Caffeine, cyclic AMP and postreplication repair of mammalian cell DNA.

The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. We tested this possibility by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA.     

Binding of glucocorticoid receptors to DNA.

DNA has been implicated as the nuclear acceptor for receptor-glucocorticoid complexes. The present study concerns the interaction of these complexes, isolated from cultured rat hepatoma cells, with purified DNA. This association is rapid, reaching a maximum within a few minutes at 0 degrees, whereas dissociation requires several hours. DNA binds neither free glucocorticoids nor those complexed with transcortin or cytosol proteins different from the receptor. Receptors which are not complexed by steroid have little or no affinity for DNA. "Activation," necessary for the binding of receptor-steroid complexes to isolated nuclei, also enhances DNA binding. The capacity of DNA for binding receptor-steroid complexes is large; saturation was not observed at the complex concentrations studied, using either crude or partially purified receptor preparations. The association of complexes with DNA is inhibited by divalent cations, at increasing ionic strengths, and by mercurial reagents. Complexes bind equally well to bacterial, bacteriophage, or rat DNA; however, there was either no or substantially reduced binding by bacterial 23 S rRNA. The binding of complexes to native DNA is roughly 3-fold greater than to denatured DNA. These characteristics are consistent with the possibility that DNA is the nuclear acceptor for receptor-glucocorticoid complexes; however, the actual composition of the acceptor sites remains unknown.     

Mechanistic Basis for Type 2 Long QT Syndrome Caused by KCNH2 Mutations that Disrupt Conserved Arginine Residues in the Voltage Sensor

KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K⁺ current (I _Kr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing I _Kr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (I _Kv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in I _Kv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing I _Kr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease I _Kr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.     

Heat stress but not inbreeding affects offensive sperm competitiveness in Callosobruchus maculatus

Environmental and genetic stress have well‐known detrimental effects on ejaculate quality, but their concomitant effect on male fitness remains poorly understood. We used competitive fertilization assays to expose the effects of stress on offensive sperm competitive ability in the beetle Callosobruchus maculatus, a species where ejaculates make up more than 5% of male body mass. To examine the effects of environmental and genetic stress, males derived from outcrosses or sib matings were heat shocked at 50°C for 50 min during the pupal stage, while their siblings were maintained at a standard rearing temperature of 28°C. Heat‐shocked males achieved only half the offensive paternity success of their siblings. While this population exhibited inbreeding depression in body size, sperm competitiveness was unaffected by inbreeding, nor did the effect of heat shock stress on sperm competitiveness depend on inbreeding status. In contrast, pupal emergence success was increased by 34% among heat‐stressed individuals, regardless of their inbreeding status. Heat‐shocked males' ejaculate size was 19% reduced, but they exhibited 25% increased mating duration in single mating trials. Our results highlight both the importance of stress in postcopulatory sexual selection, and the variability among stressors in affecting male fitness.     

Impact in quarantine of the galling weevil Lepidapion argentatum on shoot growth of French broom (Genista monspessulana), an invasive weed in the western U.S.

In weed biocontrol, there is a need for pre-release efficacy assessments for potential agents. Genista monspessulana ((L.) L. A. S. Johnson (Fabaceae), French broom) is an invasive perennial shrub in the western U.S. The galling weevil Lepidapion argentatum Gerstaecker is a potential biocontrol agent. The impact of increasing weevil density on galling damage, plant height, width, leaf damage, and relative growth rate (RGR) was assessed in greenhouse experiments on two to three-month-old seedlings infested with either one or three weevils. Infestation by three female weevils caused 48% more galls producing 27% more larvae than did infestation with one female while causing only 1% leaf damage and no difference in total leaf area. Infestation with multiple weevils caused a 55% and 29% decrease in plant height and canopy width respectively, while single-weevil infestation decreased height by 32% and width to the same degree as for multiple weevils. The RGR of seedlings infested with three weevils was three times slower than the controls, while growth was reduced 2-fold by single-weevil infestation. Reductions in plant size and growth rate induced by weevil galling could reduce plant competitive survival to reproduction and also plant population dispersal as seedlings. Our results suggest that L. argentatum has the potential to cause impact to French broom seedlings if released in the invasive range.     

Sperm competition games between sneaks and guards: a comparative analysis using dimorphic male beetles

Sperm competition is widely recognized as a pervasive force of sexual selection. Theory predicts that across species increased risk of sperm competition should favor an increased expenditure on the ejaculate, a prediction for which there is much evidence. Sperm competition games have also been developed specifically for systems in which males adopt the alternative male mating tactics of sneaking copulations or guarding females. These models have not yet been tested in a comparative context, but predict that: across species male expenditure on the ejaculate should increase with increasing probability of a sneak mating; within species, sneaks should have the greater expenditure on the ejaculate; and the disparity in expenditure between sneaks and guards should be greatest in species with moderate risk of a sneak mating, and decline toward parity in species with low or high risk. Beetles in the genus Onthophagus are often characterized by dimorphic male morphologies that reflect the alternative mating tactics of sneak (minor males) and guard (major males). We conducted a comparative analysis across 16 species of male dimorphic onthophagines, finding that testes size increased across species with increasing frequency of the minor male phenotype. Minor males generally had the greater testes size, but across species the disparity between morphs was independent of the frequency of minor males. We present data on testes allometry from two populations of O. taurus that have undergone genetic divergence in the frequency of minor males. Consistent with the comparative analysis, these data support the notion that the relative frequency of sneaks in the population influences male expenditure on the ejaculate.     

Developmental expression and stress induction of glutathione S-transferase in the spruce budworm, Choristoneura fumiferana

Developmental and stress-induced expression of Choristoneura fumiferana glutathione S-transferase (CfGST) mRNA and protein were examined using Northern blots and Western blots. High levels of CfGST mRNA and protein were detected in 1st instar larvae and diapausing 2nd instar larvae. Expression of CfGST gradually decreased during larval development from 3rd to 5th instar, after which the expression increased once again, reaching peak levels in 6th instar larvae. CfGST mRNA and protein were undetectable in the pupal stage. Exposure to low temperature did not induce an increase in CfGST expression. Feeding on balsam fir foliage resulted in an increase in the expression of CfGST as compared to larvae that fed on artificial diet. The bacterial insecticide, Bacillus thuringiensis delta-endotoxin (Bt), the non-steroidal ecdysone analog, tebufenozide, and the synthetic pyrethroid, permethrin, induced the expression of CfGST mRNA in 5th instar larvae, whereas the chitin synthesis inhibitor, diflubenzuron, did not have any such effect. These results suggest that CfGST plays an important role in detoxifying various allelochemicals and insecticides in the spruce budworm. The developmental expression pattern strongly suggests that in addition to detoxification, CfGST might be involved in other functions.