Mutation of a dibasic amino acid motif within the C terminus of the P2X7 nucleotide receptor results in trafficking defects and impaired function

Activation of the P2X(7) receptor by extracellular nucleotides modulates multiple immune functions, including inflammatory mediator production, membrane fusion events, and apoptosis. Previous studies have revealed that the C terminus of this multimeric cation channel possesses a lipid-interaction motif that has been proposed to regulate receptor function. This domain is homologous to the LPS binding region of the LPS binding protein, and we demonstrated that two basic residues (Arg(578), Lys(579)) within this motif are essential for LPS binding to P2X(7) in vitro. Because P2X(7) can influence LPS action, and because lipid interaction motifs modulate the trafficking of other ion channel-linked receptors, we hypothesized that this motif of P2X(7) is critical for receptor function and trafficking. In these studies we mutated Arg(578) and Lys(579) of P2X(7), and the expression profile, channel activity, and pore formation of the mutant were characterized in transfected human embryonic kidney 293 cells. In contrast with the wild-type receptor, the P2X(7)-R578E/K579E mutant fails to demonstrate surface immunoreactivity despite normal levels of total protein expression. This effect on the mutant receptor is unlikely to result from widespread defects in protein folding, because surface localization, determined using conformation-specific Abs, can be restored by growing the cells at 25 degrees C, conditions that slow receptor recycling. Despite surface expression at reduced temperatures, at 25 degrees C the P2X(7)-R578E/K579E mutant still exhibits greatly reduced sodium, potassium, and calcium channel activity when compared with the wild-type receptor, and cannot induce pore formation. These data suggest that the lipid interaction motif of the P2X(7) C terminus controls receptor trafficking and modulates channel activity.     

Parturition hormone in the tsetse Glossina morsitans: activity in reproductive tissues from other species and response of tsetse to identified neuropeptides and other neuroactive compounds

Parturition hormone (PH) activity is present not only in the uterus of the tsetse Glossina morsitans but also in the oviducts of Bombyx mori and Schistocerca gregaria, as well as the ejaculatory duct of S. gregaria males. Activity thus appears to be present in the reproductive ducts of diverse insect taxa. To determine whether any of the common insect neuropeptides are capable of mimicking the effect of PH, 35 identified neuropeptides and analogs were evaluated for PH activity. Modest PH activity was observed for only high doses of proctolin and a pyrokinin analog, thus suggesting that PH is unlikely to be closely related to any of the identified neuropeptides tested. While proctolin was highly effective in stimulating contractions of the S. gregaria oviduct, the extract from the tsetse uterus elicited only a weak response in this bioassay. PH activity was, however, effectively mimicked with an injection of 8 bromo-cyclic GMP, thus suggesting a potential role for this cyclic nucleotide in mediating the PH response. Pregnant females were responsive to PH, other neuropeptides and cyclic nucleotides only when females were neck-ligated. In intact females, the brain can presumably override the stimulation provided by the active compounds.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.      

Oleic acid is elevated in cell membranes during rapid cold-hardening and pupal diapause in the flesh fly, Sarcophaga crassipalpis

The integrity of cellular membranes is critical to the survival of insects at low temperatures, thus an advantage is conferred to insects that can adjust their composition of membrane fatty acids (FAs). Such changes contribute to homeoviscous adaption, a process that allows cellular membranes to maintain a liquid-crystalline state at temperatures that are potentially low enough to cause the membrane to enter the gel state and thereby lose its ability to maintain homeostasis. Flesh flies (Sarcophaga crassipalpis) were subjected to two experimental conditions that elicit low temperature tolerance: rapid cold-hardening and diapause. FAs were isolated and analyzed using gas chromatography-mass spectrometry. FAs changed in response to both rapid cold-hardening and diapause. In response to rapid cold-hardening (8 h at 4 degrees C), the proportion of oleic acid (18:1n-9) in pharate adults increased from 30% to 47% of the total FA pool. The proportion of almost every other FA was reduced. By entering diapause, pupae experienced an even greater increase in oleic acid proportion, to 58% of the total FA pool. Oleic acid not only promotes membrane fluidity at low temperature but also allows the cell membrane to maintain a liquid crystalline state if temperatures increase.     

An endosymbiotic conidial fungus, Scopulariopsis brevicaulis, protects the American dog tick, Dermacentor variabilis, from desiccation imposed by an entomopathogenic fungus

The functional role of an endosymbiotic conidial fungus (Scopulariopsis brevicaulis) prevalent within the integumental glands and hemocoel of the American dog tick (Dermacentor variabilis) was investigated to explore the nature of this tick/fungus association. D. variabilis is normally highly resistant to Metarhizium anisopliae, a widely-distributed entomopathogenic fungus, but when mature female ticks harboring S. brevicaulis were fed a solution containing a mycotoxin (Amphotericin B) to purge this mycobiont internally, the ticks inoculated with M. anisopliae displayed classic signs of pathogenicity, as evidenced by recovery of M. anisopliae from ticks by internal fungus culture, greatly accelerated net transpiration water loss rates (nearly 3x faster than ticks containing S. brevicaulis naturally) and elevation of critical equilibrium humidity (CEH) closer to saturation, implying a reduced capacity to absorb water vapor and disruption of water balance (water gain not equal water loss) that resulted in tick death. The presence of S. brevicaulis within the tick was previously puzzling: the fungus is transmitted maternally and there is no apparent harm inflicted to either generation. This study suggests that S. brevicaulis provides protection to D. variabilis ticks against M. anisopliae. Thus, the S. brevicaulis/tick association appears to be mutualistic symbiosis. Given that both organisms are of medical-veterinary importance, disruption of this symbiosis has potential for generating novel tools for disease control.