Inhibition of DNA replication fork progression and mutagenic potential of 1, N6-ethenoadenine and 8-oxoguanine in human cell extracts

Comparative mutagenesis of 1,N⁶-ethenoadenine (εA) and 8-oxoguanine (8-oxoG), two endogenous DNA lesions that are also formed by exogenous DNA damaging agents, have been evaluated in HeLa and xeroderma pigmentosum variant (XPV) cell extracts. Two-dimensional gel electrophoresis of the duplex M13mp2SV vector containing these lesions established that there was significant inhibition of replication fork movement past εA, whereas 8-oxoG caused only minor stalling of fork progression. In extracts of HeLa cells, εA was weakly mutagenic inducing all three base substitutions in approximately equal frequency, whereas 8-oxoG was 10-fold more mutagenic inducing primarily G→T transversions. These data suggest that 8-oxoG is a miscoding lesion that presents a minimal, if any, block to DNA replication in human cells. We hypothesized that bypass of εA proceeded principally by an error-free mechanism in which the undamaged strand was used as a template, since this lesion strongly blocked fork progression. To examine this, we determined the sequence of replication products derived from templates in which a G was placed across from the εA. Consistent with our hypothesis, 93% of the progeny were derived from replication of the undamaged strand. When translesion synthesis occurred, εA→T mutations increased 3-fold in products derived from the mismatched εA: G construct compared with those derived from the εA: T construct. More efficient repair of εA in the εA: T construct may have been responsible for lower mutation frequency. Primer extension studies with purified pol η have shown that this polymerase is highly error-prone when bypassing εA. To examine if pol η is the primary mutagenic translesion polymerase in human cells, we determined the lesion bypass characteristics of extracts derived from XPV cells, which lack this polymerase. The εA: T construct induced εA→G and εA→C mutant frequencies that were approximately the same as those observed using the HeLa extracts. However, εA→T events were increased 5-fold relative to HeLa extracts. These data support a model in which pol η-mediated translesion synthesis past this adduct is error-free in the context of semiconservative replication in the presence of fidelity factors such as PCNA.     

Intraguild predation by the generalist predator Dicyphus hesperus on the parasitoid Encarsia formosa

Predation was measured in the laboratory by Dicyphus hesperus females on patches of greenhouse whitefly (Trialeurodes vaporariorum) pupae. The patches contained different proportions of unparasitized pupae and those parastisized as nymphs by Encarsia formosa. Dicyphus hesperus fed readily on parasitized pupae and the number of parasitized pupae consumed increased with the proportion available indicating a capacity for intraguild predation. However, D. hesperus females did not display a preference for feeding on parasitized pupae versus unparasitized pupae. Dicyphus hesperus adults were released at three densities into greenhouse enclosures containing tomato plants harbouring greenhouse whitefly and immature E. formosa. Numbers of killed and parasitized whitefly nymphs and pupae and the total number of healthy whitefly were compared among Dicyphus density treatments. On upper leaves, the amount of predation on whitefly nymphs was higher at the highest predator density than at the lower density or in enclosures where D. hesperus was not released. On lower leaves, the number of parasitized pupae was lowest with the highest predator density indicating a negative effect of intraguild predation by D. hesperus on E. formosa. Despite this, Dicyphus density did not affect the density of whitefly in the presence of E. formosa. The results are discussed as they relate to concurrent use of D. hesperus and E. formosa for regulation of greenhouse whitefly populations.     

Male-like mudballing behavior of some female fiddler crabs (Uca tangeri)

At each low tide, male and female Uca tangeri remove mudballs from inside their burrows and place them on the surface. Previous studies have shown clear intersexual differences in mudball arrangements. However, we noticed that some females placed their mudballs in an arrangement similar to that of males. In this study, we investigated several factors that may have been responsible for this change in female mudballing behavior. We found no significant effect of the lunar cycle, female size and reproductive state, or burrow features. We briefly discuss the avoidance of sexual coercion or parasite modification of host behavior as possible factors. Our study shows that intersexual differences in mudballing behavior are more complex than previously thought. Received: October 18, 2000 / Accepted: May 7, 2001     

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.     

Isoelectric focusing of human red cell phosphoglucomutase

Summary A total of 637 individuals from the rural village of Keneba in The Gambia, West Africa, have been typed for red cell PGM using isoelectric focusing (pH 5–7) in polyacrylamide gels. Eight different phenotypes have been detected. The frequency of the four alleles at the PGM₁ locus was found to be PGM ₁ ¹⁺ 0.795, PGM ₁ ^1- 0.053, PGM ₁ ²⁺ 0.133, and PGM ₁ ^2- 0.019. A study of the PGM phenotypes in 89 families confirmed the simple Mendelian codominant inheritance of the four alleles. Comparative population data suggest that red cell PGM typing by isoelectric focusing might prove to be a useful genetic marker in anthropological studies.     

Allogeneic restriction of acquired antimicrobial resistance in the rat.

Allogeneic restriction of acquired antimicrobial resistance was demonstrated by infusing Listeria-immune thoracic duct lymphocytes (TDL) into syngeneic, semisyngeneic, or allogeneic recipients that had been infected i.v. with Listeria monocytogenes (LM). Syngeneic and semisyngeneic recipients enjoyed a high level of protection (3 to 5 log10 reduction in viable organisms within 64 hr, whereas allogeneic recipients were poorly protected (0.5 to 2.5 log10 reduction). Sharing one haplotype between TDL-donors and challenged recipients resulted in a 4- to 8-fold increase in the level of resistance transferred. Histoincompatibility reactions could be excluded as a cause of the observed allogeneic restriction. Therefore, it is likely that LM-immune TDL are unable to respond efficiently to the antigens of this organism in an allogeneic environment. But allogeneic immune TDL do confer a low level of protection that cannot be ascribed to B cell activity nor to GvH and/or HvG reactions. Evidence is provided that the modest protection afforded by allogeneic lymphocytes is conveyed by the same T cell subset that operates with much higher efficiency in genetically related rats.     

Long-term anaesthesia with alfentanil and midazolam for lung transplantation in the dog

An anaesthetic regime was developed for lung transplantation in the dog using a continuous infusion of alfentanil and midazolam. This combination of agents provided excellent analgesia and also produced loss of consciousness. Cardiovascular stability was well maintained over a 24-h period of anaesthesia following lung transplantation. Although no animals were allowed to recover from anaesthesia in the present series, the regime described is likely to be suitable for recovery anaesthesia, particularly since both of the agents used can be reversed with specific antagonists.