Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination

Progressive telomere shortening from cell division (replicative aging) provides a barrier for human tumor progression. This program is not conserved in laboratory mice, which have longer telomeres and constitutive telomerase. Wild species that do/do not use replicative aging have been reported, but the evolution of different phenotypes and a conceptual framework for understanding their uses of telomeres is lacking. We examined telomeres/telomerase in cultured cells from > 60 mammalian species to place different uses of telomeres in a broad mammalian context. Phylogeny‐based statistical analysis reconstructed ancestral states. Our analysis suggested that the ancestral mammalian phenotype included short telomeres (< 20 kb, as we now see in humans) and repressed telomerase. We argue that the repressed telomerase was a response to a higher mutation load brought on by the evolution of homeothermy. With telomerase repressed, we then see the evolution of replicative aging. Telomere length inversely correlated with lifespan, while telomerase expression co‐evolved with body size. Multiple independent times smaller, shorter‐lived species changed to having longer telomeres and expressing telomerase. Trade‐offs involving reducing the energetic/cellular costs of specific oxidative protection mechanisms (needed to protect < 20 kb telomeres in the absence of telomerase) could explain this abandonment of replicative aging. These observations provide a conceptual framework for understanding different uses of telomeres in mammals, support a role for human‐like telomeres in allowing longer lifespans to evolve, demonstrate the need to include telomere length in the analysis of comparative studies of oxidative protection in the biology of aging, and identify which mammals can be used as appropriate model organisms for the study of the role of telomeres in human cancer and aging.     

Induced pluripotent stem cells from highly endangered species

For some highly endangered species there are too few reproductively capable animals to maintain adequate genetic diversity, and extraordinary measures are necessary to prevent extinction. We report generation of induced pluripotent stem cells (iPSCs) from two endangered species: a primate, the drill, Mandrillus leucophaeus and the nearly extinct northern white rhinoceros, Ceratotherium simum cottoni. iPSCs may eventually facilitate reintroduction of genetic material into breeding populations.     

Short-term microbial and physico-chemical variability in low-temperature hydrothermal fluids near 5°S on the Mid-Atlantic Ridge

This study examines the representativeness of low-temperature hydrothermal fluid samples with respect to their chemical and microbiological characteristics. Within this scope, we investigated short-term temporal chemical and microbial variability of the hydrothermal fluids. For this purpose we collected three fluid samples consecutively from the same spot at the Clueless field near 5°S on the southern Mid-Atlantic Ridge over a period of 50 min. During sampling, the temperature was monitored online. We measured fluid chemical parameters, characterized microbial community compositions and used statistical analyses to determine significant differences between the samples. Overall, the three fluid samples are more closely related to each other than to any other tested habitat. Therefore, on a broad scale, the three collected fluid samples can be regarded as habitat representatives. However, small differences are apparent between all samples. One of the Clueless samples even displayed significant differences ( P -value < 0.01) to the other two Clueless samples. Our data suggest that the observed variations in fluid chemical and microbial compositions are not reflecting sampling artefacts but are related to short-term fluid variability due to dynamic subseafloor fluid mixing. Recorded temporal changes in fact reflect spatial heterogeneity found in the subsurface as the fluid flows through distinctive pathways. While conservative elements (Cl, Si, Na and K) indicate variable degrees of fluid-seawater mixing, reactive components, including Fe(II), O₂ and H₂S, show that chemical and microbial reactions within the mixing zone further modify the emanating fluids on short-time scales. Fluids entrain microorganisms, which modify the chemical microenvironment within the subsurface biotopes. This is the first study focusing on short-term microbial variability linked to chemical changes in hydrothermal fluids.     

Apolipoprotein(a), through its strong lysine-binding site in KIV(10'), mediates increased endothelial cell contraction and permeability via a Rho/Rho kinase/MYPT1-dependent pathway

Substantial evidence indicates that endothelial dysfunction plays a critical role in atherogenesis. We previously demonstrated that apolipoprotein(a) (apo(a); the distinguishing protein component of the atherothrombotic risk factor lipoprotein(a)) elicits rearrangement of the actin cytoskeleton in human umbilical vein endothelial cells, characterized by increased myosin light chain (MLC) phosphorylation via a Rho/Rho kinase-dependent signaling pathway. Apo(a) contains kringle (K)IV and KV domains similar to those in plasminogen: apo(a) contains 10 types of plasminogen KIV-like sequences, followed by sequences homologous to the plasminogen KV and protease domains. Several of the apo(a) kringles contain lysine-binding sites (LBS) that have been proposed to contribute to the pathogenicity of Lp(a). Here we demonstrate that apo(a)-induced endothelial barrier dysfunction is mediated via a Rho/Rho kinase-dependent signaling pathway that results in increased MYPT1 phosphorylation and hence decreased MLC phosphatase activity, thus leading to an increase in MLC phosphorylation, stress fiber formation, cell contraction, and permeability. In addition, studies using recombinant apo(a) variants indicated that these effects of apo(a) are dependent on sequences within the C-terminal half of the apo(a) molecule, specifically, the strong LBS in KIV(10). In parallel experiments, the apo(a)-induced effects were completely abolished by treatment of the cells with the lysine analogue epsilon-aminocaproic acid and the Rho kinase inhibitor Y27632. Taken together, our findings indicate that the strong LBS in apo(a) KIV(10) mediates all of our observed effects of apo(a) on human umbilical vein endothelial cell barrier dysfunction. Studies are ongoing to further dissect the molecular basis of these findings.     

Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice

Molecular mechanisms regulating the remodeling of the lymphatic vasculature from an immature plexus of vessels to a hierarchal network of initial and collecting lymphatics are not well understood. One gene thought to be important for this process is Angiopoietin-2 (Ang-2). Ang2^−/− mice have previously been reported to exhibit an abnormal lymphatic phenotype but the precise nature of the lymphatic defects and the underlying mechanisms have yet to be defined. Here we demonstrate by whole-mount immunofluorescence staining of ear skin and mesentery that lymphatic vessels in Ang2^−/− mice fail to mature and do not exhibit a collecting vessel phenotype. Furthermore, dermal lymphatic vessels in Ang2^−/− pups prematurely recruit smooth muscle cells and do not undergo proper postnatal remodeling. In contrast, Ang2 knock-out Ang1 knock-in mice do develop a hierarchal lymphatic vasculature, suggesting that activation of Tie-2 is required for normal lymphatic development. Taken together, this work pinpoints a specific lymphatic defect of Ang2^−/− mice and further defines the sequential steps in lymphatic vessel remodeling.     

A high-resolution cat radiation hybrid and integrated FISH mapping resource for phylogenomic studies across Felidae

We describe the construction of a high-resolution radiation hybrid (RH) map of the domestic cat genome, which includes 2662 markers, translating to an estimated average intermarker distance of 939 kilobases (kb). Targeted marker selection utilized the recent feline 1.9x genome assembly, concentrating on regions of low marker density on feline autosomes and the X chromosome, in addition to regions flanking interspecies chromosomal breakpoints. Average gap (breakpoint) size between cat-human ordered conserved segments is less than 900 kb. The map was used for a fine-scale comparison of conserved syntenic blocks with the human and canine genomes. Corroborative fluorescence in situ hybridization (FISH) data were generated using 129 domestic cat BAC clones as probes, providing independent confirmation of the long-range correctness of the map. Cross-species hybridization of BAC probes on divergent felids from the genera Profelis (serval) and Panthera (snow leopard) provides further evidence for karyotypic conservation within felids, and demonstrates the utility of such probes for future studies of chromosome evolution within the cat family and in related carnivores. The integrated map constitutes a comprehensive framework for identifying genes controlling feline phenotypes of interest, and to aid in assembly of a higher coverage feline genome sequence.     

Synthesis and SAR of amino acid-derived heterocyclic progesterone receptor full and partial agonists

Two classes of amino acid-derived heterocyclic progesterone receptor ligands were developed to address the metabolic issues posed by the dimethyl amide functionality of the lead compound (1). The tetrazole-derived ligands behaved as potent partial agonists, while the 1,2,4-triazole ligands behaved as potent full agonists.     

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a)

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.     

Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV (2), KIV (7), KIV (9) DeltaCys (which lacks the seventh unpaired cysteine) and KIV (10) which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV (10) to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV (10) is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by approximately 43% at a concentration of 10 microM KIV (10). The ability of the KIV (10) mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp (70) to Arg abolished binding to both lysine-Sepharose and plasmin-modified fibrinogen, while the Trp (70) -->Phe and Arg (35) -->Lys substitutions each resulted in decreased binding to these substrates. None of the KIV (10) mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV (7) contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV (10). However, apo(a) KIV (7) had no effect on fibrinolysis in vitro. Apo(a) KIV (2) and KIV (9) DeltaCys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro.