. Overexpression of A2bR in CF yielded significant decreases in basal levels of collagen and protein synthesis and correlated with increases in cAMP levels. However, at higher doses of ADO receptor agonists, significant increases in protein and collagen synthesis were observed. CF with underexpression of A2bR yielded increases in protein and collagen synthesis. In contrast, A2aR underexpression did not modify ADO-induced decreases in CF protein or collagen synthesis. In conclusion, results derived from the molecular manipulation of receptor levels indicate that A2bR are critically involved in ADO-mediated inhibition of CF functions.     

Upregulation of angiotensin II type 2 receptor expression in estrogen-induced pituitary hyperplasia

Recent evidence shows that reexpression and upregulation of angiotensin II (ANG II) type 2 (AT2) receptor in adult tissues occur during pathological conditions such as tissue hyperplasia, inflammation, and remodeling. In particular, expression of functional AT2 receptors in the pituitary and their physiological significance and regulation have not been described. In this study, we demonstrate that chronic in vivo estrogen treatment, which induces pituitary hyperplasia, enhances local AT2 expression (measured by Western blot and RT-PCR) concomitantly with downregulation of ANG II type 1 (AT1) receptors. In vivo progesterone treatment of estrogen-induced pituitary hyperplasia did not modify either the ANG II receptor subtype expression pattern or octapeptide-induced and AT1-mediated calcium signaling. Nevertheless, an unexpected potentiation of the ANG II prolactin-releasing effect was observed in this group, and this response was sensitive to both AT1 and AT2 receptor antagonists. These data are the first to document that ANG II can act at the pituitary level through the AT2 receptor subtype and that estrogens display a differential regulation of AT1 and AT2 receptors at this level.     

GFSWeb: a web tool for genome-based identification of proteins from mass spectrometric samples

The interpretation of mass spectrometry data for protein identification has become a vital component of proteomics research. However, since most existing software tools rely on protein databases, their success is limited, especially as the pace of annotation efforts fails to keep pace with sequencing. We present a publicly available, web-based version of a software tool that maps peptide mass fingerprint data directly to their genomic origin, allowing for genome-based, annotation-independent protein identification.     

Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA-destabilizing elements and protein-induced changes in ribonucleoprotein structure

Messenger RNA turnover directed by A + U-rich elements (AREs) involves selected ARE-binding proteins. Whereas several signaling systems may modulate ARE-directed mRNA decay and/or post-translationally modify specific trans-acting factors, it is unclear how these mechanisms are linked. In THP-1 monocytic leukemia cells, phorbol ester-induced stabilization of some mRNAs containing AREs was accompanied by dephosphorylation of Ser83 and Ser87 of polysome-associated p40AUF1. Here, we report that phosphorylation of p40AUF1 influences its ARE-binding affinity as well as the RNA conformational dynamics and global structure of the p40AUF1-ARE ribonucleoprotein complex. Most notably, association of unphosphorylated p40AUF1 induces a condensed RNA conformation upon ARE substrates. By contrast, phosphorylation of p40AUF1 at Ser83 and Ser87 inhibits this RNA structural transition. These data indicate that selective AUF1 phosphorylation may regulate ARE-directed mRNA turnover by remodeling local RNA structures, thus potentially altering the presentation of RNA and/or protein determinants involved in subsequent trans-factor recruitment.     

Relationships among native and introduced populations of the Argentine ant (Linepithema humile) and the source of introduced populations

The Argentine ant (Linepithema humile) is a damaging invasive species that has become established in many Mediterranean-type ecosystems worldwide. To identify likely sources of introduced populations we examined the relationships among native Linepithema populations from Argentina and Brazil and introduced populations of L. humile using mitochondrial cytochrome b sequence data and nuclear microsatellite allele frequencies. The mitochondrial phylogeny revealed that the populations in Brazil were only distantly related to both the introduced populations and the native populations in Argentina, and confirmed that populations in Brazil, previously identified as L. humile, are likely a different species. The microsatellite-based analysis provided resolution among native and introduced populations of L. humile that could not be resolved using the mitochondrial sequences. In the native range, colonies that were geographically close to one another tended to be genetically similar, whereas more distant colonies were genetically different. Most samples from the introduced range were genetically similar, although some exceptions were noted. Most introduced populations were similar to native populations from the southern Rio Parana and were particularly similar to a population from Rosario, Argentina. These findings implicate populations from the southern Rio Parana as the most likely source of introduced populations. Moreover, these data suggest that current efforts to identify natural enemies of the Argentine ant for biological control should focus on native populations in the southern Rio Parana watershed.     

Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.     

Spatiotemporal patterns of intraspecific aggression in the invasive Argentine ant

The Argentine ant, Linepithema humile, is a widespread invasive species characterized by reduced intraspecific aggression within introduced populations. To illuminate the mechanisms underlying nestmate recognition in Argentine ants, we studied the spatial and temporal fidelity of intraspecific aggression in an introduced population of Argentine ants within which intraspecific aggression does occur. We quantified variation in the presence or absence of intraspecific aggression among nests over time both in the field and under controlled laboratory conditions to gain insight into the role of environmental factors as determinants of nestmate discriminatory ability. In addition, we compared levels of intraspecific aggression between nest pairs to the similarity of their cuticular hydrocarbons to determine the potential role of these compounds as labels for nestmate discrimination. In both field and laboratory comparisons, nest pairs behaved in a consistent manner throughout the course of the experiment: pairs that fought did so for an entire year, and pairs that did not fight remained nonaggressive. Moreover, we found a negative relationship between cuticular hydrocarbon similarity and the degree of aggression between nests, suggesting that these hydrocarbons play a role in nestmate discriminatory ability. In contrast to the prevailing pattern, ants from one site showed a marked change in behaviour during the course of this study. A concomitant change was also seen in the cuticular hydrocarbon profiles of ants from this site. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.