No postcopulatory response to inbreeding by male crickets

Previous studies of the cricket Teleogryllus oceanicus have shown a paternity bias towards non-sibling males. Although non-kin-biased paternity could represent a mechanism of postcopulatory inbreeding avoidance by females, evolutionarily stable strategy (ESS) models of ejaculate evolution also predict that males should reduce their expenditure on the ejaculate when mating with their sisters. Here we provide a test of these models, finding that male crickets invest equally in matings with full-siblings, half-siblings and non-sibling females. The data suggest that in this species, males and females differ in their response to inbreeding.     

Childbearing may increase visceral adipose tissue independent of overall increase in body fat

Objective: To examine whether childbearing is associated with increased visceral adiposity and whether the increase is proportionally larger than other depots. Methods and Procedures: This prospective study examined changes in adiposity assessed via computed tomography (CT) and dual‐energy X‐ray absorptiometry among 122 premenopausal women (50 black, 72 white) examined in 1995–1996 and again in 2000–2001. During the 5‐year interval, 14 women had one interim birth and 108 had no interim births. Multiple linear regression models estimated mean (95% confidence interval (CI)) 5‐year changes in anthropometric and adiposity measures by interim births adjusted for age, race, and changes in total and subcutaneous adiposity. Results: We found no significant differences between one interim birth and no interim births for 5‐year changes in weight, BMI, total body fat, subcutaneous adipose tissue, or total abdominal adipose tissue. Visceral adipose tissue increased by 40 and 14% above initial levels for 1 birth and 0 birth groups, respectively. Having 1 birth vs. 0 births was associated with a greater increase in visceral adipose tissue of 18.0 cm² (4.8, 31.2), P < 0.01; gain of 27.1 cm² (14.5, 39.7) vs. 9.2 cm² (4.8, 13.6), and a borderline greater increase in waist girth of 2.3 cm (0, 4.5), P = 0.05; gain of 6.3 cm (4.1, 8.5) vs. 4.0 cm (3.2, 4.8), controlling for gain in total body fat and covariates. Discussion: Pregnancy may be associated with preferential accumulation of adipose tissue in the visceral compartment for similar gains in total body fat. Further investigation is needed to confirm these findings and determine whether excess visceral fat deposition with pregnancy adversely affects metabolic risk profiles among women.     

Latency associated peptide has in vitro and in vivo immune effects independent of TGF-beta1

Latency Associated Peptide (LAP) binds TGF-beta1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-beta1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-beta1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-beta1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.     

Automated quantitative analysis of epithelial cell scatter

Epithelial cell scatter is a well-known in vitro model for the study of epithelial-mesenchymal transition (EMT). Scatter recapitulates many of the events that occur during EMT, including the dissociation of multicellular structures and increased cell motility. Because it has been implicated in tumor invasion and metastasis, much effort has been made to identify the molecular signals that regulate EMT. To better understand the quantitative contributions of these signals, we have developed metrics that quantitatively describe multiple aspects of cell scatter. One metric (cluster size) quantifies the disruption of intercellular adhesions while a second metric (nearest-neighbor distance) quantifies cell dispersion. We demonstrate that these metrics delineate the effects of individual cues and detect synergies between them. Specifically, we find epidermal growth factor (EGF), cholera toxin (CT) and insulin to synergistically reduce cluster sizes and increase nearest-neighbor distances. To facilitate the rapid measurement of our metrics from live-cell images, we have also developed automated techniques to identify cell nuclei and cell clusters in fluorescence images. Taken together, these studies provide broadly applicable quantitative image analysis techniques and insight into the control of epithelial cell scatter, both of which will contribute to the understanding of EMT and metastasis.Key words: cell scatter, EMT, automated image analysis     

The microtubule-based motor Kar3 and plus end-binding protein Bim1 provide structural support for the anaphase spindle

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ~8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs.     

Idealization of pericellular fluid space geometry and dimension results in a profound underprediction of nano-microscale stresses imparted by fluid drag on osteocytes

To date, no published study has examined quantitatively the effect of geometric and dimensional idealization on prediction of the mechanical signals imparted by fluid drag to cell surfaces. We hypothesize that this idealization affects the magnitude and range of imparted forces predicted to occur at a subcellular level. Hence, we used computational fluid dynamics to predict magnitudes and spatial variation of fluid velocity and pressure, as well as shear stress, on the cell surface in two- and three-dimensional models of actual and idealized pericellular canalicular geometries. Furthermore, variation in actual pericellular space dimensions was analyzed statistically based on high-resolution transmitted electron micrographs (TEM). Accounting for the naturally occurring protrusions of the pericellular space delineating lamina limitans resulted in predictions of localized stress spikes on the cell surface, up to five times those predicted using idealized geometries. Predictions accounting for actual pericellular geometries approached those required to trigger cell activity in in vitro models. Furthermore, statistical analysis of TEM-based dimensions showed significant variation in the width of the canalicular space as well as the diameter of the cell process, both of which decrease with increasing distance from the cell body. For the first time to our knowledge, this study shows the influence of physiologic geometry per se on the nano-scale flow regimes in bone, and the profound influence of physiologic geometry on force magnitudes and variations imparted locally to cells through load-induced fluid flow.     

The biflavonoid isoginkgetin is a general inhibitor of Pre-mRNA splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.     

Purinergic and nitrergic junction potential in the human colon

The aim of the present work is to investigate a putative junction transmission [nitric oxide (NO) and ATP] in the human colon and to characterize the electrophysiological and mechanical responses that might explain different functions from both neurotransmitters. Muscle bath and microelectrode techniques were performed on human colonic circular muscle strips. The NO donor sodium nitroprusside (10 microM), but not the P2Y receptor agonist adenosine 5'-O-2-thiodiphosphate (10 microM), was able to cause a sustained relaxation. NG-nitro-L-arginine (L-NNA) (1 mM), a NO synthase inhibitor, but not 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate tetraammonium salt (MRS 2179) (10 microM), a P2Y antagonist, increased spontaneous motility. Electrical field stimulation (EFS) at 1 Hz caused fast inhibitory junction potentials (fIJPs) and a relaxation sensitive to MRS 2179 (10 microM). EFS at higher frequencies (5 Hz) showed biphasic IJP with fast hyperpolarization sensitive to MRS 2179 followed by sustained hyperpolarization sensitive to L-NNA; both drugs were needed to fully block the EFS relaxation at 2 and 5 Hz. Two consecutive single pulses induced MRS 2179-sensitive fIJPs that showed a rundown. The rundown mechanism was not dependent on the degree of hyperpolarization and was present after incubation with L-NNA (1 mM), hexamethonium (100 microM), MRS 2179 (1 microM), and NF023 (10 microM). We concluded that single pulses elicit ATP release from enteric motor neurons that cause a fIJP and a transient relaxation that is difficult to maintain over time; also, NO is released at higher frequencies causing a sustained hyperpolarization and relaxation. These differences might be responsible for complementary mechanisms of relaxation being phasic (ATP) and tonic (NO).