Structural Requirements for Function of the Crkl Adapter Protein in Fibroblasts and Hematopoietic Cells

Crkl is an adapter protein and phosphotyrosine-containing substrate implicated in transformation by the bcr-abl oncogene and in signaling by cytokines. When phosphorylated, Crkl binds through its Src homology 2 (SH2) domain to other tyrosine phosphoproteins such as paxillin and Cbl. Overexpression of Crkl in fibroblasts induces transformation. Here we examine the role of Crkl in hematopoietic cells and find that overexpression of Crkl confers a signal leading to increased adhesion to fibronectin. In both fibroblasts and hematopoietic cells, individual mutations or deletions of each SH2 and SH3 domain abrogated transformation and adhesion, respectively, indicating that interactions with other proteins such as Cbl and paxillin (SH2 domain) and Abl, Sos, and C3G (N-terminal SH3 domain) are essential for biological activity. In vivo and in vitro tryptic phosphopeptide mapping studies show that Crkl is phosphorylated on multiple tyrosine residues when overexpressed or when activated by Bcr-Abl. Mutation at tyrosine 207, a residue conserved in c-Crk, abrogates all in vivo tyrosine phosphorylation of Crkl. Despite this loss of phosphotyrosine, mutation at this site enhanced Crkl function as measured by complex formation with SH2 binding proteins, signal transduction to Jun Kinase, and fibroblast transformation. These observations implicate Crkl in cellular adhesion and demonstrate that Y207 functions as a negative regulatory site.     

Transient association of MCM complex proteins with the nuclear matrix during initiation of mammalian DNA replication

The minichromosome maintenance complex (MCM2-7) is the putative DNA helicase ineukaryotes, and essential for DNA replication. By applying serial extractions to mammaliancells synchronized by release from quiescence, we reveal dynamic changes to thesub-nuclear compartmentalization of MCM2 as cells pass through late G1 and early S phase,identifying a brief window when MCM2 becomes transiently attached to the nuclear-matrix.The data distinguish 3 states that correspond to loose association with chromatin prior toDNA replication, transient highly stable binding to the nuclear-matrix coincident withinitiation, and a post-initiation phase when MCM2 remains tightly associated withchromatin but not the nuclear-matrix. The data suggests that functional MCM complexloading takes place at the nuclear-matrix.     

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19(MOLF/Ei)), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.     

CYST FORMATION IN THE FRESHWATER DINOFLAGELLATE CERATIUM HIRUNDINELLA (DINOPHYCEAE)1

Cyst formation in Ceratium hirundinella (O. F. Müll.) Bergh was studied by light and electron microscopy, using material from several lakes and reservoirs and also laboratory cultures. Cells preparing to encyst build up large quantities of starch and lipid and at the same time reduce their other cell components. The cyst is released from the theca as a naked cell bounded by a double membrane. The most commonly found cyst deposits a layer of electron-dense granules containing silicon on the outer membrane and lays down a cellulose-like material between the two membranes. Cysts without the electron-dense granules are commonly formed in cultures but rarely found in lakes. These cysts appear less resistant to decay and do not show the reorganization of cell contents for dormancy. It is suggested that C. hirundinella has both a resting cyst, forming part of the life cycle, and a temporary cyst stage.     

Single-locus species delimitation: a test of the mixed Yule-coalescent model, with an empirical application to Philippine round-leaf bats

Prospects for a comprehensive inventory of global biodiversity would be greatly improved by automating methods of species delimitation. The general mixed Yule-coalescent (GMYC) was recently proposed as a potential means of increasing the rate of biodiversity exploration. We tested this method with simulated data and applied it to a group of poorly known bats (Hipposideros) from the Philippines. We then used echolocation call characteristics to evaluate the plausibility of species boundaries suggested by GMYC. In our simulations, GMYC performed relatively well (errors in estimated species diversity less than 25%) when the product of the haploid effective population size (N(e)) and speciation rate (SR; per lineage per million years) was less than or equal to 10(5), while interspecific variation in N(e) was twofold or less. However, at higher but also biologically relevant values of N(e) × SR and when N(e) varied tenfold among species, performance was very poor. GMYC analyses of mitochondrial DNA sequences from Philippine Hipposideros suggest actual diversity may be approximately twice the current estimate, and available echolocation call data are mostly consistent with GMYC delimitations. In conclusion, we consider the GMYC model useful under some conditions, but additional information on N(e), SR and/or corroboration from independent character data are needed to allow meaningful interpretation of results.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Mammals of the northern Philippines: tolerance for habitat disturbance and resistance to invasive species in an endemic insular fauna

Aim Island faunas, particularly those with high levels of endemism, usually are considered especially susceptible to disruption from habitat disturbance and invasive alien species. We tested this general hypothesis by examining the distribution of small mammals along gradients of anthropogenic habitat disturbance in northern Luzon Island, an area with a very high level of mammalian endemism. Location Central Cordillera, northern Luzon Island, Philippines. Methods  Using standard trapping techniques, we documented the occurrence and abundance of 16 endemic and two non‐native species along four disturbance gradients where habitat ranged from mature forest to deforested cropland. Using regression analysis and AIC_c for model selection, we assessed the influence of four predictor variables (geographic range, elevational range, body size and diet breadth) on the disturbance tolerance of species. Results Non‐native species dominated areas with the most severe disturbance and were rare or absent in mature forest. Native species richness declined with increasing disturbance level, but responses of individual species varied. Elevational range (a measure of habitat breadth) was the best predictor of response of native species to habitat disturbance. Geographic range, body size and diet breadth were weakly correlated. Main conclusions The endemic small mammal fauna of northern Luzon includes species adapted to varying levels of natural disturbance and appears to be resistant to disruption by resident alien species. In these respects, it resembles a diverse continental fauna rather than a depauperate insular fauna. We conclude that the long and complex history of Luzon as an ancient member of the Philippine island arc system has involved highly dynamic ecological conditions resulting in a biota adapted to changing conditions. We predict that similar responses will be seen in other taxonomic groups and in other ancient island arc systems.