Interfacial adsorption and activity of pancreatic lipase-related protein 2 onto heterogeneous plant lipid model membranes.

Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called "bulk", digestion. The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes.

Modulation of gastric lipase adsorption onto mixed galactolipid-phospholipid films by addition of phytosterols.

The rapid and preferential adsorption of a gastric lipase recombinant dog gastric lipase (rDGL) in heterogeneous films of phospholipids and triacylglycerols has previously been unveiled using Langmuir films analyzed by tensiometry, ellipsometry and Langmuir-Blodgett transfer coupled to atomic force microscopy. Here we invest the adsorption behavior of rDGL in heterogeneous galactolipid and mixed galactolipid-phospholipid or galactolipid-phospholipid-phytosterol films representative of plant membrane. Again rDGL, preferentially got adsorbed at the expanded lipid phases of the films underlining the genericity of such adsorption behavior. The addition of phytosterols to these mixtures resulted in the creation of defects, favoring the adsorption of rDGL at the fluid phases, but also improving the adsorption capacities of the lipase at the phase boundaries and towards the defects in the condensed phase. rDGL, like all gastric lipases, does not show any activity on galactolipids and phospholipids but its adsorption impacts their lateral organization and may change the adsorption and activity of other lipolytic enzymes in the course of digestion.

Interfacial organization and phase behavior of mixed galactolipid-DPPC-phytosterol assemblies at the air-water interface and in hydrated mesophases.

The structural behavior of model assemblies composed of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the two main galactolipids found in plants, was investigated at the air/water interface and in aqueous dispersion. To approach the composition of the natural photosynthetic membranes, tunable Langmuir model membrane of galactolipids (GL) were used, and were complexified to form either heterogenous binary or ternary assemblies of GL, phospholipids (PL), and phytosterols (pS). The impact of pS, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or both on the structural properties of GL membrane was studied. The nature of the interactions between the different molecules was investigated using biophysical characterizations (ellipsometry, tensiometry, atomic force microscopy). In addition, the phase behavior was determined by SAXS analysis on the model assemblies in aqueous dispersions. Results revealed the good interfacial stability of these specific plant membrane lipids. The morphology of the GL film was characteristic of a fluid phase, with an interfacial roughness induced by the intercalation of monogalactosyl and digalactosyl polar heads of MGDG and DGDG, respectively. A phase heterogeneity in the monolayer was induced by the addition of DPPC and/or pS, which resulted in the modification of galactolipid organization and headgroup interactions. These structural changes were confirmed by SAXS analysis, showing more favorable interactions between MGDG and DPPC than between DGDG and DPPC in aqueous dispersion. This phenomenon was exacerbated in the presence of pS.

Interfacial Activity of Lipoprotein (a) Isoforms with a Variable Number of Kringle IV Type 2 Repeats: A New Indicator of Cardiovascular Risk?

OBJECTIVE: Lipoprotein (a) [Lp(a)] is an LDL-like particle constituted by lipids, apolipoprotein B100 and apolipoprotein (a) [apo(a)], a multidomain glycoprotein whose molecular mass is dependent on the genetically encoded number of Kringle IV type 2 (KIV-2) repeats. Because Lp(a) isoforms have been associated with cardiovascular risk (CVR), we have investigated if their interfacial properties can contribute to distinguish between low and high-risk groups and thus be used as a new CVR indicator. METHODS: Four Lp(a) variants, each carrying a different apo(a) isoform (K20, K24, K25, and K29), were purified from plasma of homozygous donors and their interfacial properties characterized using ellipsometry and surface pressure techniques. RESULTS: Ellipsometry measurements revealed that these isoforms had a similar propensity to form adsorbed layers at hydrophobic-hydrophilic interfaces, but surface pressure enabled to clearly separate them into two groups: K20 and K24 on one side, and K25 and K29 on the other side. CONCLUSION: Though K24 and K25 differ only by a single KIV-2 domain, their sharp difference in surface pressure suggests a critical threshold between the two Lp(a) forms, providing insights into the use of condensed matter approaches to monitor CVR. Our findings may represent a new laboratory window to assist medical decisions and to develop precision medicine treatments, practices, and products for CVR, which can be extended to other cardiovascular disease conditions.

Protein Transport upon Advection at the Air/Water Interface: When Charge Matters.

The formation of dense protein interfacial layers at a free air-water interface is known to result from both diffusion and advection. Furthermore, protein interactions in concentrated phases are strongly dependent on their overall positive or negative net charge, which is controlled by the solution pH. As a consequence, an interesting question is whether the presence of an advection flow of water toward the interface during protein adsorption produces different kinetics and interfacial structure of the adsorbed layer, depending on the net charge of the involved proteins and, possibly, on the sign of this charge. Here we test a combination of the following parameters using ovalbumin and lysozyme as model proteins: positive or negative net charge and the presence or absence of advection flow. The formation and the organization of the interfacial layers are studied by neutron reflectivity and null-ellipsometry measurements. We show that the combined effect of a positive charge of lysozyme and ovalbumin and the presence of advection flow does induce the formation of interfacial multilayers. Conversely, negatively charged ovalbumin forms monolayers, whether advection flow is present or not. We show that an advection/diffusion model cannot correctly describe the adsorption kinetics of multilayers, even in the hypothesis of a concentration-dependent diffusion coefficient as in colloidal filtration, for instance. Still, it is clear that advection is a necessary condition for making multilayers through a mechanism that remains to be determined, which paves the way for future research.

Stability to oxidation and interfacial behavior at the air/water interface of minimally-processed versus processed walnut oil-bodies.

Oil bodies (OB), the form of triacylglycerol storage in seeds, are interesting natural assemblies for nutritional applications. In walnuts, OB contain an important amount of polyunsaturated fatty acids that could be interesting food ingredients but may be prone to oxidation. The oxidative and interfacial behavior of walnut OB, either minimally-processed or after processing, were compared with processed complex walnut juice. The good oxidative stability of minimally-processed OB over 10 days (PV ≤ 8.4 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) and of processed walnut complex matrixes over 20 days (PV ≤ 4.8 meq O2/kg, TBARS = 1.4 mmol eq MDA/kg) was evidenced. In comparison, processing of OB promoted their oxidation. The interfacial studies led to the proposition of a new model of adsorption for minimally-processed OB that will be useful to design functional emulsion or foam in which OB act as emulsifiers.

Physico-chemical behaviors of human and bovine milk membrane extracts and their influence on gastric lipase adsorption.

Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions.

Branched lipid chains to prepare cationic amphiphiles producing hexagonal aggregates: supramolecular behavior and application to gene delivery.

A ramified lipid alcohol, 2-hexyldecanol, was used as a hydrophobic moiety to prepare cationic amphiphiles on a gram scale in 3 to 4 steps, featuring either a trimethylammonium 5, dimethylhydroxyethylammonium 6 or N-methylimidazolium 7 polar head group. Compression isotherms at the air-water interface reveal that all these cationic amphiphiles collapse at a relatively low pressure indicating a weak stabilization of the monolayer via hydrophobic interactions. Ellipsometry measurements point out the presence of a thin monolayer at low lateral pressure whereas thickening of the monolayer occurs at higher pressure with a high percentage of variation of the thickness, thus demonstrating an adaptability to the constraints. 31P NMR spectroscopy of the hydrated cationic amphiphiles clearly shows that these cationic amphiphiles self-assemble in water to form hexagonal phases, irrespective of the nature of their polar head group. Furthermore, a comparison of molecular structures suggests that compounds 5-7 self-organize into an inverted hexagonal phase (HII). These cationic amphiphiles, alone or in the presence of DOPE, were evaluated for the transfection of three human-derived cell lines (i.e. A549, 16HBE and HeLa). The three compounds demonstrated high transfection efficacies in every cell line tested, 7/DOPE being the most efficient.

Synthesis of Pyridoclax Analogues: Insight into Their Druggability by Investigating Their Physicochemical Properties and Interactions with Membranes.

Pyridoclax is considered a promising anticancer drug, acting as a protein-protein interaction disruptor, with potential applications in the treatment of ovarian, lung, and mesothelioma cancers. Eighteen sensibly selected structural analogues of Pyridoclax were synthesized, and their physicochemical properties were systematically assessed and analyzed. Moreover, considering that drug-membrane interactions play an essential role in understanding the mode of action of a given drug and its eventual toxic effects, membrane models were used to investigate such interactions in bulk (liposomes) and at the air-water interface. The measured experimental data on all original oligopyridines allowed the assessment of relative differences in terms of physicochemical properties, which could be determinant for their druggability, and hence for drug development.

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery.

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.

Substitution of unsaturated lipid chains by thioether-containing lipid chains in cationic amphiphiles: physicochemical consequences and application for gene delivery.

The hydrophobic moiety of cationic amphiphiles plays an important role in the transfection process because its structure has an impact on both the type of the supramolecular assembly and the dynamic properties of these assemblies. The latter have to exhibit a compromise between stability and instability to efficiently compact then deliver DNA into target cells. In the present work, we report the synthesis of new cationic amphiphiles featuring a thioether function at different positions of two 18-atom length lipid chains and we study their physicochemical properties (anisotropy of fluorescence and compression isotherms) with analogues possessing either oleyl (C18:1) or stearyl (C18:0) chains. We show that the fluidity of cationic lipids featuring a thioether function located close to the middle of each lipid chain is intermediate between that of oleyl- and stearyl-containing analogues. These properties are also supported by the compression isotherm assays. When used as carriers to deliver a plasmid DNA, thioether-containing cationic amphiphiles demonstrate a good ability to transfect human-derived cell lines, with those incorporating such a moiety in the middle of the chain being the most efficient. This work supports the use of a thioether function as a possible alternative to unsaturation in aliphatic lipid chains of cationic amphiphiles to modulate physicochemical behaviours and in turn biological activities such as gene delivery ability.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

Dystrophin Hot-Spot Mutants Leading to Becker Muscular Dystrophy Insert More Deeply into Membrane Models than the Native Protein.

Dystrophin (DYS) is a membrane skeleton protein whose mutations lead to lethal Duchenne muscular dystrophy or to the milder Becker muscular dystrophy (BMD). One third of BMD "in-frame" exon deletions are located in the region that codes for spectrin-like repeats R16 to R21. We focused on four prevalent mutated proteins deleted in this area (called RΔ45-47, RΔ45-48, RΔ45-49, and RΔ45-51 according to the deleted exon numbers), analyzing protein/membrane interactions. Two of the mutants, RΔ45-48 and RΔ45-51, led to mild pathologies and displayed a similar triple coiled-coil structure as the full-length DYS R16-21, whereas the two others, RΔ45-47 and RΔ45-49, induced more severe pathologies and showed "fractional" structures unrelated to the normal one. To explore lipid packing, small unilamellar liposomes (SUVs) and planar monolayers were used at various initial surface pressures. The dissociation constants determined by microscale thermophoresis (MST) were much higher for the full-length DYS R161-21 than for the mutants; thus the wild type protein has weaker SUV binding. Comparing surface pressures after protein adsorption and analysis of atomic force microscopy images of mixed protein/lipid monolayers revealed that the mutants insert more into the lipid monolayer than the wild type does. In fact, in both models every deletion mutant showed more interactions with membranes than the full-length protein did. This means that mutations in the R16-21 part of dystrophin disturb the protein's molecular behavior as it relates to membranes, regardless of whether the accompanying pathology is mild or severe.

Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation.

The enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interactions.

Cholesterol strongly affects the organization of lipid monolayers studied as models of the milk fat globule membrane: Condensing effect and change in the lipid domain morphology.

The biological membrane that surrounds the milk fat globules exhibits phase separation of polar lipids that is poorly known. The objective of this study was to investigate the role played by cholesterol in the organization of monolayers prepared as models of the milk fat globule membrane (MFGM). Differential scanning calorimetry and X-ray diffraction experiments allowed characterization of the gel to liquid crystalline phase transition temperature of lipids, Tm ~35°C, in vesicles prepared with a MFGM lipid extract. For temperature below Tm, atomic force microscopy revealed phase separation of lipids at 30 mN·m(-1) in Langmuir-Blodgett monolayers of the MFGM lipid extract. The high Tm lipids form liquid condensed (LC) domains that protrude by about 1.5 nm from the continuous liquid expanded (LE) phase. Cholesterol was added to the MFGM extract up to 30% of polar lipids (cholesterol/milk sphingomyelin (MSM) molar ratio of 50/50). Compression isotherms evidenced the condensing effect of the cholesterol onto the MFGM lipid monolayers. Topography of the monolayers showed a decrease in the area of the LC domains and in the height difference H between the LC domains and the continuous LE phase, as the cholesterol content increased in the MFGM lipid monolayers. These results were interpreted in terms of nucleation effects of cholesterol and decrease of the line tension between LC domains and LE phase in the MFGM lipid monolayers. This study revealed the major structural role of cholesterol in the MFGM that could be involved in biological functions of this interface (e.g. mechanisms of milk fat globule digestion).

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the Escherichia coli outer membrane.

Lysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein.

Moderate conformational impact of citrate on ovotransferrin considerably increases its capacity to self-assemble at the interface.

We have compared the behavior of ovotransferrin at the air-solution interface in the presence of a monovalent ion (acetate), or a divalent ion (citrate), the latter being known to induce conformational changes of this protein upon interaction with its iron-binding sites. We have characterised the adsorption layer at the air-water interface in terms of homogeneity, surface concentration excess and rheological properties at pH 4.0. Besides we have investigated the bulk conformation in the presence of the two anions. In the presence of citrate only, interfacial layers display well-defined domains of higher overall surface concentration suggesting multilayers adsorption. Citrate also induces higher helical content and stabilizes the protein against thermal denaturation. Hence we propose that these changes are involved in the propensity of ovotransferrin to self-assemble at the air-water interface resulting in thick and heterogeneous interfacial layer.

Cholesterol favors the anchorage of human dystrophin repeats 16 to 21 in membrane at physiological surface pressure.

Dystrophin (DYS) is a filamentous protein that connects the cytoskeleton and the extracellular matrix via the sarcolemma, conferring resistance to muscular cells. In this study, interactions between the DYS R16-21 fragment and lipids were examined using Langmuir films made of anionic and zwitterionic lipids. The film fluidity was modified by the addition of 15% cholesterol. Whatever the lipid mixture examined, at low surface pressure (20 mN/m) few differences appeared on the protein insertion and the presence of cholesterol did not affect the protein/lipid interactions. At high surface pressure (30 mN/m), the protein insertion was very low and occurred only in zwitterionic films in the liquid-expanded phase. In anionic films, electrostatic interactions prevented the protein insertion outright, and caused accumulation of the protein on the hydrophilic part of the monolayer. Addition of cholesterol to both lipid mixtures drastically modified the protein-lipid interactions: the DYS R16-21 insertion increased and its organization in the monolayer appeared to be more homogeneous. The presence of accessible cholesterol recognition amino-acid consensus sequences in this fragment may enhance the protein/membrane binding at physiological lateral pressure. These results suggest that the anchorage of dystrophin to the membrane in vivo may be stabilized by cholesterol-rich nano-domains in the inner leaflet of sarcolemma.

In vitro characterization of proliferation and differentiation of trout satellite cells.

Fish satellite cells have been extracted from various species, but the myogenic characteristics of these cells in culture remain largely unknown. We show here that 60%-70% of the adherent cells are myogenic based on their immunoreactivity for the myogenic regulatory factor MyoD. In DMEM containing 10% fetal calf serum (FCS), trout myoblasts display rapid expression of myogenin (18% of myogenin-positive cells at day 2) combined with rapid fusion into myotubes (50% of myogenin-positive nuclei and 30% nuclei in myosin heavy chain [MyHC]-positive cells at day 7). These kinetics of differentiation are reminiscent of the behavior of fetal myoblasts in mammals. However, not all the myogenic cells differentiate; this subpopulation of cells might correspond to the previously named "reserve" cells. More than 90% of the BrdU-positive cells are also positive for MyoD, indicating that myogenic cells proliferate in vitro. By contrast, less than 1% of myogenin-positive cells are positive for BrdU suggesting that myogenin expression occurs only in post-mitotic cells. In order to maximize either the proliferation or the differentiation of cells, we have defined new culture conditions based on the use of a proliferation medium (F10+10%FCS) and a differentiation medium (DMEM+2%FCS). Three days after switching the medium, the differentiation index (% MyHC-positive nuclei) is 40-fold higher than that in proliferation medium, whereas the proliferation index (% BrdU-positive nuclei) is three-fold lower. Stimulation of cell proliferation by insulin-like growth factor 1 (IGF1), IGF2, and FGF2 is greater in F10 medium. The characterization of these extracted muscle cells thus validates the use of this in vitro system of myogenesis in further studies of the myogenic activity of growth factors in trout.