Vertical structure of seed banks and the impact of depth of burial on recruitment in two temporary marshes

The structure of the seed bank (including Chara oospores), in relation to depth within the sediment and disturbance, was studied in two Rhône delta temporary marshes for two years. The seeds of all species were concentrated in the top 2 cm of sediment with very low numbers beeing found below 4 cm. When an exclosure eliminated disturbances of the sediment by animals, the vertical repartition of seeds at site 2 was more pronounced than outside the exclosure.In experiment 1, the emergence capacity of seeds from different depths and buried under layers of sterile equivalent to those in the field was measured. Depending of the species, 22 to 98% of the seeds germinated from unburied seeds in the top 2 cm. Only 1% of the oospores of Chara (from site 2) at 2 to 4 cm depth in the sediment emerged.In experiment 2, surface seed bank samples were placed under 0, 2 or 4 cm sterile sediment depth. The samples contained numerous recent seeds and the emergence percentage reached 41% (for Ruppia maritima). Only the seeds of Zannichellia spp failed to germinate from a depth of 2 cm or more. The emergence percentage from 2 cm depth or more was always lower than at the surface. These experiments showed that both burial and ageing of seeds decrease germination capacity.The majority of the active seeds located at the surface germinate when the marsh is flooded. Seeds located between 2 and 4 cm can be brought back to the surface by disturbances and play the role of a reserve involved in maintenance of populations that go without seed production for one or some years.     

Cumulus cells secrete a meiosi-inducing substance by stimulation with forskolin and dibutyric cyclic adenosine monophosphate

The role of the cumulus cells in initiating the resumption of meiosis after exposure to forskolin and dbcAMP was studied in the mouse. The resumption of meiosis was monitored by the percentage of germinal vesicle breakdown (GVBD) and polar body formation (PB). The cumulus-enclosed oocytes (CEO) and denuded oocytes (DO) were cultured with and without hypoxanthine (HX) in the culture medium. Three types of experiments were performed: (1) Effect of forskolin on spontaneous resumption of meiosis, i.e. cultures without HX, and two experiments in which HX is present throughout the culture: (2) Effect of transient exposure to forskolin or dibutyric-cyclic adenosinemonophosphate (dbcAMP) on GVBD prior to continued culture without forskolin or dbcAMP (oocyte priming). (3) Priming of CEO with forskolin for 2 hr, separation of cumulus cells and oocytes, followed by coculture of rejoined cumulus cells and oocytes, or coculture of the cumulus cells and new, unprimed DO. (1) Forskolin inhibited a spontaneous resumption of meiosis in a dose-dependent manner during the first 5 hr of culturing. After 22 hr all controls and CEO resumed meiosis, whereas only half of the DO did. (2) At least 1 hr of priming the CEO with forskolin is needed to induce GVBD and PB formation, but forskolin inhibited the resumption of meiosis when present for 24 hr. Similar results were obtained with a high concentration of dbcAMP. (3) A separation and rejoining of oocytes and cumulus cells after priming induced the resumption of meiosis in a significantly greater number of oocytes than in the control oocytes which were not primed. The GVBD of unstimulated DO also increased significantly when cocultured with cumulus cells from primed CEO. The percentage of GVBD in unprimed DO and in DO isolated from primed CEO was the same. We suggest that within 1–2 hr, forskolin and cAMP stimulate cumulus cells to produce a diffusible meiosis-inducing substance which overcomes HX-inhibition and induces oocyte maturation, including both GVBD and PB formation. The CEO must be primed for more than 2 hr before the resumption of meiosis in DO isolated from such CEO is induced. Oocyte-cumulus connections are crucial as far as initiating the production of a meiosis-inducing substance is concerned. Oocyte-cumulus connections are not needed for transferring this substance to the oocyte. © 1994 Wiley-Liss, Inc.     

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells

Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.     

Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation

Tissue inhibitor of metalloproteinase-2 (TIMP-2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP-2 on incorporation of ³H-thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half-maximal effective concentration of TIMP-2 was 107 ± 12 nM (S.D.). In contrast, TIMP-1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP-2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP-2. Cell adhesion to tissue culture platic was slightly stimulated by TIMP-2, and cell migration was inhibited with short-term exposure to TIMP-2, but neither process was affected by longer-term exposure. The ability of TIMP-2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP-2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor receptors lose ligand binding ability as WI-38 cells progress from short-term to long-term quiescence

WI-38 cells, density arrested for short periods of time, can be stimulated to re-enter the cell cycle by epidermal growth factor (EGF) alone. However, cells density arrested for longer periods have a prolonged prereplicative phase when serum stimulated and cannot be stimulated by EGF alone. Radio-ligand binding studies performed on WI-38 cells showed that actively growing cells bind [¹²⁵I]EGF at relatively low levels that increase to a maximum as the cells become contact inhibited. As the cells enter a state of deeper quiescence, EGF binding falls to one-third to one-fifth the short-term growth arrested levels, remaining constant thereafter. The EGF-receptor complexes internalize more slowly in long-term growth arrested cells, and the rate of ligand association to the receptor is lower than short-term growth arrested cells. The amount of EGF receptor protein in lysates of equal numbers of both short- and long-term quiescent cells remains the same. These results suggest that the failure of long-term growth arrested cells to respond to EGF is not due to dramatic changes in the amount of receptor protein during prolonged quiescence but more likely to an alteration in the ability of these receptors to bind ligand and/or activate the EGF signal transduction pathway. © 1993 Wiley-Liss, Inc.     

Effects of Motility and Adsorption Rate Coefficient on Transport of Bacteria through Saturated Porous Media

Three strains of Pseudomonas fluorescens with different motility rates and adsorption rate coefficients were injected into porous-medium reactors packed with l-mm-diameter glass spheres. Cell breakthrough, time to peak concentration, tailing, and cell recovery were measured at three interstitial pore velocities (higher than, lower than, and much lower than the maximal bacterial motility rate). All experiments were done with distilled water to reduce the effects of growth and chemotaxis. Contrary to expectations, motility did not result in either early breakthrough or early time to peak concentration at flow velocities below the motility rate. Bacterial size exclusion effects were shown to affect breakthrough curve shape at the very low flow velocity, but no such effect was seen at the higher flow velocity. The tendency of bacteria to adsorb to porous-medium surfaces, as measured by adsorption rate coefficients, profoundly influenced transport characteristics. Cell recoveries were shown to be correlated with the ratio of advective to adsorptive transport in the reactors. Adsorption rate coefficients were found to be better predictors of microbial transport phenomena than individual characteristics, such as size, motility, or porous-medium hydrodynamics.     

The Effect of Extracellular Matrix Molecules on the in Vitro Behavior of Bovine Endothelial Cells

Extracellular matrix (ECM) is an important mediator of endothelial functions such as adhesion, spreading, migration, proliferation, and maintenance of differentiated functions. Attachment of cultured cells to tissue culture polystyrene (TCPS) is dependent on vitronectin which adsorbs onto the surface from the serum in the culture medium. Vitronectin (VN) will adsorb efficiently to TCPS even if the latter has been coated with another matrix molecule and blocked with albumin. This means that studies of the interactions of cells with individual coated ECM molecules will be confounded by the presence of adsorbed VN if serum is present in the culture medium. In this study, the adhesion, spreading, growth, and output of endogenous matrix molecules by bovine corneal endothelial (BCE) cells were measured on five different matrix substrates using medium which had been depleted of vitronectin to avoid such confounding effects. The same cell adhesion and spreading maxima were achieved on vitronectin, fibronectin (FN), laminin (LM), and types I and IV collagen (col I, col IV). The coating concentrations required to achieve these maxima, however, differed among the substrates, LM needing considerably higher concentrations than the other substrates for both maximal adhesion and spreading and FN needing higher concentrations for cell spreading. When cells were continuously passaged on each of the five substrates coated at concentrations optimal for cell spreading, no differences in cell proliferation rates or cell morphology were observed. Significant differences, however, were observed in the subcellular output of endogenous matrix molecules (FN, LM, col IV, and thrombospondin) between the different substrates. Col I was a poor substrate for the production of all ECM molecules tested over the 10 passages of the experiment, whereas col IV was a consistently good substrate. LM and FN substrates displayed differential effects on the output of different ECM molecules. VN was unique in that BCE cells at early passage on this substrate produced high levels of endogenous matrix molecules, whereas with continued passage on this substrate, a progressive decline in ECM secretion was observed. These results show that incorporation of individual molecules into the ECM by BCE cells in culture is significantly affected by the nature of the substratum. They further suggest that passage of endothelial cells in media containing serum (which results in coating of VN onto the substrate) may result in a progressive reduction of ECM output.