ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

Maturational costs of reproduction due to clutch size and ontogenetic conflict as revealed in the invisible fraction

Reproduction is thought to entail costs, but assessing survival costs associated with maturation as organisms express reproductive genes for the first time is problematic because many will die prior to expressing a phenotype. We quantified selection acting on this invisible fraction by measuring selection on predicted breeding values for clutch size estimated from a multigenerational pedigree of side-blotched lizards in which clutch size was heritable (h2=0.25+/-0.04). The survival effects of predicted breeding values for clutch size during maturation, however, differed between males and females indicating ontogenetic conflict. Increased mortality during maturation was associated with lower predicted breeding values for clutch size for females, but higher predicted breeding values for males who do not produce a clutch. Genetic correlations between clutch size and male and female survival were consistent with calculated selection differentials. Experimental yolk removal did not affect progeny survival during maturation, indicating that selection on maturing progeny was not due to confounding yolk-volume maternal effects, and hormone manipulations confirmed clutch size as the target of viability selection during maturation. Such episodes of selection prior to phenotypic expression of the trait will have important consequences for the evolution of reproductive investment.     

In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

Single-Cell Deconvolution of Fibroblast Heterogeneity in Mouse Pulmonary Fibrosis

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Effects of Coxiella burnetii on MAPKinases phosphorylation

Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-γ production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity.     

The dynamics of forest tent caterpillar outbreaks across east‐central Canada

An analysis of forest tent caterpillar Malacosoma disstria defoliation records from Ontario and Quebec over the period 1938–2002 indicates that outbreaks recur periodically and somewhat synchronously among regions of the two provinces. Cluster analysis revealed that the most strongly periodic, large‐scale, synchronized fluctuations occurred within three regions: northwestern Ontario, eastern Ontario/western Quebec and southeastern Quebec. Defoliation in the vast surrounding hinterlands tended to be infrequent and sporadic, loosely tracking defoliation in the core outbreak regions. One small cluster in northeastern Ontario stood out as anomalous, as a result of an increasing trend in the duration of periodic defoliation episodes, marked by an unprecedented double‐wave of defoliation that persisted from 1992 to 1999. This is the precise area where, in the early 2000s, trembling aspen Populus tremuloides stems were mapped as being in a state of decline of unprecedented severity and extent. Our results suggest forest tent caterpillar has the potential to cause significant impacts on forest health and, hence, carbon budgets in east‐central Canada and that the forest tent caterpillar deserves more attention as a model system of forest insect disturbance ecology.     

Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases

Platelet endothelial cell adhesion molecule-1 (CD31) is a 130-kDa glycoprotein receptor present on the surface of platelets, neutrophils, monocytes, certain T-lymphocytes, and vascular endothelial cells. CD31 is involved in adhesion and signal transduction and is implicated in the regulation of a number of cellular processes. These include transendothelial migration of leukocytes, integrin regulation, and T-cell function, although its function in platelets remains unclear. In this study, we demonstrate the ability of the platelet agonists collagen, convulxin, and thrombin to induce tyrosine phosphorylation of CD31. Furthermore, we show that this event is independent of platelet aggregation and secretion and is accompanied by an increase in surface expression of CD31. A kinase capable of phosphorylating CD31 was detected in CD31 immunoprecipitates, and its activity was increased following activation of platelets. CD31 tyrosine phosphorylation was reduced or abolished by the Src family kinase inhibitor PP2, suggesting a role for these enzymes. In accordance with this, each of the Src family members expressed in platelets, namely Fyn, Lyn, Src, Yes, and Hck, was shown to co-immunoprecipitate with CD31. The involvement of Src family kinases in this process was confirmed through the study of mouse platelets deficient in Fyn.     

Is Working More Costly than Waiting in Monkeys?

We studied how value for instrumental action is discounted by predicted effort and delay. The monkeys were trained to perform instrumental trials that required a bar release when a visual target changed from red-to-green. There were two trial conditions. In delay trials, after the monkeys performed one instrumental trial correctly a reward was delivered 0–7 seconds later. In work trials, the monkeys had to perform 0, 1, or 2 additional instrumental trials to obtain a reward. The lengths of trials in delay matched the time it took to complete work trials. The length of delay or number of trials was indicated by a visual cue presented throughout the trial. Our hypothesis was that the monkeys would all show temporal discounting of reward in the delay trials, and that in the work trials the monkeys’ performance might reflect an additional cost due to working. The error rate increased linearly as remaining cost increased for all 8 monkeys. For 4 monkeys the error rate was significantly larger in work trials than in delay trials (effort sensitive monkeys). For the other 4 monkeys there was no significant difference in error rate (effort insensitive monkeys). Since the error rate has an inverse relation with value for action, these results suggest that value is discounted hyperbolically by effort as well as by delay. Error rates generally increased as the testing sessions progressed and the total reward accumulated (i.e., effect of reward devaluation). The relative impact of delay and effort on error rates was reasonably stable within subjects. Thus, within the monkey population there seems to be a significant dichotomy in the sensitivity governing whether working is more costly than waiting, possibly arising from a constitutional or genetic trait.