Recognizing the forest for the trees: testing temporal patterns of cladogenesis using a null model of stochastic diversification

Computer simulations are developed and employed to examine the expected temporal distributions of nodes under a null model of stochastic lineage bifurcation and extinction. These Markovian models of phylogenetic process were constructed so as to permit direct comparisons against empirical phylogenetic trees generated from molecular or other information available solely from extant species. For replicate simulated phylads with n extant species, cumulative distribution functions (cdf's) of branching times were calculated, and compared (using the Kolmogorov-Smirnov test statistic D) to those from three published empirical trees. Molecular phylogenies for columbine plants and avian cranes showed statistically significant departures from the null expectations, in directions indicating recent and ancient species' radiations, respectively, whereas a molecular phylogeny for the Drosophila virilis species group showed no apparent historical clustering of branching events. Effects of outgroup choice and phylogenetic frame of reference were investigated for the columbines and found to have a predictable influence on the types of conclusions to be drawn from such analyses. To enable other investigators to statistically test for nonrandomness in temporal cladogenetic pattern in empirical trees generated from data on extant species, we present tables of mean cdf's and associated probabilities under the null model for expected branching times in phylads of varying size. The approaches developed in this report complement and extend those of other recent methods for employing null models to assess the statistical significance of pattern in evolutionary trees.      

Determinants of high-affinity binding and receptor activation in the N-terminus of CCL-19 (MIP-3 beta)

CC chemokine receptor 7 (CCR-7) is expressed on mature dendritic cells and T-cells. Its ligands, CCL-19 (MIP-3beta) and CCL-21 (SLC), play an important role in the migration of these cells to secondary lymphoid organs where they are predominantly expressed. For most chemokines, the N-terminal domain preceding the first two conserved cysteines is involved in stabilizing the active conformation of its cognate receptors. We have chemically synthesized N-terminal analogues of CCL-19 with the aid of a native chemical ligation method to investigate structure function requirements of this ligand domain by performing ligand binding, GTP-gammaS binding, and chemotaxis assays. Successive truncations of the N-terminus of CCL-19 reduced the affinity of the receptor for the ligand in a size-dependent manner. Furthermore, Ala substitutions of Asn(3), Asp(4), and Asp(7) show that the side chains of these residues are important for high-affinity binding of CCL-19 to CCR-7. The effects observed were mirrored in both GTP-gammaS binding and chemotaxis assays, highlighting the functional importance of this ligand domain. We also describe two partial agonists of CCR-7 ([Nle(72)]CCL-19(6-77) and Ac-[Nle(72)]CCL-19(7-77)), and identify the first analogue of CCL-19 (Ac-[Nle(72)]CCL-19(8-77)) that acts as a functional antagonist in vitro (K(B) approximately 350 nM for GTP-gammaS binding assays). As mutations of both Glu(6) and Asp(7) to Ala did not dissociate effects on ligand binding from receptor activation, it is likely that the backbone of these two residues is crucial for agonist activity.