Pierre Couillard

Absence of a complete block to polyspermy after fertilization of Mytilus galloprovincialis (Mollusca, Pelecypoda) oocytes

Mytilus galloprovincialis oocytes undergo monospermic fertilizations (1 sperm nucleus/oocyte) over a wide range of sperm-oocyte ratios beyond which the number of penetrating sperm increases either linearly or exponentially over 10 min. Artificial activation of oocytes by KCl or the ionophore A 23187, up to the polar body extrusion stage, allows successful fertilizations upon a subsequent insemination. No organized and complete detachment of supernumerary oocyte-bound sperm is detected after fertilization. Reducing the external Na+ concentration promotes a higher rate of fertilizations. These results suggest that no complete block to polyspermy is established in this species but that a partial block, Na+ dependent, might be sufficient to ensure monospermic fertilizations under natural conditions.

The effects of vasopressin and related peptides on osmoregulation in Amoeba proteus

We describe the effects of arginine-vasopressin (AVP) and five related peptides on the contractile vacuole, the osmoregulatory organelle of the fresh water Amoeba proteus. Arginine-vasopressin, lysine-vasopressin, and SKF 101926, a synthetic antagonist of vasopressin, cause a significant increase in the rate of output of the contractile vacuole. Deamino-vasopressin (dAVP), oxytocin, and arginine-vasotocin have no such activity, although dAVP interferes with the action of AVP when present in equimolar concentration. Relatively high concentrations are required and the effect of active peptides is readily reversible. When the normal, hypotonic medium (a synthetic pond water) is replaced by isotonic sucrose, the action of AVP on the vacuole is abolished. Thus vasopressin is believed to act by increasing permeability of the Amoeba plasma membrane to water.

The amphiphilic action of vasopressin and analogues on the plasma membrane of Amoeba proteus

Arginine (AVP) and lysine vasopressin induce a weak but statistically significant increase in the water permeability of Amoeba proteus plasmalemma. Vasotocin and deaminovasopressin, which share the hydroosmotic properties of AVP on classical vertebrate systems, are without effects on Amoeba while SKF 101926, a synthetic AVP antagonist, is even more effective than the parent compound. Theophyllin and dibutyryl-cAMP do not affect AVP action on Amoeba. Lithium, oxytocin, and carbachol are also without effect. Thus, it is unlikely that either V2 (cAMP) or V1 (phosphatidylinositol choline) receptors are involved. A clear correlation has been found between the amphiphilic character of tested peptides and their effect on Amoeba water permeability. Classical amphiphilic peptides, melittin, mastoparan, and fragment 1-8 of alpha-neoendorphin, also increased water permeability in Amoeba. It is known that vasopressin can interact with artificial lipid membranes, increasing their permeability to water. We propose that amphiphilic members of the AVP family interact directly with the lipid phase of the Amoeba membrane. Their incorporation within the lipid bilayer may cause local disruptions or may create micellar water channels as shown for other amphiphilic proteins. Our observations provide a model for the early evolution of peptide hormone systems, preceding the appearance of specific membrane receptors and associated second messenger amplifying mechanisms.

Photoreception in Protozoa, An Overview

I remember Lynn Margulis showing in a seminar, that the lowly Prokaryotes, Bacteria and blue-green Algae, had invented most everything in Biochemistry, well before we, the Eukaryotes, came onto the scene. Photosynthesis, glycolysis, protein synthesis, dicarboxylic acid cycle, oxidative phosphorylation, you name it and some microbe has it!

Direct membrane effects of morphine and endorphins on amoeba proteus

Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1-8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.

External ions and direct membrane effects of enkephalins on amoeba proteus

We have shown that amphiphilic hormones like vasopressins and endorphins increase water permeability and activate the contractile vacuole (CV) in Amoeba by direct action on the plasma membrane. Using our standard CV assay, the effects of nine opioids, morphine, naloxone and 7 enkephalin derivatives, have been compared in normal, ion-containing growth medium (Chalkleys) and in glass-distilled water. While the absence of external ions does not affect the activity of molecules with net positive charges, opioids with no net charge are devoid of action in glass-distilled water. This shows that, in addition to amphiphilicity, which permits insertion into the lipid core of the membrane, electrostatic interactions with ions and with negative charges of phospholipids membrane and glycocalyx, the thick glycoproteic cell coat of Amoeba, are important in the direct action of these compounds.

A passively-driven mechanical model of the ciliary axoneme

A three-dimensional, macroscopic model of the ciliary axoneme has been designed and constructed. It allows students to visualize relative displacement of peripheral microtubular doublets at any degree of imposed bending of the cilium. Direct measurement of doublet sliding at desired points is also possible. An example of such measurements is given and compared with corresponding geometrically obtained data.