Telomere dynamics in wild banded mongooses: Evaluating longitudinal and quasi-longitudinal markers of senescence.

Telomere length and the rate of telomere shortening have been suggested as particularly useful physiological biomarkers of the processes involved in senescent decline of somatic and reproductive function. However, longitudinal data on changes in telomere length across the lifespan are difficult to obtain, particularly for long-lived animals. Quasi-longitudinal studies have been proposed as a method to gain insight into telomere dynamics in long-lived species. In this method, minimally replicative cells are used as the baseline telomere length against which telomere length in highly replicative cells (which represent the current state) can be compared. Here we test the assumptions and predictions of the quasi-longitudinal approach using longitudinal telomere data in a wild cooperative mammal, the banded mongoose, Mungos mungo. Contrary to our prediction, telomere length (TL) was longer in leukocytes than in ear cartilage. Longitudinally, the TL of ear cartilage shortened with age, but there was no change in the TL of leukocytes, and we also observed many individuals in which TL increased rather than decreased with age. Leukocyte TL but not cartilage TL was a predictor of total lifespan, while neither predicted post-sampling survival. Our data do not support the hypothesis that cross-tissue comparison in TL can act as a quasi-longitudinal marker of senescence. Rather, our results suggest that telomere dynamics in banded mongooses are more complex than is typically assumed, and that longitudinal studies across whole life spans are required to elucidate the link between telomere dynamics and senescence in natural populations.

Lifetime fitness consequences of early-life ecological hardship in a wild mammal population.

Early-life ecological conditions have major effects on survival and reproduction. Numerous studies in wild systems show fitness benefits of good quality early-life ecological conditions ("silver-spoon" effects). Recently, however, some studies have reported that poor-quality early-life ecological conditions are associated with later-life fitness advantages and that the effect of early-life conditions can be sex-specific. Furthermore, few studies have investigated the effect of the variability of early-life ecological conditions on later-life fitness. Here, we test how the mean and variability of early-life ecological conditions affect the longevity and reproduction of males and females using 14 years of data on wild banded mongooses (Mungos mungo). Males that experienced highly variable ecological conditions during development lived longer and had greater lifetime fitness, while those that experienced poor early-life conditions lived longer but at a cost of reduced fertility. In females, there were no such effects. Our study suggests that exposure to more variable environments in early life can result in lifetime fitness benefits, whereas differences in the mean early-life conditions experienced mediate a life-history trade-off between survival and reproduction. It also demonstrates how early-life ecological conditions can produce different selection pressures on males and females.

Variable ecological conditions promote male helping by changing banded mongoose group composition.

Ecological conditions are expected to have an important influence on individuals' investment in cooperative care. However, the nature of their effects is unclear: both favorable and unfavorable conditions have been found to promote helping behavior. Recent studies provide a possible explanation for these conflicting results by suggesting that increased ecological variability, rather than changes in mean conditions, promote cooperative care. However, no study has tested whether increased ecological variability promotes individual-level helping behavior or the mechanisms involved. We test this hypothesis in a long-term study population of the cooperatively breeding banded mongoose, Mungos mungo, using 14 years of behavioral and meteorological data to explore how the mean and variability of ecological conditions influence individual behavior, body condition, and survival. Female body condition was more sensitive to changes in rainfall leading to poorer female survival and pronounced male-biased group compositions after periods of high rainfall variability. After such periods, older males invested more in helping behavior, potentially because they had fewer mating opportunities. These results provide the first empirical evidence for increased individual helping effort in more variable ecological conditions and suggest this arises because of individual differences in the effect of ecological conditions on body condition and survival, and the knock-on effect on social group composition. Individual differences in sensitivity to environmental variability, and the impacts this has on the internal structure and composition of animal groups, can exert a strong influence on the evolution and maintenance of social behaviors, such as cooperative care.

Age-related declines and disease-associated variation in immune cell telomere length in a wild mammal.

Immunosenescence, the deterioration of immune system capability with age, may play a key role in mediating age-related declines in whole-organism performance, but the mechanisms that underpin immunosenescence are poorly understood. Biomedical research on humans and laboratory models has documented age and disease related declines in the telomere lengths of leukocytes ('immune cells'), stimulating interest their having a potentially general role in the emergence of immunosenescent phenotypes. However, it is unknown whether such observations generalise to the immune cell populations of wild vertebrates living under ecologically realistic conditions. Here we examine longitudinal changes in the mean telomere lengths of immune cells in wild European badgers (Meles meles). Our findings provide the first evidence of within-individual age-related declines in immune cell telomere lengths in a wild vertebrate. That the rate of age-related decline in telomere length appears to be steeper within individuals than at the overall population level raises the possibility that individuals with short immune cell telomeres and/or higher rates of immune cell telomere attrition may be selectively lost from this population. We also report evidence suggestive of associations between immune cell telomere length and bovine tuberculosis infection status, with individuals detected at the most advanced stage of infection tending to have shorter immune cell telomeres than disease positive individuals. While male European badgers are larger and show higher rates of annual mortality than females, we found no evidence of a sex difference in either mean telomere length or the average rate of within-individual telomere attrition with age. Our findings lend support to the view that age-related declines in the telomere lengths of immune cells may provide one potentially general mechanism underpinning age-related declines in immunocompetence in natural populations.

Histopathological effects of the Yen-Tc toxin complex from Yersinia entomophaga MH96 (Enterobacteriaceae) on the Costelytra zealandica (Coleoptera: Scarabaeidae) larval midgut.

Yersinia entomophaga MH96, which was originally isolated from the New Zealand grass grub, Costelytra zealandica, produces an orally active proteinaceous toxin complex (Yen-Tc), and this toxin is responsible for mortality in a range of insect species, mainly within the Coleoptera and Lepidoptera. The genes encoding Yen-Tc are members of the toxin complex (Tc) family, with orthologs identified in several other bacterial species. As the mechanism of Yen-Tc activity remains unknown, a histopathological examination of C. zealandica larvae was undertaken in conjunction with cultured cells to identify the effects of Yen-Tc and to distinguish the contributions that its individual subunit components make upon intoxication. A progressive series of events that led to the deterioration of the midgut epithelium was observed. Additionally, experiments using a cell culture assay system were carried out to determine the cellular effects of intoxication on cells after topical application and the transient expression of Yen-Tc and its individual components. While observations were broadly consistent with those previously reported for other Tc family members, some differences were noted. In particular, the distinct stepwise disintegration of the midgut shared features associated with both apoptosis and necrotic programmed cell death pathways. Second, we observed, for the first time, a contribution of toxicity from two chitinases associated with the Yen-Tc complex. Our findings were suggestive of the activities encoded within the subunit components of Yen-Tc targeting different sites along putative programmed cell death pathways. Given the observed broad host range for Yen-Tc, these targeted loci are likely to be widely shared among insects.

The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

The insect toxin complex of Yersinia.

Many members of the Yersinia genus encode homologues of insect toxins first observed in bacteria that are insect pathogens such as Photorhabdus, Xenorhabdus and Serratia entomophila. These bacteria secrete high molecular weight insecticidal toxins comprised of multiple protein subunits, termed the Toxin Complexes or Tc's. In Photorhabdus three distinct Tc subunits are required for full oral toxicity in insects, that include the [A], [B] and [C] types, although the exact stochiometry remains unclear. The genomes of Photorhabdus strains encode multiple tc loci, although only two have been shown to exhibit oral and injectable activity against the Hawk Moth, Manduca sexta. The exact role of the remaining homologues is unclear. The availability of bacterial genome sequences has revealed the presence of tc gene homologues in many different species. In this chapter we review the tc gene homologues in Yersinia genus. We discuss what is known about the activity of the Yersinia Tc protein homologues and attempt to relate this to the evolution of the genus and of the tca gene family.

The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain.

Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.

Structure of murine angiogenin: features of the substrate- and cell-binding regions and prospects for inhibitor-binding studies.

Angiogenin is an unusual member of the pancreatic ribonuclease superfamily that induces blood-vessel formation and is a promising anticancer target. The three-dimensional structure of murine angiogenin (mAng) has been determined by X-ray crystallography. Two structures are presented: one is a complex with sulfate ions (1.5 Angstroms resolution) and the other a complex with phosphate ions (1.6 Angstroms resolution). Residues forming the putative B(1), P(1) and B(2) subsites occupy positions similar to their hAng counterparts and are likely to play similar roles. The anions occupy the P(1) subsite, sulfate binding conventionally and phosphate adopting two orientations, one of which is novel. The B(1) subsite is obstructed by Glu116 and Phe119, with the latter assuming a less invasive position than its hAng counterpart. Hydrophobic interactions between the C-terminal segment and the main body of the protein are more extensive than in hAng and may underly the lower enzymatic activity of the murine protein. Elsewhere, the structure of the H3-B2 loop supports the view that hAng Asn61 interacts directly with cell-surface molecules and does not merely stabilize adjacent regions of the hAng structure. mAng crystals appear to offer small-molecule inhibitors a clear route to the active site and may even withstand a reorientation of the C-terminal segment that provides access to the cryptic B(1) subsite. These features represent considerable advantages over crystalline hAng and bAng.

Crystallographic studies on structural features that determine the enzymatic specificity and potency of human angiogenin: Thr44, Thr80, and residues 38-41.

Human angiogenin (Ang) is a potent inducer of blood vessel formation and is a member of the pancreatic ribonuclease superfamily. Its enzymatic activity is unusually weak and biased toward cleavage after cytidine nucleotides. As part of an ongoing investigation into the structural basis of Ang's characteristic activity, we have determined the crystal structures of three Ang variants having novel activity. (i) The structure of T44D-Ang indicates that Asp44 can participate directly in pyrimidine binding and that the intrinsic hydrogen-bonding capability of this residue largely governs the pyrimidine specificity of this variant. Unexpectedly, the mutation also causes the most extensive disruption of the C-terminus seen in any Ang variant thus far. This allows the side chain of Arg101 to penetrate the B(1) site, raising the possibility that it participates in substrate binding as occurs in ribonuclease 4. (ii) The structure of T80A-Ang supports the view that Thr80 plays little role in maintaining the obstructive conformation of the C-terminus and that its participation in a hydrogen bond with Thr44 selectively weakens the interaction between Thr44 and N3 of cytosine. (iii) ARH-II is an angiogenin/RNase A chimera in which residues 38-41 of Ang are replaced with the corresponding residues (38-42) of RNase A. Its structure suggests that the guest segment influences catalysis by subtle means, possibly by reducing the pK(a) of the catalytic lysine. The loss of angiogenic activity is not attributable to disruption of known cell-binding or nuclear translocation sites but may be a consequence of the chimera's enhanced ribonucleolytic activity.

The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen.

The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli. Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome. The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B. However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1. HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death. Here we show that E. coli expressing Mcf2 can, like E. coli expressing Mcf1, kill insects. Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells. The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains.

Guest-host crosstalk in an angiogenin-RNase A chimeric protein.

Angiogenin and ribonuclease A share 33% sequence identity but have distinct functions. Angiogenin is a potent inducer of angiogenesis that is only weakly ribonucleolytic, whereas ribonuclease A is a robust ribonuclease that is not angiogenic. A chimera ("ARH-I"), in which angiogenin residues 58-70 are replaced with residues 59-73 of ribonuclease A, has intermediate ribonucleolytic potency and no angiogenic activity. Here we report a crystal structure of ARH-I that reveals the molecular basis for these characteristics. The ribonuclease A-derived (guest) segment adopts a structure largely similar to that in ribonuclease A, and successfully converts this region from a cell-binding site to a purine-binding site. At the same time, its presence causes complex changes in the angiogenin-derived (host) portion that account for much of the increased ribonuclease activity of ARH-I. Guest-host interactions of this type probably occur more generally in protein chimeras, emphasizing the importance of direct structural information for understanding the functional behavior of such molecules.