Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.     

Granivory in California sage scrub: implications for common plant invaders and ecosystem conservation

Seed predation may influence community assembly and invasion dynamics when seed predators preferentially select some seed species over others. However, the role of different seed predators and their preferences for seeds in multiple ecological contexts, including the endangered California sage scrub ecosystem, remain unresolved making predictions about which processes limit or promote invasions difficult. In addition, selection criteria, specifically how seed size and biogeographic origin (native versus invasive) influences selection, requires further research across multiple granivore guilds. We examined seed predator preference for common native and invasive seeds across small and large seed classes using seed dish experiments with motion-sensor video observation. We also quantified the relative importance of ants, birds, and mammals as seed predators in sage scrub. Community-wide, granivores preferred the small-seeded invasive mustard Brassica nigra, avoided the large-seeded invasive grass Bromus rubens and the native large-seeded shrub Encelia californica, and did not show significant selection or avoidance for the small-seeded native shrub Salvia apiana. Birds, notably California towhees (Melozone crissalis), were the most frequent granivore while rodents and ants rarely removed seeds. This study reveals that birds are important seed predators in California sage scrub and have the potential to contribute to biotic resistance to mustard invasions (Brassica nigra and potentially others).

     

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca²⁺ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160–168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g·ml^?1), and a dense one with a peak density of 1.12 g·ml^?1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca²⁺ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.     

Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation

The biochemical events that lead to thrombin-stimulated release of von Willebrand factor and prostacyclin synthesis in cultured endothelial cells are examined. Treatment of human umbilical vein endothelial cells with thrombin results in an instantaneous increase in phospholipid methylation which can be blocked by 3-deazaadenosine, a methyltransferase inhibitor. 3-Deazaadenosine also blocks the thrombin-induced Ca2+ influx into endothelial cells and the release of von Willebrand factor, indicating that these processes are coupled. The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) and the Ca2+ ionophore A23187 both bypass the phospholipid methylation and directly stimulate Ca2+ influx and von Willebrand factor release. In contrast to the stimulus-induced von Willebrand factor release, the thrombin-induced prostacyclin synthesis cannot be blocked by 3-deazaadenosine. Similarly, incubation of endothelial cells with EDTA has no influence on the thrombin-induced prostacyclin synthesis, and PMA has no stimulatory effect on prostacyclin synthesis. These observations indicate that thrombin induces different metabolic responses in endothelial cells: phospholipid methylation followed by a Ca2+ influx, which subsequently leads to release of von Willebrand factor, and liberation of arachidonic acid from phospholipids for prostacyclin formation, which is independent of phospholipid methylation and Ca2+ influx.     

Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.     

The effect of combined hypergravity and micro-grooved surface topography on the behaviour of fibroblasts

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and micro-grooved substrata (groove depth: 1 mum, width: 1, 2, 5, 10 microm), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and alpha(1)- and beta(1)-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces.     

Impacts of invasive annuals on soil carbon and nitrogen storage in southern California depend on the identity of the invader

Non‐native plant invasions can alter nutrient cycling processes and contribute to global climate change. In southern California, California sage scrub (hereafter sage scrub), a native shrub‐dominated habitat type in lowland areas, has decreased to <10% of its original distribution. Postdisturbance type‐conversion to non‐native annual grassland, and increasingly to mustard‐dominated invasive forbland, is a key contributor to sage scrub loss. To better understand how type‐conversion by common invasive annuals impacts carbon (C) and nitrogen (N) storage in surface soils, we examined how the identity of the invader (non‐native grasses, Bromus spp.; and non‐native forbs, Brassica nigra), microbial concentrations, and soil properties interact to influence soil nutrient storage in adjacent native and invasive habitat types at nine sites along a coast to inland gradient. We found that the impact of type‐conversion on nutrient storage was contingent upon the invasive plant type. Sage scrub soils stored more C and N than non‐native grasslands, whereas non‐native forblands had nutrient storage similar to or higher than sage scrub. We calculate that >940 t C km^?2 and >60 t N km^?2 are lost when sage scrub converts to grass‐dominated habitat, demonstrating that grass invasions are significant regional contributors to greenhouse gas emissions. We found that sites with greater total C and N storage were associated with high cation exchange capacities and bacterial concentrations. Non‐native grassland habitat type was a predictor of lower total C, and soil pH, which was greatest in invasive habitats, was a predictor of lower total N. We demonstrate that modeling regional nutrient storage requires accurate classification of habitat type and fine‐scale quantification of cation exchange capacity, pH, and bacterial abundance. Our results provide evidence that efforts to restore and conserve sage scrub enhance nutrient storage, a key ecosystem service reducing atmospheric CO₂ concentrations.