A TR3/Nur77 peptide-based high-throughput fluorescence polarization screen for small molecule Bcl-B inhibitors

Nuclear receptor TR3/Nur77/NR4A1 binds several antiapoptotic Bcl-2-family proteins (Bcl-B, Bcl-2, Bfl-1) in a non-BH3-dependent manner. A 9-amino-acid peptide derived from full-length TR3 with polyarginine tail (TR3-r8) recapitulates TR3's binding specificity, displaying high affinity for Bcl-B. TR3-r8 peptide was used to screen for small molecule Bcl-B inhibitors. A fluorescence polarization assay (FPA) employing fluorescein isothiocyanate (FITC)-labeled TR3-r8 peptide (FITC-TR3-r8) and Bcl-B protein was optimized, with nonfluorescent TR3-r8 serving to demonstrate reversible, competitive binding. Approximately 50,000 compounds were screened at 3.75 mg/L, yielding 145 reproducible hits with >/=50% FITC-TR3-r8 displacement (a confirmed hit rate of 0.29%). After dose-response analyses and counterscreening with an unrelated FITC-based FPA, 6 candidate compounds remained. Nuclear magnetic resonance (NMR) showed that 2 of these compounds bound Bcl-B, but not glutathione S-transferase (GST) control protein. One Bcl-B-binding compound was unable to displace FITClabeled BH3 peptides from Bcl-B, confirming a unique binding mechanism compared with traditional antagonists of antiapoptotic Bcl-2-family proteins. This compound bound Bcl-B with K(d) 1.94 +/- 0.38 microM, as determined by isothermal titration calorimetry. Experiments using Bcl-B overexpressing HeLa cells demonstrated that this compound induced Bcl-B-dependent cell death. The current FPA represents a screen that can identify noncanonical inhibitors of Bcl-2-family proteins.     

Landscape and Climatic Variations Shaped Secondary Contacts amid Barn Owls of the Western Palearctic

The combined actions of climatic variations and landscape barriers shape the history of natural populations. When organisms follow their shifting niches, obstacles in the landscape can lead to the splitting of populations, on which evolution will then act independently. When two such populations are reunited, secondary contact occurs in a broad range of admixture patterns, from narrow hybrid zones to the complete dissolution of lineages. A previous study suggested that barn owls colonized the Western Palearctic after the last glaciation in a ring-like fashion around the Mediterranean Sea, and conjectured an admixture zone in the Balkans. Here, we take advantage of whole-genome sequences of 94 individuals across the Western Palearctic to reveal the complex history of the species in the region using observational and modeling approaches. Even though our results confirm that two distinct lineages colonized the region, one in Europe and one in the Levant, they suggest that it predates the last glaciation and identify a secondary contact zone between the two in Anatolia. We also show that barn owls recolonized Europe after the glaciation from two distinct glacial refugia: a previously identified western one in Iberia and a new eastern one in Italy. Both glacial lineages now communicate via eastern Europe, in a wide and permeable contact zone. This complex history of populations enlightens the taxonomy of Tyto alba in the region, highlights the key role played by mountain ranges and large water bodies as barriers and illustrates the power of population genomics in uncovering intricate demographic patterns.     

Centric fusion differences among Oryx dammah, O. gazella, and O. leucoryx (Artiodactyla, Bovidae)

G- and C-banded karyotypes of the genus Oryx were compared using the standard karyotype of Bos taurus. Chromosomal complements were 2n = 56 in O. gazella gazella, 2n = 58 in O. g. beisa and O. g. callotis, 2n = 56-58 in O. dammah, and 2n = 57-58 in O. leucoryx. The number of autosomal arms in all karyotypes was 58. Nearly all variation in diploid number was the result of three independent centric fusions, but one 2n = 57 specimen of O. g. gazella deviated from the normal complement of 2n = 56 due to XXY aneuploidy. A 2;17 centric fusion was fixed in O. g. gazella, whereas O. g. beisa and O. g. callotis lacked this fusion and had indistinguishable karyotypes. Oryx dammah was polymorphic for a 2;15 centric fusion, and O. leucoryx was polymorphic for an 18;19 centric fusion. The five Oryx taxa shared a fixed 1;25 centric fusion; the small acrocentric element involved in the 1;25 fusion was identified by fluorescence in situ hybridization using a cosmid specific to Bos chromosome 25. The X and Y chromosomes were also conserved among the five taxa. Oryx g. gazella differed from the other Oryx species because of the fixed 2;17 centric fusion. This difference reflects an apparently longer period of geographic isolation between O. g. gazella and other populations of Oryx, and it is consistent with the classification of O. gazella and O. beisa as distinct species (see Kingdon, 1997). The lack of monobrachial relationships among the Oryx taxa indicates that sterility barriers between species have not developed. Viability of hybrid offspring constitutes a threat to captive breeding programs designed for endangered species conservation; in the case of Oryx, the 2;15, 2;17, and 18;19 metacentrics could serve as marker chromosomes for assessing hybridization between certain Oryx taxa.     

Genealogical surveys show a high rate of non-paternal surname transmission with regional differences in Argentina

Surnames are a vertically transmitted cultural trait that in Argentina follows the paternal line of descent when the paternity is known. There was a lack of empirical information regarding non-paternal surname transmissions among the general population, so we performed 2,550 genealogical interviews, which included 6,954 surname passes, in different regions of this country. We compared the proportion of non-paternal transmissions between the propositus and parental generation and found no significant difference between them (p < 0.01). Inter-population comparisons allowed us to describe 4 regional groups. We also drew models and simulations to estimate how many generations it would take to find that only half of the population maintained the paternal transmission. The lowest proportion of non-paternal transmission was 7.3%, estimating 9 generations (between 225 and 315 years) to find that, at most, half its population keeps following the paternal transmission; the highest proportion was 23%, taking 3 generations (75–105 years). Our results show a high proportion of unrecognized paternities among the general population, a very quick loss of association between male lineages and surnames, and regional proportions with significant differences between each other.     

Influence of unilateral castration and increased plane of nutrition on sexual development of Holstein bulls. I. Growth and sperm production

Sixty-five Holstein bull calves were used to study the effects of unilateral castration (UC) and increased plane of nutrition on the growth and development of the reproductive system. Bulls were slaughtered at 1 wk., 2, 4, 8 and 16 months. Half of each slaughter group above one week was unilaterally castrated at 7 days of age. Half of the bulls remaining at 6 months of age received 90% of their recommended daily TDN allowance while the remainder received 120%. Compensatory hypertrophy was evident as early as 2 months and the degree of compensation increased for the duration of the experiment (Age x UC, P<.01). By 16 months of age the remaining testis of UC animals was 73% heavier than the average testis weight of intact bulls. While epididymal weight was significantly increased by UC, seminal vesicle weight was not. UC bulls produced significantly more sperm per testis than intact bulls both from the onset of puberty to slaughter and for the 16 week period prior to slaughter. Testis sperm concentration was similar in UC and intact bulls. UC at one weel of age caused greater testis growth and greater sperm production per testis, but did not promote earlier puberty.     

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.     

Structural determinants of caspase-9 inhibition by the vaccinia virus protein, F1L

In multicellular organisms, apoptosis is a powerful method of host defense against viral infection. Apoptosis is mediated by a cascade of caspase-family proteases that commit infected cells to a form of programmed cell death. Therefore, to replicate within host cells, viruses have developed various strategies to inhibit caspase activation. In the mitochondrial cell-death pathway, release of cytochrome c from mitochondria into the cytosol triggers assembly of the oligomeric apoptosome, resulting in dimerization and activation of the apical caspase-9 (C9), and in turn its downstream effector caspases, leading to apoptosis. We previously showed that the vaccinia virus-encoded Bcl-2-like protein, F1L, which suppresses cytochrome c release by binding Bcl-2 family proteins, is also a C9 inhibitor. Here, we identify a novel motif within the flexible N-terminal region of F1L that is necessary and sufficient for interaction with and inhibition of C9. Based on functional studies and mutagenesis, we developed an atomic model of the complex in which F1L inhibits C9 by engaging the active site in the reverse orientation with respect to substrate peptides, in a manner analogous to that of XIAP-mediated inhibition of caspases-3 and -7. These studies offer new insights into the mechanism of apoptosome inhibition by F1L as well as novel probes to understand the molecular bases of apoptosome regulation and turnover. They also suggest how the two distinct functionalities of F1L (inhibition of C9 and suppression of pro-apoptotic Bcl-2 family proteins) may operate in a cellular setting.     

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.     

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer

The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.