Michel Lavoie

An LHRH agonist inhibits FSH-induced cyclic AMP accumulation and steroidogenesis in porcine granulosa cells in culture

Abstract The direct action of a potent LHRH agonist, [D-Ser-(TBU) 6 , des-Gly-NH 2 10 ]LHRH ethylamide was studied in porcine granulosa cells in primary culture. The LHRH agonist inhibits by 30 to 55% the stimulatory effect of FSH on cyclic AMP accumulation up to 9 hours of incubation. In the absence of FSH, a more transient inhibitory effect is observed on cyclic AMP levels. The LHRH agonist inhibits both basal and FSH-induced progesterone secretion. The present data suggest that ovarian LHRH receptors are negatively coupled to adenylate cyclase.

Cadmium detoxification strategies in two phytoplankton species: metal binding by newly synthesized thiolated peptides and metal sequestration in granules.

The aim of this study was to evaluate whether intracellular detoxification mechanisms could explain, at least partially, the different sensitivity to Cd of two freshwater green algae, Chlamydomonas reinhardtii and Pseudokirchneriella subcapitata. Subcellular Cd distribution and the synthesis of metal-binding thiolated peptides were thus examined in both algae exposed to a range of free [Cd(2+)] from 0.7 to 253 nM. Cadmium partitioning among five subcellular fractions (cellular debris, granules, organelles, heat-denaturable proteins - HDP, and heat-stable proteins - HSP) was determined after differential centrifugation of algal homogenates. Thiolated-peptides, phytochelatins (PC(n)) and precursors, were analyzed by HPLC with pre-column monobromobimane derivatization. Cadmium accumulation per cell was 2-4 times greater for C. reinhardtii than for P. subcapitata, yet C. reinhardtii was more resistant to Cd with an EC(50) of 273 nM Cd(2+) [244-333 nM Cd(2+) CI(95%)]) compared to 127 nM Cd(2+) [111-143 nM Cd(2+) CI(95%)] for P. subcapitata. Although [Cd] generally increased in the organelle fractions when free [Cd(2+)] increased in the experimental media, their relative contributions to the total Cd cellular content decreased, suggesting that partial protection of some metal sensitive sites was achieved by the initiation of cellular detoxification mechanisms. An increase in the proportion of Cd in the granules fraction was observed for C. reinhardtii between 6 and 15 nM Cd(2+) (i.e., at [Cd(2+)]<the threshold for growth inhibition) suggesting the involvement of granules in protecting against the occurrence of toxic effects in C. reinhardtii. Both species also produced also high levels of PC(n), but with longer oligomers for C. reinhardtii. Unknown thiolated compounds (X(n)), which were not canonical or hydroxymethyl PC(n), were also found in both algae but at much higher concentrations for C. reinhardtii than for P. subcapitata. This difference in thiol synthesis could also be involved in the higher Cd resistance of C. reinhardtii with respect to P. subcapitata. This study demonstrates the importance of metal detoxification strategies in explaining the Cd sensitivity of different algal species.

A study of LH-RH receptors in the pituitary gland of the winter flounder (Pseudopleuronectes americanus Walbaum)

A study using an iodinated [D-Ser(tBu)6,Pro9-NHEt]LH-RH (Buserelin), demonstrated the presence of a single class of high-affinity (KD = 2.90 nM), high-capacity LH-RH binding sites in pituitaries obtained from sexually mature male and female winter flounder. Displacement curves for unlabeled Buserelin and other preparations of mammalian and fish LH-RH, but a lack of competition for structurally unrelated peptide hormones, indicated the hormone specific nature of the fish pituitary LH-RH receptor preparation. Compared with native mammalian LH-RH and salmon LH-RH, Buserelin and an analog of salmon LH-RH, [D-Arg6,Trp7,Leu8,Pro9-NHEt]LH-RH, had significantly higher binding affinities for the flounder pituitary receptor correlating with results of previous studies demonstrating the superagonist biological activity of LH-RH analogs in trout and goldfish.

A mine of information: Benthic algal communities as biomonitors of metal contamination from abandoned tailings

Various biomonitoring approaches were tested in the field to assess the response of natural periphythic algal communities to chronic metal contamination downstream from an abandoned mine tailings site. The accumulation of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) as well as the production of phytochelatins, the presence of diatom taxa known to tolerate high metal concentrations, diatom diversity and the presence of teratologies were determined. We observed highly significant relationships between intracellular metal and calculated free metal ion concentrations. Such relationships are often observed in laboratory studies but have been rarely validated in field studies. These results suggest that the concentration of metal inside the field-collected periphyton, regardless of its species composition, is a good indicator of exposure and is an interesting proxy for bioavailable metal concentrations in natural waters. The presence of teratologies and metal-tolerant taxa at our contaminated sites provided a clear indication that diatom communities were responding to this metal stress. A multi-metric approach integrating various bioassessment methods could be used for the field monitoring of metal contamination and the quantification of its effects.

Extending the Biotic Ligand Model to Account for Positive and Negative Feedback Interactions between Cadmium and Zinc in a Freshwater Alga

Low concentrations of essential trace metals such as zinc (Zn) were recently shown to strongly modulate cadmium (Cd) uptake in the freshwater alga Chlamydomonas reinhardtii. Here we studied the mechanisms of Cd and Zn acquisition by this alga, using metal uptake kinetics experiments. Cadmium uptake rates fitted a three transport site model characterized by the affinity constants K(Cd–1)(Cd) = 10(5.0), K(Cd–2)(Cd) = 10(7.6), and K(Cd–3)(Cd) = 10(8.8). Similar uptake kinetics were obtained for Zn with K(Zn–1)(Zn) = 10(5.0), K(Zn–2)(Zn) = 10(7.4), and K(Zn–3)(Zn) > 10(9). Competitive binding experiments suggest that Zn and Cd share the same three transport systems. The capacities of the transport systems were modulated by as much as 10-fold following preacclimation to high or low Zn(2+) and Cd(2+) concentrations. We conclude that the strong protective effect of Zn on Cd accumulation is mainly due to the reduction of the maximal uptake rate of the high-affinity Zn–2 (or Cd–2) transport system. A biotic ligand model was developed to incorporate the effects of both chemical speciation and physiological regulation of Cd transport systems. The model successfully predicts the experimentally measured steady-state Cd content of C. reinhardtii in the presence of low or high [Zn(2+)].

Influence of essential elements on cadmium uptake and toxicity in a unicellular green alga: The protective effect of trace zinc and cobalt concentrations

Within the biotic ligand model (BLM) construct, major cations are considered to be simple competitors for metal binding to uptake sites and may offer some protection against metal-induced toxicity, but the influence of essential trace elements and cell preconditioning to different micronutrient concentrations on metal uptake and toxicity is considered negligible. To test these underlying assumptions, we monitored Cd uptake and toxicity in a green alga (Chlamydomonas reinhardtii) after long-term exposures (60 h) to a range of environmentally realistic free Zn(2+) , Co(2+) , Fe(3+) , Mn(2+) , Ca(2+) , and Cu(2+) concentrations buffered with nitrilotriacetic acid. A 200-fold increase in free [Mn(2+) ] as well as a 100-fold increase in free [Fe(3+) ] did not affect Cd uptake or toxicity, whereas a 50-fold increase in free [Ca(2+) ] effectively offered some protection, as predicted by the BLM. However, a 10-fold increase in free [Cu(2+) ] significantly enhanced Cd toxicity by a factor of approximately 2, whereas a 100-fold increase in free [Zn(2+) ] and [Co(2+) ] from 10(-11) to 10(-9) M significantly decreased Cd uptake and toxicity by more than twofold. These effects did not change with prior algal acclimation to different essential micronutrient concentrations. Low essential trace metal concentrations may strongly affect the uptake and toxicity of Cd in freshwater algae and should be taken into consideration in future developments of the BLM.

Allelopathic interactions of linoleic acid and nitric oxide increase the competitive ability of Microcystis aeruginosa.

The frequency and intensity of cyanobacterial blooms are increasing worldwide with major societal and economic costs. Interactions between toxic cyanobacteria and eukaryotic algal competitors can affect toxic bloom formation, but the exact mechanisms of interspecies interactions remain unknown. Using metabolomic and proteomic profiling of co-cultures of the toxic cyanobacterium Microcystis aeruginosa with a green alga as well as of microorganisms collected in a Microcystis spp. bloom in Lake Taihu (China), we disentangle novel interspecies allelopathic interactions. We describe an interspecies molecular network in which M. aeruginosa inhibits growth of Chlorella vulgaris, a model green algal competitor, via the release of linoleic acid. In addition, we demonstrate how M. aeruginosa takes advantage of the cell signaling compound nitric oxide produced by C. vulgaris, which stimulates a positive feedback mechanism of linoleic acid release by M. aeruginosa and its toxicity. Our high-throughput system-biology approach highlights the importance of previously unrecognized allelopathic interactions between a broadly distributed toxic cyanobacterial bloom former and one of its algal competitors.

Characteristics of the β-adrenergic receptor in the rat ventral prostate using [125I]cyanopindolol

The binding characteristics of the beta-adrenergic receptor in the rat ventral prostate homogenate have been studied using the highly potent beta-adrenergic antagonist [125I]cyanopindolol (CYP) as ligand. The bound ligand was separated from the free moiety by precipitation with polyethylene glycol (PEG-6000). This technique is simple, accurate, fast and more advantageous than filtration of the hormone-receptor complex on glass fiber filters or direct centrifugation. [125I]CYP binds to a single class of high affinity sites at an apparent KD value of 23 pM. Using 0.1 microM (-)propranolol to determine non-specific binding, a number of sites of 600 fmol/mg protein were measured. The observed order of potency of adrenergic agonists (KD values) in competing for [125I]CYP binding was: (-)isoproterenol (25 nM) greater than (-)epinephrine (74 nM) much greater than (-)norepinephrine (1900 nM). Detailed study of the binding potency of a large series of beta 1- and beta 2-adrenergic agonists and antagonists showed the presence of a typical beta 2-subtype adrenergic receptor in the rat ventral prostate. The best estimate indicates that the proportion of beta 2-adrenergic receptors in rat ventral prostate is more than 95% of the total population of beta-adrenergic receptors in this tissue. The high selectivity and density of beta 2-adrenergic receptors in rat ventral prostate suggest a physiological role of circulating and/or locally secreted catecholamines in the control of prostatic growth and function.

Diclofop-methyl affects microbial rhizosphere community and induces systemic acquired resistance in rice

Diclofop-methyl (DM), a widely used herbicide in food crops, may partly contaminate the soil surface of natural ecosystems in agricultural area and exert toxic effects at low dose to nontarget plants. Even though rhizosphere microorganisms strongly interact with root cells, little is known regarding their potential modulating effect on herbicide toxicity in plants. Here we exposed rice seedlings (Xiushui 63) to 100μg/L DM for 2 to 8days and studied the effects of DM on rice rhizosphere microorganisms, rice systemic acquired resistance (SAR) and rice-microorganisms interactions. The results of metagenomic 16S rDNA Illumina tags show that DM increases bacterial biomass and affects their community structure in the rice rhizosphere. After DM treatment, the relative abundance of the bacterium genera Massilia and Anderseniella increased the most relative to the control. In parallel, malate and oxalate exudation by rice roots increased, potentially acting as a carbon source for several rhizosphere bacteria. Transcriptomic analyses suggest that DM induced SAR in rice seedlings through the salicylic acid (but not the jasmonic acid) signal pathway. This response to DM stress conferred resistance to infection by a pathogenic bacterium, but was not influenced by the presence of bacteria in the rhizosphere since SAR transcripts did not change significantly in xenic and axenic plant roots exposed to DM. The present study provides new insights on the response of rice and its associated microorganisms to DM stress.

Analyzing Arabidopsis thaliana root proteome provides insights into the molecular bases of enantioselective imazethapyr toxicity

Imazethapyr (IM) is a widely used chiral herbicide that inhibits the synthesis of branched-chain amino acids (BCAAs). IM is thought to exert its toxic effects on amino acid synthesis mainly through inhibition of acetolactate synthase activity, but little is known about the potential effects of IM on other key biochemical pathways. Here, we exposed the model plant Arabidospsis thaliana to trace S- and R-IM enantiomer concentrations and examined IM toxicity effects on the root proteome using iTRAQ. Conventional analyses of root carbohydrates, organic acids, and enzyme activities were also performed. We discovered several previously unknown key biochemical pathways targeted by IM in Arabidospsis. 1,322 and 987 proteins were differentially expressed in response to R- and S-IM treatments, respectively. Bioinformatics and physiological analyses suggested that IM reduced the BCAA tissue content not only by strongly suppressing BCAA synthesis but also by increasing BCAA catabolism. IM also affected sugar and starch metabolism, changed the composition of root cell walls, increased citrate production and exudation, and affected the microbial community structure of the rhizosphere. The present study shed new light on the multiple toxicity mechanisms of a selective herbicide on a model plant.