What ecological factors can affect albumen corticosterone levels in the clutches of seabirds? Timing of breeding, disturbance and laying order in rockhopper penguins (Eudyptes chrysocome chrysocome).

Female birds deposit corticosterone into their eggs. Elevated concentrations of this hormone may interfere with the development of their offspring, and mothers should thus regulate corticosterone levels deposited into the eggs adaptively. However, if females are unable to regulate deposition, then the corticosterone concentration in eggs should reflect that in female plasma and should be influenced by stressors to the females. We measured corticosterone levels in the albumen of rockhopper penguins, and assessed their relationship with hatching order, human disturbance and laying date. Rockhopper penguins (Eudyptes chrysocome chrysocome) lay two eggs, of which the second egg (B-egg) is larger and hatches faster than the first egg (A-egg). The chick hatching from the B-egg is also much more likely to survive than its sibling. Albumen corticosterone concentrations were lower in B-eggs. However, as B-eggs contained more albumen than A-eggs, the total corticosterone deposited in the albumen was not significantly different between the two eggs. Daily disturbance by human observers during albumen production did not influence albumen corticosterone levels. Laying date had an effect on total albumen corticosterone through a higher albumen mass. However, we observed a high individual component in the composition of eggs from the same clutch. Thus, more work is required to explore the hypotheses of passive versus active transfer to eggs and to understand the adaptive value of contrary effects on the amount and concentration of corticosterone.

Full-scale model of glycolysis in Saccharomyces cerevisiae.

We present a powerful, general method of fitting a model of a biochemical pathway to experimental substrate concentrations and dynamical properties measured at a stationary state, when the mechanism is largely known but kinetic parameters are lacking. Rate constants and maximum velocities are calculated from the experimental data by simple algebra without integration of kinetic equations. Using this direct approach, we fit a comprehensive model of glycolysis and glycolytic oscillations in intact yeast cells to data measured on a suspension of living cells of Saccharomyces cerevisiae near a Hopf bifurcation, and to a large set of stationary concentrations and other data estimated from comparable batch experiments. The resulting model agrees with almost all experimentally known stationary concentrations and metabolic fluxes, with the frequency of oscillation and with the majority of other experimentally known kinetic and dynamical variables. The functional forms of the rate equations have not been optimized.

DNA breaks and cell cycle arrest induced by okadaic acid in Caco-2 cells, a human colonic epithelial cell line.

Okadaic acid (OA) is a shellfish toxin produced by dinoflagellates, in mussels. It is a potent tumour promoter and represents a potential threat to human health even at low concentrations. OA targets mainly the gastrointestinal tract in acute poisoning, causing diarrhoea. Therefore the present investigations were designed to study the ability of okadaic acid to induce cytotoxicity and DNA lesions in a human colonic cell line (Caco-2). Incubation of Caco-2 cells with OA (3.75-60 ng/ml, i.e. 4.6 x 10(-3)-7.5 x 10(-2) microM) causes a significant reduction in cell viability. Moreover, okadaic acid inhibits protein and DNA synthesis with, respectively, IC50 of 16 and 6.5 ng/ml after 24 h incubation. It also provokes cell cycle arrest, characterised by an increase in the number of S phase cells, correlated with a significant decrease in G0/G1 phase cells at high concentration. One of the main results obtained in these investigations is the apoptosis induced by OA in Caco-2 cells of intestinal origin, shown by DNA laddering in agarose gel electrophoresis (250-1000 base pairs). OA also induces clastogenic effects evaluated by DNA fragmentation analysis using the method of Higuchi and Aggarwal (52% for 60 ng/ml) and comet assay (increase of the frequency of comets and their tails length). Therefore, the cell death induced by OA seems clearly to be concentration-dependent after 24 h of incubation. The cytotoxic properties of okadaic acid and its ability to damage DNA result in cell death, mainly by apoptosis. Since consumption of shellfish contaminated with acceptable okadaic acid concentrations exposes colonic cells to harmful concentrations of this toxin, the possibility that OA would display its toxic effects on intestinal cells in vivo should be evaluated in human primary intestinal cells and human intestinal slices for cytotoxic effects, DNA fragmentation and apoptosis.

Synchronization of glycolytic oscillations in a yeast cell population.

The mechanism of active phase synchronization in a suspension of oscillatory yeast cells has remained a puzzle for almost half a century. The difficulty of the problem stems from the fact that the synchronization phenomenon involves the entire metabolic network of glycolysis and fermentation, and consequently it cannot be addressed at the level of a single enzyme or a single chemical species. In this paper it is shown how this system in a CSTR (continuous flow stirred tank reactor) can be modelled quantitatively as a population of Stuart-Landau oscillators interacting by exchange of metabolites through the extracellular medium, thus reducing the complexity of the problem without sacrificing the biochemical realism. The parameters of the model can be derived by a systematic expansion from any full-scale model of the yeast cell kinetics with a supercritical Hopf bifurcation. Some parameter values can also be obtained directly from analysis of perturbation experiments. In the mean-field limit, equations for the study of populations having a distribution of frequencies are used to simulate the effect of the inherent variations between cells.

Combined effects of okadaic acid and cadmium on lipid peroxidation and DNA bases modifications (m5dC and 8-(OH)-dG) in Caco-2 cells.

Okadaic acid (OA) is a marine toxin, a tumour promoter and an inducer of apoptosis. It mainly inhibits protein-phosphatases, protein synthesis and enhances lipid peroxidation. Cadmium (Cd) is known to be carcinogenic in animals and humans (group 1 according to the International Agency for Research on Cancer (IARC) classification). Cd also induces oxidative stress in living organisms. Since they are sometimes found simultaneously in mussels, we have evaluated in the present investigation, the lipid peroxidation, as malondialdehyde (MDA) production, in the variation of the ratios of 8-(OH)-dG/10(5)dG and m5dC/(dC + m5dC) induced by OA and/or Cd in Caco-2 cells. When cells were treated exclusively by OA (15 ng/ml) or Cd (0.625 and 5 microg/ml) for 24 h, protein synthesis was inhibited (by 42 +/- 5%, 18 +/- 13%, and 90 +/- 4% respectively) while MDA production was 2,235 +/- 129, 1710 +/- 20, and 11,496 +/-1,624 pmol/mg protein respectively. In addition, each toxicant induced modified bases in DNA; increases in oxidised bases and methylated dC. The combination of OA and cadmium was more cytotoxic and caused more DNA base modifications; the ratio m(5)dC/(m(5)dC + dC) was increased from 3 +/- 0.15 to 9 +/- 0.15 and the ratio 8-(OH)-dG/10(5) dG also (from 36 +/- 2 to 76 +/- 6). The combination of OA and Cd also increased the level of MDA (1,6874 +/- 2,189 pmole/mg protein). The present results strongly suggest that DNA damage resulting from the oxidative stress induced by these two toxicants may significantly contribute to increasing their carcinogenicity via epigenetic processes.

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells.

Fumonisin B1 (FB1), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB1 also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB1 was first obtained when C6 glioma cells were incubated with fumonisin B1 (3-27 microM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB1 at 9 and 18 microM induces respectively 50 +/- 2% and 40 +/- 1% of cells with a comet with an increased tail length of 93 +/- 9 microm and 102 +/- 17 microm respectively. Under these concentrations, FB1 induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 microM) for 24 h before FB1 (18 microM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB1.

Epigenetic properties of fumonisin B(1): cell cycle arrest and DNA base modification in C6 glioma cells.

Fumonisin B(1) produced by the fungus Fusarium moniliforme is a member of a new class of sphinganine analogue mycotoxins that occur widely in the food chain. Epidemiological studies associate FB(1) with human oesophageal cancer in China and South Africa. FB(1) also causes acute pulmonary edema in pigs and equine leucoencephalomalacia. This disease is thought to be a consequence of inhibition by FB(1) of cellular ceramide synthesis in cells. To investigate further on this pathogenesis, the effect of FB(1) was studied on cell viability (3 to 54 microM of FB(1)), protein (2.5 to 20 microM of FB(1)) and DNA syntheses (2.5 to 50 microM of FB(1)), and cellular cycle (3 to 18 microM of FB(1)) of rat C6 glioma cells after 24 h incubation. The results of the viability test show that FB(1) induces 10 +/- 2% and 47 +/- 4% cell death with, respectively, 3 and 54 microM, in C6 cells. This cytotoxicity induced by FB(1) was efficiently prevented when the cells were preincubated 24 h with vitamin E (25 microM). FB(1) displays epigenetic properties since it induced hypermethylation of the DNA (9-18 microM). Inhibition of protein synthesis was observed with FB(1) with an IC(50) of 6 microM showing that C6 glioma cells are very sensitive to FB(1); however, the synthesis of DNA was only slightly inhibited, up to 20 microM of FB1. The flow cytometry showed that the number of cells in phase S decreased significantly as compared to the control p = 0.01 from 18. 7 +/- 2.5% to 8.1 +/- 1.1% for 9 microM FB(1). The number of cells in phase G(2)/M increased significantly as compared to the control (p

Sustained oscillations in living cells.

Glycolytic oscillations in yeast have been studied for many years simply by adding a glucose pulse to a suspension of cells and measuring the resulting transient oscillations of NADH. Here we show, using a suspension of yeast cells, that living cells can be kept in a well defined oscillating state indefinitely when starved cells, glucose and cyanide are pumped into a cuvette with outflow of surplus liquid. Our results show that the transitions between stationary and oscillatory behaviour are uniquely described mathematically by the Hopf bifurcation. This result characterizes the dynamical properties close to the transition point. Our perturbation experiments show that the cells remain strongly coupled very close to the transition. Therefore, the transition takes place in each of the cells and is not a desynchronization phenomenon. With these two observations, a study of the kinetic details of glycolysis, as it actually takes place in a living cell, is possible using experiments designed in the framework of nonlinear dynamics. Acetaldehyde is known to synchronize the oscillations. Our results show that glucose is another messenger substance, as long as the glucose transporter is not saturated.

Synergistic effects of some metals contaminating mussels on the cytotoxicity of the marine toxin okadaic acid.

Okadaic acid (OA), a marine toxin is cytotoxic and promotes tumours in mouse skin. It is a specific and potent inhibitor of protein synthesis and also inhibits phosphatases A1 and A2 in vitro. In the present study, we investigated the influence of metals found at acceptable levels in mussels as environmental pollutants on the cytotoxicity of OA in Vero cells. Among the metals found in mussels (Mytilus edulis), the most represented, in terms of molar quantities per gram of dried weight are aluminium (230 nmol/g), copper (58 nmol/g), lead (16 nmol/g), mercury (14 nmol/g) and cadmium (7.4 nmol/g). A solution containing these five metals Al(3+), Cu(2+), Pb(2+), Hg(2+) and Cd(2+) combined at the concentrations detected in mussels, stimulated protein synthesis (+25%, P < 0.01), whereas different dilutions of this solution in the presence of okadaic acid (15 ng/ml, i.e. 18.7 x 10(-9) M) increased the percentage of protein synthesis inhibition from 35 to 79%. The metals also increased the lactate dehydrogenase (LDH) release into the medium and the lipid peroxidation induced by this algal toxin. In addition, these metals reduced the cell viability for an incubation period of 24 h especially at the two higher concentrations. These results indicate that metals (Al(3+), Cu(2+), Pb(2+), Hg(2+), Cd(2+)) in concentration ranges largely below the acceptable levels, synergistically increase the cytotoxicity of low concentrations of OA in cultured cells.