Case-control study of birth characteristics and the risk of hepatoblastoma

Background: Hepatoblastoma is a malignant embryonal tumor typically diagnosed in children younger than five years of age. Little is known on hepatoblastoma etiology. Methods: We matched California Cancer Registry records of hepatoblastomas diagnosed in children younger than age 6 from 1988 to 2007 to birth records using a probabilistic record linkage program, yielding 261 cases. Controls (n = 218,277), frequency matched by birth year to all cancer cases in California for the same time period, were randomly selected from California birth records. We examined demographic and socioeconomic information, birth characteristics, pregnancy history, complications in pregnancy, labor and delivery, and abnormal conditions and clinical procedures relating to the newborn, with study data taken from birth certificates. Results: We observed increased risks for hepatoblastoma among children with low [1500–2499 g, Odds Ratio (OR) = 2.02, 95% confidence interval (CI) 1.29–3.15] and very low birthweight (<1500 g, OR = 15.4, 95% CI 10.7–22.3), preterm birth <33 weeks (OR = 7.27, 95% CI 5.00, 10.6), small size for gestational age (OR = 1.75, 95% CI 1.25–2.45), and with multiple birth pregnancies (OR = 2.52, 95% CI 1.54–4.14). We observed a number of pregnancy and labor complications to be related to hepatoblastoma, including preeclampsia, premature labor, fetal distress, and congenital anomalies. Conclusion: These findings confirm previously reported associations with low birthweight and preeclampsia. The relation with multiple birth pregnancies has been previously reported and may indicate a relation to infertility treatments.     

Foraging success under uncertainty: search tradeoffs and optimal space use

Understanding the structural complexity and the main drivers of animal search behaviour is pivotal to foraging ecology. Yet, the role of uncertainty as a generative mechanism of movement patterns is poorly understood. Novel insights from search theory suggest that organisms should collect and assess new information from the environment by producing complex exploratory strategies. Based on an extension of the first passage time theory, and using simple equations and simulations, we unveil the elementary heuristics behind search behaviour. In particular, we show that normal diffusion is not enough for determining optimal exploratory behaviour but anomalous diffusion is required. Searching organisms go through two critical sequential phases (approach and detection) and experience fundamental search tradeoffs that may limit their encounter rates. Using experimental data, we show that biological search includes elements not fully considered in contemporary physical search theory. In particular, the need to consider search movement as a non‐stationary process that brings the organism from one informational state to another. For example, the transition from remaining in an area to departing from it may occur through an exploratory state where cognitive search is challenged. Therefore, a more comprehensive view of foraging ecology requires including current perspectives about movement under uncertainty.     

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.     

Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.     

An extensive interaction interface between thrombin and factor V is required for factor V activation

The interaction interface between human thrombin and human factor V (FV), necessary for complex formation and cleavage to generate factor Va, was investigated using a site-directed mutagenesis strategy. Fifty-three recombinant thrombins, with a total of 78 solvent-exposed basic and polar residues substituted with alanine, were used in a two-stage clotting assay with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV activation were identified and mapped to anion-binding exosite I (ABE-I), anion-binding exosite II (ABE-II), the Leu(45)-Asn(57) insertion loop, and the Na(+) binding loop of thrombin. Three ABE-I mutants (R68A, R70A, and Y71A) and the ABE-II mutant R98A had less than 30% of WT activity. The thrombin Na(+) binding loop mutants, E229A and R233A, and the Leu(45)-Asn(57) insertion loop mutant, W50A, had a major effect on FV activation with 5, 15, and 29% of WT activity, respectively. The K52A mutant, which maps to the S' specificity pocket, had 29% of WT activity. SDS-polyacrylamide gel electrophoresis analysis of cleavage reactions using the thrombin ABE mutants R68A, Y71A, and R98A, the Na(+) binding loop mutant E229A, and the Leu(45)-Asn(57) insertion loop mutant W50A showed a requirement for both ABEs and the Na(+)-bound form of thrombin for efficient cleavage at the FV residue Arg(709). Several basic residues in both ABEs have moderate decreases in FV activation (40-60% of WT activity), indicating a role for the positive electrostatic fields generated by both ABEs in enhancing complex formation with complementary negative electrostatic fields generated by FV. The data show that thrombin activation of FV requires an extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S' subsite are required for optimal cleavage, and the Na(+)-bound form of thrombin is important for its procoagulant activity.     

Peroxisome proliferator-activated receptor gamma inhibits transforming growth factor beta-induced connective tissue growth factor expression in human aortic smooth muscle cells by interfering with Smad3

Activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) after balloon injury significantly inhibits VSMC proliferation and neointima formation. However, the precise mechanisms of this inhibition have not been determined. We hypothesized that activation of PPAR gamma in vascular injury could attenuate VSMC growth and matrix production during vascular lesion formation. Since connective tissue growth factor (CTGF) is a key factor regulating extracellular matrix production, abrogation of transforming growth factor beta (TGF-beta)-induced CTGF production by PPAR gamma activation may be one of the mechanisms through which PPAR gamma agonists inhibit neointima formation after vascular injury. In this study, we demonstrate that the PPAR gamma natural ligand (15-deoxyprostaglandin J(2)) and a synthetic ligand (GW7845) significantly inhibit TGF-beta-induced CTGF production in a dose-dependent manner in HASMCs. In addition, suppression of CTGF mRNA expression is relieved by pretreatment with an antagonist of PPAR gamma (GW9662), suggesting that the inhibition of CTGF expression is mediated by PPAR gamma. To elucidate further the molecular mechanism by which PPAR gamma inhibits CTGF expression, an approximately 2-kilobase pair CTGF promoter was cloned. We found that PPAR gamma activation inhibits TGF-beta-induced CTGF promoter activity in a dose-dependent manner, and suppression of CTGF promoter activity by PPAR gamma activation is completely rescued by overexpression of Smad3, but not by Smad4. Furthermore, PPAR gamma physically interacts with Smad3 but not Smad4 in vitro in glutathione S-transferase pull-down experiments. Taken together, the data suggest that PPAR gamma inhibits TGF-beta-induced CTGF expression in HASMCs by directly interfering with the Smad3 signaling pathway.     

Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857-1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.     

Short-term treatment with estrone oleate in liposomes (Merlin-2) does not affect the expression of the ob gene in Zucker obese rats

Young female Zucker fa/fa rats of 370-430 g were implanted with osmotic minipumps releasing 3.5 mol/dayúkg of estrone oleate in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a sustained loss of appetite, and a decrease in body weight of 3.5%, which contrasts with the 8.2% increase in controls during the period studied. Plasma insulin, glucose and urea decreased, and liver glycogen increased with Merlin-2 treatment. Plasma ACTH and corticosterone increased to a maximum at the end of the experiment. The expression of the ob gene in adipose tissue was unchanged, and plasma leptin levels were also unchanged by treatment. Estrone levels increased more than 1500-fold, and estrone oleate rose 100-fold during treatment. The fact that estrone oleate had no effect on the leptin levels or expression in obese rats, in contrast with the marked inhibition observed in the lean suggests that the functionality of the leptin receptor is essential for estrone oleate inhibition of the ob gene. This also suggests that leptin may control ob gene expression in white adipose tissue and that estrone oleate may activate this process. The slimming effect of estrone oleate is, thus, not directly dependent on leptin, since both normoleptinemic and hyperleptinemic animals lose fat following treatment nor are the effects on appetite and energy expenditure mediated by leptin. However, leptin levels and the expression of the ob gene are directly linked with estrone oleate function. A possible involvement of leptin in estrone oleate action is postulated. The results support the participation of estrone oleate in the control of body weight and hint at the complexity of its regulation by leptin and glucocorticoids.     

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.     

Required parental investment and mating patterns: a quantitative analysis in the context of evolutionarily stable strategies

Much social psychological research has been dedicated to understanding mating strategies from the standpoint of genetic-fitness payout (e.g., Simpson and Gangestad, 2000). The current work is designed to provide a coherent, quantitative model for predicting different classes of mating strategies in both males and females. Specifically, the framework developed in this paper is an elaboration of Dawkins' (1989) quantitative assessment of different male and female mating strategies. Dawkins suggests that the prevalence of different strategies employed should be predictable in terms of evolutionary stable strategies. In the current work, a quantitative analysis predicting the prevalence of different mating strategies within each sex was conducted. The mathematical functions derived suggest that variability in the costs associated with raising offspring affects the expected prevalence of mating strategies differently for males and females. According to the present model, variability in female strategies should be less affected by changes in parental investment (PI) than variability in male strategies. Important predictions regarding male and female mating strategies across cultures are discussed.