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A single muscle of Wistar female rats, either soleus or peroneus longus, was immobilized by fixing its cut distal tendon to the bone during 8 weeks. We observed a transitory weight loss in both muscles; the mean fiber cross-sectional area (CSA) showed a reduction at day 30, followed by an increase at day 60. The time course of the activation of the immobilized muscle was evaluated by recording the chronic electromyographic (EMG) activity during short periods (1 min every other day) of treadmill locomotion. During immobilization, the integrated EMG amplitude of the soleus increased, reaching a maximum at 4 weeks, but remained close to control values during 8 weeks for the peroneus. The median frequency (MF) of the power density spectrum of the soleus EMG was not statistically different between immobilized and control muscles, while MF of the immobilized peroneus EMG was permanently higher than that of control muscles. This suggests two different modes of adaptation in motor unit command, depending on the muscle profile, which could be concomitant with the restoration of muscle fibers CSA after 8 weeks.

Using the macro-patch clamp technique, we show that cardiac myocytes isolated from the heart of the oyster Crassostrea gigas possess several types of voltage-activated ionic currents. (1) A classical non-inactivating potassium current of the IK type that is inhibited by tetraethyl ammonium and shows an outward rectification and a slow activation. (2) A potassium current of the IA type that shows rapid activation and inactivation, and is blocked by 4-amino pyridine or preliminary depolarisation. (3) A potassium calcium-dependent current that is inhibited by charybdotoxin, activated by strong depolarisations and shows a large conductance. (4) A calcium inward current of the L-type that is inhibited by verapamil, cobalt and high concentrations of cadmium. This current is identified in most cells, but a T-type calcium current and classical fast sodium current are only identified in few cells, and only after a strong hyperpolarizing pulse. This suggests that these channels are normally inactivated in cultured cells and are not involved in the spontaneous activity of these cells. When they exist, the fast sodium channel is blocked by tetrodotoxin. The L-type calcium conductance is increased by serotonin. The identification in cultured oyster atrial cells of classical ionic currents, which are observed in most vertebrate species but only in a few species of molluscs, demonstrates that these cells are an interesting model. Moreover the viability and the electrophysiological properties of these cells are not significantly modified by freezing and thawing, thus increasing their usefulness in various bioassays.

The aim of this study was to evaluate how a modification in the mechanical conditions under which a muscle is used could induce changes in the characteristics and the spinal drive of its motor units (MU). The distal tendon of the soleus muscle of Wistar rats was transferred to the distal stump of the plantaris muscle tendon. The EMG activity of the soleus was chronically recorded for 8 weeks, every other day, during a 1-min treadmill walk. After spinal ventral root splitting, individual MU contractile properties were measured in control soleus (102 MUs) or in transposed soleus muscles after 4 weeks (41 MUs) or 8 weeks (28 MUs). Muscle/body weight ratio did not vary after transposition, nor did MU tetanic forces. A decrease in MU twitch contraction times and in their half relaxation times was observed at weeks 4 and 8. MU tension-frequency curves varied significantly after tendon transfer, becoming closer to the curves of the fast MUs of the control group. During locomotion, we observed no change in the amplitude of rectified-filtered electromyographic activity, but a significant decrease in mean burst duration and an increase in the median frequency of the power density spectrum. Tendon transposition of the soleus muscle brought about adaptations in MU contractile properties and soleus spinal control.

Spontaneous beating cells can be isolated from the heart of the oyster (Crassostrea gigas) and cultured for more than two months. They form adherent contractile networks in culture conditions. They show muscarinic and beta-adrenergic reactivity thus showing that they are functional cardio-myocytes: Acetylcholine induced a dose dependent decrease in spontaneous beating rate via an increase in potassium conductance, this effect being blocked by atropine. Epinephrine induced a dramatic increase in calcium conductance which was blocked by high concentrations of propranolol but not by sotalol and reversed by verapamil. Tributyltin and cadmium induced a dose and time dependent decrease mainly in inward ionic conductances, leading to a decrease or even a total suppression of the beating rate. Present study indicates that this model could be used as a sensitive test to study the effects of some marine pollutants at the cellular level in molluscs.

The comet assay is a single cell gel electrophoresis test currently used as a qualitative and quantitative genotoxicity test. However, some of the results from this comet assay and current knowledge on apoptosis lead us to suspect the presence of some false positive results. The aim of this study was to ascertain if apoptotic cells can yield comet images that might distort the interpretation of the results. Using Jurkat cells, that hardly express Fas antigen, and apoptosis induction with anti-Fas antibody, it was possible to show that apoptosis can generate typical comet pictures as soon as the cells enter the apoptosis process. Therefore, comet images cannot be interpreted as a genotoxicity indicator when an apoptosis risk is present. Yopro-1 staining, that is also nearly immediate after apoptosis induction, can be used to balance comet assay results.

The aim of this work was to determine mass transfer coefficients in the cases of ultrasonically-assisted and classical stirred dead-end ultrafiltration. A comparative study was then performed, and mass transfer coefficients obtained under ultrasonic conditions are described by an empirical model. This correlation results from an analogy with what is observed using a stirred cell and involves the ultrasonic power as the main parameter. The hydrodynamics are assumed to depend on the intensity of the ultrasound effects illustrated by the agitation arising within the cell. This agitation is due to convective currents as well as physical effects due to cavitation. The concentration polarization phenomenon is therefore affected by this action of ultrasonic waves.

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight-line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/10(8) cells and from 0.6 to 6.0 nmole/10(8) cells, respectively. Moreover, 31P-NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/10(9) spermatozoa/min. FCCP, an uncoupler of oxidative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3-, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3- altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxidative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo-osmotic medium, mitochondrial oxidative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [14C]leucine, [3H]thymidine, and [14C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.

A dissociation protocol for Pecten maximus heart has been established that makes it possible to obtain functional primary cultures routinely (9, 10); freezing assays of isolated cells were performed with the aim of making it possible to cultivate these cells in vitro after thawing to provide a constant standardized source of cells for applied research. Various parameters such as the nature and the concentration of the cryoprotectant, the cooling rate, and the incubation time of cells with the cryoprotective agent were evaluated. Best results were obtained by freezing cells in 12% dimethyl sulfoxide (Me2SO) in Leibovitz L15 medium at a cooling rate of approximately 2-3 degreesC/min. Thawed cells in culture attached to the substrate and survived for at least 3 weeks. They exhibited similar morphology and synthesised proteins, DNA, and lipids in vitro at levels close to those observed in fresh cells. To our knowledge, it is the first time that cultures have been obtained from cryopreserved marine invertebrate cells. Copyright 1998 Academic Press.

Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas. Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes. Two culture media were used to establish primary cell cultures and tested as transfection media. Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions. In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species. Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters.