Comparative sensitivity to the fungicide tebuconazole of biofilm and plankton microbial communities in freshwater ecosystems.

Stream and lake ecosystems in agricultural watersheds are exposed to fungicide inputs that can threaten the structure and functioning of aquatic microbial communities. This research analyzes the impact of the triazole fungicide tebuconazole (TBZ) on natural biofilm and plankton microbial communities from sites presenting different degrees of agricultural contamination. Biofilm and plankton communities from less-polluted (LP) and polluted (P) sites were exposed to nominal concentrations of 0 (control), 2 and 20 µg TBZ L(-1) in 3-week microcosm experiments. Descriptors of microbial community structure (bacterial density and chlorophyll-a concentration) and function (bacterial respiration and production and photosynthesis) were analyzed to chart the effects of TBZ and the kinetics of TBZ attenuation in water during the experiments. The results showed TBZ-induced effects on biofilm function (inhibition of substrate-induced respiration and photosynthetic activity), especially in LP-site communities, whereas plankton communities experienced a transitory stimulation of bacterial densities in communities from both LP and P sites. TBZ attenuation was stronger in biofilm (60-75%) than plankton (15-18%) experiments, probably due to greater adsorption on biofilms. The differences between biofilm and plankton responses to TBZ were likely explained by differences in community structure (presence of extracellular polymeric substances (EPS) matrix) and microbial composition. Biofilm communities also exhibited different sensitivity levels according to their in-field pre-exposure to fungicide, with P-site communities demonstrating adaptation capacities to TBZ. This study indicates that TBZ toxicity to non-targeted aquatic microbial communities essentially composed by microalgae and bacteria was moderate, and that its effects varied between stream and lake microbial communities.

Systems biology of cellular rhythms.

Rhythms abound in biological systems, particularly at the cellular level where they originate from the feedback loops present in regulatory networks. Cellular rhythms can be investigated both by experimental and modeling approaches, and thus represent a prototypic field of research for systems biology. They have also become a major topic in synthetic biology. We review advances in the study of cellular rhythms of biochemical rather than electrical origin by considering a variety of oscillatory processes such as Ca++ oscillations, circadian rhythms, the segmentation clock, oscillations in p53 and NF-κB, synthetic oscillators, and the oscillatory dynamics of cyclin-dependent kinases driving the cell cycle. Finally we discuss the coupling between cellular rhythms and their robustness with respect to molecular noise.

Modelling the dual role of Per phosphorylation and its effect on the period and phase of the mammalian circadian clock.

Circadian clocks are regulated at the post-translational level by a variety of processes among which protein phosphorylation plays a prominent, although complex, role. Thus, the phosphorylation of different sites on the clock protein PER by casein kinase I (CKI) can lead to opposite effects on the stability of the protein and on the period of circadian oscillations. Here the authors extend a computational model previously proposed for the mammalian circadian clock by incorporating two distinct phosphorylations of PER by CKI. On the basis of experimental observations the authors consider that phosphorylation at one site (denoted here PER-P1) enhances the rate of degradation of the protein and decreases the period, while phosphorylation at another site (PER-P2) stabilises the protein, enhances the transcription of the Per gene, and increases the period. The model also incorporates an additional phosphorylation of PER by the Glycogen Synthase Kinase 3 (GSK3). The authors show that the extended model incorporating the antagonistic effects of PER phosphorylations by CKI can account for observations pertaining to (i) the decrease in period in the Tau mutant, because of an increase in phosphorylation by CKI leading to PER-P1, and (ii) the familial advanced sleep phase syndrome (FASPS) in which the period is shortened and the phase of the oscillations is advanced when the rate of phosphorylation leading to PER-P2 is decreased. The model further accounts for the increase in period observed in the presence of CKI inhibitors that decrease the rate of phosphorylation leading to both PER-P1 and PER-P2. A similar increase in period results from inhibition of GSK3. [Includes supplementary material].

Dynamics of sulfate-reducing microorganisms (dsrAB genes) in two contrasting mudflats of the Seine estuary (France).

By combining molecular biology and biochemical approaches, the dynamics of sulfate-reducing microorganisms (SRM) was investigated in the sediments of the Seine estuary (France). Both intertidal mixing-zone and freshwater mudflats were sampled during a 1-year period; the quantification of SRM was realized by using competitive polymerase chain reaction (PCR) based on dsrAB gene amplification, previously described by Leloup et al. (2004), and sulfate reduction rate (SRR) was determined via the SO4(2-) radiotracer method. Throughout the year, abundance of dsrAB genes and SRR were predominantly high in the top 15 cm of the sediment. A seasonal dynamic was observed; a predominance of activity was noted during the early summer, and seems to be mainly controlled by physical-chemical parameters (temperature and dissolved organic carbon concentration) and topographic evolution of the mudflat (erosion/deposit erosion).

A molecular explanation for the long-term suppression of circadian rhythms by a single light pulse.

With the use of a molecular model for circadian rhythms in Drosophila based on transcriptional regulation, we show how a single, critical pulse of light can permanently suppress circadian rhythmicity, whereas a second light pulse can restore the abolished rhythm. The phenomena occur via the pulsatile induction of either protein degradation or gene expression in conditions in which a stable steady state coexists with stable circadian oscillations of the limit cycle type. The model indicates that suppression by a light pulse can only be accounted for by assuming that the biochemical effects of such a pulse much outlast its actual duration. We determine the characteristics of critical pulses suppressing the oscillations as a function of the phase at which the rhythm is perturbed. The model predicts how the amplitude and duration of the biochemical changes induced by critical pulses vary with this phase. The results provide a molecular, dynamic explanation for the long-term suppression of circadian rhythms observed in a variety of organisms in response to a single light pulse and for the subsequent restoration of the rhythms by a second light pulse.

Theoretical models for circadian rhythms in Neurospora and Drosophila.

We examine theoretical models proposed for the molecular mechanism of circadian rhythms in Drosophila. The models are based on the negative feedback exerted by a complex between the PER and TIM proteins on the expression of the per and tim genes. We show that a similar model can account for circadian oscillations in Neurospora, where the protein FRQ negatively regulates the expression of the frq gene. The effect of light on the circadian rhythms is included by considering that it elicits a rise in the rate of TIM degradation in Drosophila, whereas in Neurospora it enhances the rate of frq transcription. The models account for the occurrence of sustained circadian oscillations in continuous darkness in Drosophila and Neurospora. Numerical simulations further indicate that the periodic forcing of circadian oscillations by light-dark cycles can result either in the entrainment to the external periodicity or in aperiodic oscillations (i.e. chaos), depending on the magnitude of the periodic changes in the light-controlled parameter.

Modeling the molecular regulatory mechanism of circadian rhythms in Drosophila.

Thanks to genetic and biochemical advances on the molecular mechanism of circadian rhythms in Drosophila, theoretical models closely related to experimental observations can be considered for the regulatory mechanism of the circadian clock in this organism. Modeling is based on the autoregulatory negative feedback exerted by a complex between PER and TIM proteins on the expression of per and tim genes. The model predicts the occurrence of sustained circadian oscillations in continuous darkness. When incorporating light-induced TIM degradation, the model accounts for damping of oscillations in constant light, entrainment of the rhythm by light-dark cycles of varying period or photoperiod, and phase shifting by light pulses. The model further provides a molecular dynamical explanation for the permanent or transient suppression of circadian rhythmicity triggered in a variety of organisms by a critical pulse of light. Finally, the model shows that to produce a robust rhythm the various clock genes must be expressed at the appropriate levels since sustained oscillations only occur in a precise range of parameter values. BioEssays 22:84-93, 2000.

Physical properties and self-setting mechanism of calcium phosphate cements from calcium bis-dihydrogenophosphate monohydrate and calcium oxide.

An apatitic calcium phosphate cement was developed from calcium bis-dihydro-genophosphate monohydrate (or monocalcium phosphate monohydrate, MCPM) and calcium oxide (CaO). The powder had a Ca/P molar ratio of 1.67, and the liquid was either pure water or 0.25 M-1 M sodium phosphate buffer, pH 7.4. The influence of the powder-to-liquid (P/L) ratio on the setting time and the mechanical strength were studied. The best results were obtained for the 1 M phosphate buffer with a P/L ratio of 1.53; the setting time was 7 min and the compressive strength was 25 MPa after 24 h and 33 MPa after 11 d. The mechanism and kinetics of the setting reaction were investigated by X-ray diffraction, differential scanning calorimetry, 31P magic angle spinning-nuclear magnetic resonance and infrared spectrometry. The setting reaction was found to be biphasic: in the first step, during the mixing time, MCPM reacted with CaO immediately to give calcium hydrogenophosphate dihydrate (or dicalcium phosphate dihydrate, DCPD) which, in the second step, reacted more slowly with the remaining CaO to give hydroxyapatite. The conversion of the starting materials to hydroxyapatite was complete within 24 h when the liquid was water, but was slower and incomplete with the phosphate buffers. Of the starting materials, 30% remained after 3 d.

Limit cycle models for circadian rhythms based on transcriptional regulation in Drosophila and Neurospora.

We examine theoretical models for circadian oscillations based on transcriptional regulation in Drosophila and Neurospora. For Drosophila, the molecular model is based on the negative feedback exerted on the expression of the per and tim genes by the complex formed between the PER and TIM proteins. For Neurospora, similarly, the model relies on the feedback exerted on the expression of the frq gene by its protein product FRQ. In both models, sustained rhythmic variations in protein and mRNA levels occur in continuous darkness, in the form of limit cycle oscillations. The effect of light on circadian rhythms is taken into account in the models by considering that it triggers degradation of the TIM protein in Drosophila, and frq transcription in Neurospora. When incorporating the control exerted by light at the molecular level, we show that the models can account for the entrainment of circadian rhythms by light-dark cycles and for the damping of the oscillations in constant light, though such damping occurs more readily in the Drosophila model. The models account for the phase shifts induced by light pulses and allow the construction of phase response curves. These compare well with experimental results obtained in Drosophila. The model for Drosophila shows that when applied at the appropriate phase, light pulses of appropriate duration and magnitude can permanently or transiently suppress circadian rhythmicity. We investigate the effects of the magnitude of light-induced changes on oscillatory behavior. Finally, we discuss the common and distinctive features of circadian oscillations in the two organisms.

A model for circadian rhythms in Drosophila incorporating the formation of a complex between the PER and TIM proteins.

The authors present a model for circadian oscillations of the Period (PER) and Timeless (TIM) proteins in Drosophila. The model for the circadian clock is based on multiple phosphorylation of PER and TIM and on the negative feedback exerted by a nuclear PER-TIM complex on the transcription of the per and tim genes. Periodic behavior occurs in a large domain of parameter space in the form of limit cycle oscillations. These sustained oscillations occur in conditions corresponding to continuous darkness or to entrainment by light-dark cycles and are in good agreement with experimental observations on the temporal variations of PER and TIM and of per and tim mRNAs. Birhythmicity (coexistence of two periodic regimes) and aperiodic oscillations (chaos) occur in a restricted range of parameter values. The results are compared to the predictions of a model based on the sole regulation by PER. Both the formation of a complex between PER and TIM and protein phosphorylation are found to favor oscillatory behavior. Determining how the period depends on several key parameters allows us to test possible molecular explanations proposed for the altered period in the per(l) and per(s) mutants. The extended model further allows the construction of phase-response curves based on the light-induced triggering of TIM degradation. These curves, established as a function of both the duration and magnitude of the effect of a light pulse, match the phase-response curves obtained experimentally in the wild type and per(s) mutant of Drosophila.

Chemical characterization of in vivo aged zinc polycarboxylate dental cements.

The chemical composition of zinc polycarboxylate dental cements aged in vivo was studied. Thirty samples aged from one to 17 years were investigated using X-ray diffraction, infrared spectroscopy, thermogravimetric analysis and differential scanning calorimetry. Evidence for the presence of zinc oxide, amorphous zinc polycarboxylate and water of hydration was found. No correlation with age concerning either the chemical structure of the components or their relative amounts was found. Zinc polycarboxylate dental cements show very good chemical stability on long-term use.

Chemical and structural changes in zinc polycarboxylate cements after immersion in dilute organic acid solutions.

The behaviour of zinc polycarboxylate cements in contact with dilute aqueous solutions of organic acids at concentrations close to those existing in buccal medium, was studied. The organic acids were acetic, citric, tartaric and lactic acids, at 0.01 M and 0.001 M. The elution of zinc and magnesium was 10-1000 times greater in acid than in pure water, and correlated with the concentrations and the dissociation constants, pK1, of the acids tested. In all cases, important water losses were observed. In the 0.01 M acids, the cement structure collapsed to form a viscous, compact and homogeneous layer on the cement surface. In this layer, the polymeric carboxylic chains were regenerated from the zinc and magnesium polycarboxylate cement. Comparison with pure water showed that even the smallest concentration of the weak acids greatly modified the cement behaviour. This could explain the well-known differences in erosion processes between theoretical erosion predicted by standard specification tests and the in vivo situation.

Temperature compensation of circadian rhythms: control of the period in a model for circadian oscillations of the per protein in Drosophila.

The factors affecting the period are examined in a model for circadian oscillations of the period protein (PER) in Drosophila. The model for the circadian clock is based on multiple phosphorylation of PER and on the negative feedback exerted by PER on the transcription of the period (per) gene. The results are used to address the possible bases of the relative invariance of the period of oscillations with respect to temperature. Such a phenomenon, referred to as temperature compensation, represents one of the most conspicuous properties of circadian rhythms.

Influence of thyroid status on water metabolism and survival of normal and dehydrated desert rodents Meriones libycus.

The relationship between thyroid status and resistance to water deprivation in a desert rodent, Meriones libycus, has been studied in normal, radiothyroidectomized (Tx), and thyroidectomized T4-supplemented (1.5 microg T4/day) (Tx + T4) animals. In animals given free access to water, 1 month after thyroidectomy water influx and efflux decreased 3-fold. This decrease was partially corrected after 5 days of T4 administration. Thyroidectomy did not modify urinary osmolality nor affect survival. In dehydrated animals, the body weight decreased (about 15%) over 2 weeks in all groups and then stabilized. Water flux decreased sharply in normal or Tx + T4 animals during the 1st week and then stabilized. A further decrease of water flux occurred in hypothyroid animals, which continued over 4 weeks, when fluxes were half those of normal or Tx + T4 animals. The urinary osmolality increased equally sharply in the three groups, at least during the first 5 days of dehydration when sampling was possible. Whereas dehydrated normal and Tx + T4 animals survived at least 7 weeks, 70% of Tx animals had died after 4 weeks and none survived more than 7 weeks. The daily metabolic energy intake was estimated from water flux and metabolic water of the dietary barley. After 4 weeks, when water influx represented only metabolic water from food, metabolic energy intake decreased 2.5-fold in hypothyroid compared with normal or Tx + T4 animals. This low metabolic energy intake led to a trend of body dehydration, hypothermia, and death. Thus, although an effect of thyroidectomy on survival of hydrated animals beyond 4 weeks cannot be excluded, we infer that thyroid hormones play a significant role in the survival of desert rodents under conditions of hydric stress.

Thyroid hormones and putative nuclear T3 receptors in tissues of the ascidian, Phallusia mammillata cuvier.

Thyroid hormones were measured by radioimmunoassay in blood plasma and in extracts (butanol/chloroform/ammonia) of pharynx, alimentary canal, and tunic of Phallusia mammillata. Other animals were injected with [125I]T3 and its distribution in the same tissues was determined from 6 to 48 hr after injection. Last, the saturable binding of [125I]T3 to salt-extracted nuclear proteins in the pharynx and alimentary canal was studied in vitro. T4 was found in all tissues examined and in the same order of magnitude (2.7 to 8.4 ng/g) whereas plasma concentration was low (0.2 ng/ml). Tissue T3 concentrations were always much lower than T4 tissue concentrations, being highest in alimentary canal (0.8-1.1 ng/g) and very low in the tunic as well as in plasma, in which T3 was generally below 0.02 ng/ml. The tissue distribution of [125I]T3 was correlated with T3 concentrations. Tissue/plasma ratios were approximately 10 in the alimentary canal, 5 in the pharynx, and 0.18 in the tunic. Saturable binding of T3 to nuclear proteins in the alimentary canal and pharynx was demonstrated. The affinity (Kd) was similar to that found in tissues from other chordates but the maximal binding capacity was much lower. The very low levels of plasma T3 and low T3/T4 ratios may indicate that the endostyle releases primarily T4 into the body fluid. On the other hand, the high levels of T3 and the high T3/T4 ratios in the alimentary canal suggest that this metabolically active target tissue is the main site of the process of deiodination of T4 into T3, a process which has been previously shown in P. mammillata in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine is necessary for the action of growth hormone in acclimation to seawater of brown (Salmo trutta) and rainbow trout (Oncorhynchus mykiss).

Brown (BT) and rainbow trout (RT) in freshwater (FW) were treated with ovine growth hormone (GH), GH + iopanoic acid (IOP), and GH + IOP plus triiodothyronine (T3) for RT only. After 1 week of treatment, trout were transferred to 30 o/oo SW and treatment continued. In FW, GH treatment increased significantly plasma T3 level (BT) and T3/T4 ratio (BT and RT) by stimulating T4 to T3 deiodination. In the GH + IOP group, the plasma T3 levels and T3/T4 ratio fell significantly as T4 to T3 deiodination was inhibited. In GH + IOP + T3-treated RT, plasma T3 and T3/T4 ratios increased significantly relative to other groups. No mortality occurred and plasma osmolarity (PO) was not altered by any treatment in FW. After transfer to SW, all IOP + GH trout died within 2 (BT) or 3 days (RT). All GH-treated or control BT survived to the end of the experiment (6 days). RT survival rates tended to be improved in GH and GH + IOP + T3 groups relative to controls. Correlatively on day 1 the PO increase was significantly higher in IOP + GH groups (BT and RT) than in the other groups and significantly lower in GH and GH + IOP + T3 treated RT than in controls from days 1 to 6. These data confirm the requirement of T3 and deiodination of T4 to T3 for the development of hypoosmoregulatory mechanisms in SW as previously shown (Lebel and Leloup 1992). Furthermore, the suppression of the hypoosmoregulatory effect of GH, when conversion of T4 to T3 was inhibited by IOP and the reversal when T3 was added to IOP + GH treatment suggests that GH osmoregulatory action in SW acts via the simulation of T4-5' monodeiodination which increases T3 production.

[Triiodothyronine is needed for the acclimatization of brown trout (Salmo trutta) or rainbow trout (Oncorhynchus mykiss) to seawater]. / La triiodothyronine est nécessaire à l'acclimatation à l'eau de mer de la truite fario (Salmo trutta) ou arc en ciel (Oncorhynchus mykiss).

Brown and rainbow trout, held in freshwater at 13 +/- 1 degrees, were injected, every 3 days, with iopanoic acid (IOP: 5 mg/100 g body wt), an inhibitor of deiodination of thyroxine (T4) to triiodothyronine (T3). One group of IOP-treated rainbow trout was immersed in T3 (20 micrograms/l water). In IOP trout, plasma T3 fell to very low levels by day 7, while changes in T4 levels were less marked. In IOP + T3 trout plasma T3 increased fivefold, plasma T4 being unchanged. No mortality occurred and plasma osmolarity (OP) was not altered by any treatment. After direct transfer to seawater (30/1000), IOP trout were unable to acclimate to salinity: all died within 2 or 3 days, while the survival at day 3 was 100% in control brown trout and 45 and 74% in control and IOP + T3 rainbow trout respectively. OP increased more in IOP and less in IOP + T3 than in controls. There was a significant inverse correlation between T3, but not T4, plasma level, at the time of transfer and the OP 1 day later. In conclusion, although T3 does not play a significant role in osmoregulation in freshwater, T3 and therefore the deiodination of T4 into T3, were required for the development of hypo-osmoregulatory capacity involved in acclimation of trout to seawater.

Triiodothyronine binding to putative solubilized nuclear thyroid hormone receptor in liver and gill of the brown trout (Salmo trutta) and the European eel (Anguilla anguilla).

The binding of 3,5,3'-triiodothyronine (T3) to salt-extracted nuclear protein of liver and gill from trout and eel was studied in vitro. [125I]T3 binding depended on the temperature and protein concentration. Binding equilibria were achieved in the two tissues of each species between 15 and 24 hr at 4 degrees. The binding was reversible in the presence of excess unlabeled T3. Scatchard analysis showed a single class of high affinity and low capacity T3 binding species considered, 2.71 x 10(-10) M for eel liver and gill. Association (k + 1) and dissociation (k-1) rate constants, calculated from the kinetics of hormone association, were respectively 8.3 x 10(8) M.hr-1 and 0.216 hr-1 for trout liver and 8.3 x 10(8) M.hr-1 and 0.257 hr-1 for trout gill. Their ratio, the equilibrium dissociation constant for liver (2.60 x 10(-10)M) and for gill 3.10 x 10(-10) M), was in good agreement with the apparent Kd from the Scatchard plot. Half-times (t1/2) of dissociation of T3 calculated from association curves, liver (3.2 hr), and gill (2.7 hr) were in reasonable agreement with corresponding values determined directly, liver (7.7 hr) and gill (11.5 hr). These data are consistent with a reversible bimolecular process to describe the binding of T3 to nuclear extracts. The maximal binding capacity (MBC) was lower in gill than in liver. MBC values were similar in liver of trout, 163, and eel, 234, but were different in gill, 82 for trout and 29 fmol/mg protein for eel.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of vertebrate prolactins and growth hormones on thyroxine 5'-monodeiodination in the eel (Anguilla anguilla): a potential bioassay for growth hormone.

Growth hormones (GHs) and prolactins (Prls) purified from representatives of each vertebrate class from bony fish onwards were tested for their ability to stimulate in vivo peripheral deiodination of labeled thyroxine (T4*) into triiodo-L-thyronine (T3*) in the eel. Plasma T3*/T4* ratio was used as parameter. All GHs significantly increased T3*/T4*, the magnitude of the response being unrelated to the phylogenic position of species. No significant stimulation was shown with the various Prl, with the exception of ovine Prl, suggesting a heterosomatotropic effect of this preparation in the eel. Furthermore, both tilapia and ovine GH produced a dose-related effect on plasma T3*, T4*, and T3*/T4*. The stimulation of the peripheral deiodination of T4* into T3* estimated in vivo in the eel could become a specific, sensitive, and rapid fish bioassay for GH.

The effect of severe starvation and captivity stress on plasma thyroxine and triiodothyronine concentrations in an antarctic bird (emperor penguin).

The effect of confinement and severe starvation on the plasma thyroxine (T4) and triiodothyronine (T3) concentrations was determined in emperor penguins (Aptenodytes forsteri). During their annual cycle, emperor penguins fast freely for periods of up to 4 months and may thus represent a unique subject to study endocrine adaptations to fasting. Plasma T4 concentrations progressively decreased following capture and confinement of naturally fasting penguins, and within 15-20 days stabilized at levels three times lower than in free-living penguins. A transient fourfold increase in plasma T3 concentration developed within the day following confinement in parallel with a rise in daily body mass loss. Both plasma T3 concentration and mass loss subsided to normal levels within 15 days. The decrease in plasma T4 concentration is in accordance with the well-known inhibitory effect of stress on thyroid function in birds and mammals, whereas the transient increase in plasma T3 concentration seems related to enhancement of energy expenditure as a consequence of restlessness. Starvation severe enough to exhaust fat stores and to activate protein catabolism induced a 6- and 5 to 10-fold fall in plasma T4 and T3, respectively. This is in marked contrast with maintenance of plasma thyroid levels during long-term natural fasting associated with protein sparing (R. Groscolas and J. Leloup (1986) Gen. Comp. Endocrinol. 63, 264-274). Surprisingly, there was a final reincrease in plasma T4 concentration in very lean penguins. These results suggest that the effect of starvation on plasma thyroid hormones seems to depend on how much protein catabolism is activated and demonstrate the acute sensitivity of thyroid hormone balance to stress in penguins.