Comparison of 125I-Angiotensin III and 125I-Angiotensin II Binding to Rat Brain Membranes

The binding of 125I-angiotensin III (125I-ANG III) to rat brain membranes was examined and compared with that of 125I-angiotensin II (125I-ANG II). Degradation of each ligand, as monitored by HPLC, was effectively inhibited using fragments of ANG III and ANG II known to have little affinity for angiotensin binding sites. Three classes of 125I-ANG III-binding sites were observed based on affinity (KD = 0.13, 1.83, and 10.16 nM) and capacity (Bmax = 1.30, 18.41, and 67.2 fmol/mg protein, respectively). Two classes of 125I-ANG II-binding sites of high affinity (KD = 0.11 and 1.76 nM) and low capacity (Bmax = 1.03 and 18.86 fmol/mg protein, respectively) were also identified. Cross-displacement studies confirmed that the two highest-affinity 125I-ANG III-binding sites and the 125I-ANG II-binding sites were the same. On the other hand, the binding of 125I-ANG III to the low-affinity 125I-ANG III-binding site could not be inhibited with ANG II. These data imply that previously measured differences in the biological potency of cerebroventricularly applied ANG III and ANG II probably do not result from differential binding of these peptides to central angiotensin receptors.     

Reconstructing the Evolutionary Radiation of Dasyurine Marsupials with Cytochrome b, 12S rRNA, and Protamine P1 Gene Trees

We report complete sequences of the cytochrome b, 12S rRNA, and protamine P1 genes from 18 of the 21 extant species of dasyurine marsupials (family Dasyuridae). Partial sequences are included for Pseudantechinus ningbing, but no data are available for Ps. mimulus or Phascolosorex doriae. Phylogenetic analyses of these sequences yield compatible gene trees with limited resolution. Simultaneous parsimony analysis of all three genes suggests the following: (a) a basal polytomy of eight lineages; (b) a sister-group relationship between phascolosoricine genera (Neophascogale and Phascolosorex, which are monophyletic) and Dasyums + Sarcophilus; (c) monophyly of Dasyurus viverrinus, D. albopunctatus, D. geoffroii, and D. spartacus apart from other quolls and Sarcophilus; and (d) sister-pairing of D. geoffroii and D. spartacus Previous attempts at unraveling the phylogenetic history of dasyurines have produced strikingly inconsistent results, due in part to differences in character systems examined, interpretations of character homology and independence, and analytical methods employed. We provide some evidence that the basal polytomy is the result of rapid cladogenesis and suggest that this episode of dasyurine evolution is temporally correlated with the onset of aridification following the New Guinean uplift of 15 million years ago.     

Delayed Cerebroventricular Metabolism of [125I]Angiotensins in the Spontaneously Hypertensive Rat

This study was designed to evaluate the hypothesis that impaired brain angiotensin signal termination contributes to the sustained blood pressure elevations noted in the genetically hypertensive rat model of human essential hypertension. A technique that combined the intracerebroventricular injection of [125I]angiotensins, followed by focused microwave fixation to stop all peptidase activity and subsequent HPLC analyses, was used for determining half-lives of [125I]angiotensin II and [125I]angiotensin III in the ventricular space. The results indicate that the spontaneously hypertensive rat evidenced significantly longer half-lives for intracerebroventricularly injected [125I]angiotensin II over those measured for the Wistar-Kyoto and Sprague-Dawley normotensive rat strains: 45.0, 27.2, and 25.0 s, respectively. This was also true for intracerebroventricularly administered [125I]angiotensin III: 19.5, 11.4, and 9.0 s, respectively. These results support the notion that a dysfunction in central aminopeptidase activity in the spontaneously hypertensive rat may result in prolonged half-lives of endogenously synthesized angiotensins II and III, which are known to serve as ligands at central angiotensin receptors responsible for the control of cardiovascular function. The extended half-lives of these ligands may contribute to the sustained elevations in blood pressure observed in this animal model.     

Live fast,die young: the life cycle of the brooding feather star Aporometra wilsoni (Echinodermata: Crinoidea)

We present here the first documentation of the entire life cycle of a crinoid. A population of the diminutive feather star Aporometra wilsoni, which broods larvae, was sampled near Adelaide, in the Gulf St Vincent, South Australia, every fortnight between February 2004 and February 2005. Body size, sex, and reproductive status were recorded for 30–50 individuals collected on each occasion. The population showed a sex ratio of 1:1, with unequivocal male and female specimens found in 10 of 13 months. The average size (arm length) of individuals increased until June, when it stabilized and females began to brood larvae. Females were found brooding larvae until November, when adults began senescing. By January, the majority of the population consisted of small recruits. The entire life cycle of this small ovoviviparous crinoid occurs over a single year, a life cycle that is unique among echinoderms.     

Design of a brain-penetrant CDK4/6 inhibitor for glioblastoma

CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2? breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MW = 285 and TPSA = 66 Ų), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpK_a = 8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse K_p,uu = 0.20–0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.     

Females competing to reproduce: dominance matters but testosterone may not

The associations among aggression, testosterone (T), and reproductive success have been well studied, particularly in male birds. In many species, males challenged with simulated or real territorial intrusions increase T and levels of aggression, outcomes linked to higher dominance status and greater reproductive success. For females, the patterns are less clear. Females behave aggressively towards one another, and in some species, females respond to a social challenge with increases in T, but in other species they do not. Prior work on female dark-eyed juncos (Junco hyemalis) had shown that experimental elevation of T increases social status and intrasexual aggression. Here, we conducted two experiments designed to answer three questions: Are endogenous concentrations of T associated with dominance status in captive female juncos? Does dominance status influence readiness to breed in female juncos? And do captive females increase T in response to a challenge? In the first experiment, we introduced two females to a breeding aviary, allowed them to form a dominance relationship and then introduced a male. We found that dominant females were more likely to breed than subordinates, but that dominance status was not predicted by circulating T. In the second experiment, we allowed a resident male and female to establish ownership of a breeding aviary (territory) then introduced a second, intruder female. We found that resident females were aggressive towards and dominant over intruders, but T did not increase during aggressive interactions. We suggest that during the breeding season, intrasexual aggression between females may influence reproductive success, but not be dependent upon fluctuations in T. Selection may have favored independence of aggression from T because high concentrations of T could interfere with normal ovulation or produce detrimental maternal effects.     

Genetics of symbiosis in Lotus japonicus: recombinant inbred lines, comparative genetic maps, and map position of 35 symbiotic loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.     

Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

Desaturases and related enzymes perform O₂-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.Desaturase enzymes perform dehydrogenation reactions that result in the introduction of double bonds into fatty acids that are initiated by the energy-demanding abstraction of a hydrogen from a methylene group (1–3). To achieve this, desaturase enzymes recruit and activate molecular oxygen with the use of an active-site diiron cluster (4). The diiron center is common to a variety of proteins, including methane monooxygenase, ribonucleotide reductase, rubrerythrins, and a variety of oxidase enzymes (5). Valuable insights regarding the tuning of diiron centers with respect to diverse chemical reactivity (6) have been made via comparisons of the diiron centers of diiron-containing enzymes (7); however, differences in amino acid sequence, multiple protein-protein interactions, and reaction outcomes complicate the analysis. The study of fatty-acid desaturases and related enzymes presents a unique opportunity for performing enzyme structure-function studies because relatively close homologs perform diverse reactions on similar substrates (8, 9).Desaturase enzymes have evolved independently twice (10); the acyl-ACP² desaturases are soluble enzymes found in the plastids of higher plants, whereas the more widespread class of integral membrane desaturases is found in endomembrane systems in prokaryotes and eukaryotes (9). In addition to forming distinct homology groups, their diiron centers possess distinct primary ligation spheres (11). The availability of crystal structures for acyl-ACP desaturases (12) makes this system amenable to detailed structure-function studies. Crystal structures are available for the 18:0 Δ⁹-desaturase³ (12, 13) from Ricinus communis (castor) and a bifunctional desaturase from Hedera helix (ivy) (14, 15). These desaturases are homodimeric proteins, with each monomer folded into a compact single domain composed of nine helices. The diiron active site of these enzymes is buried within a core four-helix bundle and is positioned alongside a deep, bent, narrow hydrophobic cavity in which the substrate is bound during catalysis. It is a textbook example of a lock-and-key type of binding site in which the bound fatty acid moiety is poised for formation of the cis-fatty acid product.Nobel Laureate Konrad Bloch observed, “The stereospecific removal of hydrogen in the formation of oleate, although predictable on principle grounds would seem to approach the limits of the discriminatory power of enzymes” (16). Bloch''s statement underscores that desaturase enzymes perform highly regio- and stereo-selective reactions on long-chain fatty acids composed of essentially equivalent methylene chains that lack distinguishing landmarks close to the site of desaturation. We will review structural features of the diiron active site of the acyl-ACP desaturases in the context of those of other diiron enzymes, discuss recent insights into the evolution of acyl-ACP desaturases, and summarize recent discoveries relating to the evolution of selectivity and functional diversity within desaturase enzyme families.