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The maison des adolescents scheme (mda), 20 years already! It was created because a lot of the existing programs dedicated to teenagers and their families were unrecognized and poorly articulated. In order to make them more identifiable, Claire Brisset, défenseure des enfants (child advocate), recommended the creation of a unique place inspired by the experience of a teenager's house opened in 1999 in Le Havre. Teenagers and their families could find information and support, have their situation evaluated, offer care and be referred to other professionals if necessary. In addition to this mandate, the MDA scheme also coordinates all the different operators working in this field in their area. Today, there are 120 MDA in France. Their mission is to prevent any teenagers' malaise or angst, anywhere in France, in urban as well as in rural areas. Since the creation of the first MDA in Le Havre in 1999, the situation has improved a lot. But there is still a need to improve the territorial coverage and the financial endowment of these schemes, in order for them to be able to fulfil this beautiful ambition to better the wellbeing of France's youth.Maisons des adolescents, 20 ans déjà ! Elles sont nées du constat que beaucoup de dispositifs sont dédiés aux adolescents et à leur famille mais qu’ils sont trop souvent peu connus et mal articulés entre eux. Pour les rendre plus accessibles, Claire Brisset, défenseure des enfants, préconise la création d’un dispositif faisant fonction de porte d’entrée unique pour les jeunes et les familles, s’inspirant de l’expérience havraise d’une maison de l’adolescent ouverte dès 1999. En plus de cette mission d’accueil, d’évaluation, d’accompagnement, de soins et d’orientation, elle propose également de leur confier la mission de coordonner les acteurs de l’adolescence de leur territoire d’intervention. Près de 120 MDA existent aujourd’hui. Elles déploient leurs missions sur l’ensemble du territoire national et ultra-marin, avec une vocation à prévenir les mal-être adolescents partout et pour tous, en milieu urbain comme en milieu rural. Si du chemin a été fait depuis la création de la première au Havre en 1999, il reste à bien couvrir l’ensemble des territoires d’action de ces MDA et à les doter à la mesure de la belle ambition d’améliorer le bien-être de la jeunesse de France….

Aim Since 1999, the development of more than 100 "Maisons des Adolescents" (MDAs) has enabled young persons to gain access to specific care in integrated youth-friendly facilities in France. To contribute to the development and standardization of international youth-friendly health care services, this review provides insight into the French MDA facilities. Methods This socio-historical analysis includes a systematic review of articles referring to the MDAs (selection through title and/or abstract), ministry reports and newspaper articles, from 1999 to 2018. Results If the various medical programmes of MDAs depend on the priorities of local teams rather as well as on official regulations, all MDAs offer the following essential services: a "Health and Prevention Space" open daily; multidisciplinary consultations; a mobile team visiting youth hospitalized in medical units; a mobile team able to meet adolescents at their homes; an open centre for art workshops; refresher and remedial courses for school work; network meetings and parent support groups. The MDAs from the start addressed an age group (young people aged 11-21 years) rather than an illness. They thus provide primary prevention for adolescents according to the World Health Organization definition of health as "a state of complete physical, mental and social well-being." This medical and political movement was shaped by the epistemological background of its first leaders. Conclusion Although more cohort studies to evaluate their early interventions would be useful, the success of the MDA network is already widely acknowledged by users, professionals and policy makers.

ResumeL’orientation d’un adolescent vers un lieu specifique de soin n’est jamais simple. Les Maisons des Adolescents possedent des atouts qui sont developpes a travers les modalites meme du travail mene par les accompagnants sociaux. Nous proposons de souligner ici la maniere dont ces derniers, dans leur accueil, appellent a un transfert tant sur l’institution que sur ses membres. L’accent est egalement mis sur un temps institutionnel fort, celui de la reunion clinique, qui permet la co-elaboration de l’experience clinique originale de ces professionnels, ainsi que la mise au travail de chacun des membres de l’equipe aupres des adolescents accueillis.Adolescence, 2012, 30, 2, 349-357.

International audience; Previous research in our laboratory has already shown the importance of the role played by ubiquitous calpains during myoblast migration. The aim of this study was to investigate calpain expression during myoblast migration and, to enhance this phenomenon via calpain stimulation. Ubiquitous calpains are members of a large family of calcium-dependent cysteine proteases. They play an important role in numerous biological and pathological phenomena, such as signal transduction, apoptosis, cell-cycle regulation, cell spreading, adhesion, invasion, myogenesis, and motility. Myoblast migration is a crucial step in myogenesis, as it is necessary for myoblast alignment and fusion to form myotubes. This study started by examining changes in calpain expression during migration, then investigated the possibility of activating myoblast migration via the stimulation of calpain expression and/or activity. The migration rate of myoblasts overexpressing mu- or milli-calpain was quantified. The results showed that calpain overexpression dramatically inhibited myoblast migration. Growth-factor treatments were then used to enhance myoblast migration. The results showed that treatment with IGF-1, TGF-beta1, or insulin induced a major increase in migration and caused a significant increase in m-calpain expression and activity. The increase in migration was totally inhibited by adding calpeptin, a calpain-specific inhibitor. These findings suggest that milli-calpain is involved in growth factor-mediated migration.

Cell migration is a fundamental cellular function particularly during skeletal muscle development. Ubiquitous calpains are well known to play a pivotal role during muscle differentiation, especially at the onset of fusion. In this study, the possible positive regulation of myoblast migration by calpains, a crucial step required to align myoblasts to permit them to fuse, was investigated. Inhibition of calpain activity by different pharmacological inhibitors argues for the involvement of these proteinases during the migration of myoblasts. Moreover, a clonal cell line that fourfold overexpresses calpastatin, the endogenous inhibitor of calpains, and that exhibits deficient calpain activities was obtained. The results showed that the migratory capacity of C2C12 and fusion into multinucleated myotubes were completely prevented in these clonal cells. Calpastatin-overexpressing myoblasts unable to migrate were characterized by rounded morphology, the loss of membrane extensions, the disorganization of stress fibers and exhibited a major defect in new adhesion formation. Surprisingly, the proteolytic patterns of desmin, talin, vinculin, focal adhesion kinase (FAK) and ezrin, radixin, moesin (ERM) proteins are the same in calpastatin-overexpressing myoblasts as compared to control cells. However, an important accumulation of myristoylated alanine-rich C kinase substrate (MARCKS) was observed in cells showing a reduced calpain activity, suggesting that the proteolysis of this actin-binding protein is calpain-dependent and could be involved in both myoblast adhesion and migration.

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27Kip1 in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27Kip1 was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27Kip1 accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27Kip1 that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27Kip1 expression. In vitro, p27Kip1 was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27Kip1 degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27Kip1 degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27Kip1 was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27Kip1 could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.

Previous studies have led us to hypothesize that m-calpain plays a pivotal role in myoblast fusion through its involvement in cell membrane and cytoskeleton component reorganization. To support this hypothesis, a convenient and simple myoblast culture model using frozen embryonic myoblasts was developed, which resolved a number of problems inherent to cell primary culture. Biological assays on cultured myoblasts using different media to define the characteristics of the fusion process were first conducted. Proteinase was detectable before the initiation of the fusion process and was closely correlated to the phenomenon of fusion under each culture condition studied. In addition, the study of calpastatin showed that the initiation of fusion does not require a decrease in the level of this endogenous inhibitor of calpains and also confirmed that calpastatin may be implicated in the determination of the end of fusion. On the other hand, analysis of the evolution of myogenic factors revealed that myogenins, MyoD and Myf5, increase very significantly during the formation of multinucleated myotubes. Moreover, the antisense technique against myogenin is capable of preventing the process of fusion by 50%, confirming the pivotal role of this factor in the early stages of differentiation. The possible role of myogenic regulator factors on m-calpain gene expression is discussed.

Previously we isolated a μ-calpain/PKCα complex from skeletal muscle which suggested tight interactions between the Ca2+-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKCα and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture. Our results show that calpains and PKCα are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKCα content in the particulate fraction and the appearance of PKMα fragment. These results suggest a possible calpain dependent down-regulation process of PKCαa in our model at the time of intense fusion. In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of pkcα in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKCα, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and μ-calpains). Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKCα.

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C2C12) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.

d -β-Hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a membrane enzyme, has been purified to homogeneity from dromedary ( Camelus dromedarius ) liver mitochondria. Our new purification method consisted of the solubilization of mitochondrial membranes by Triton X 100 and purification of BDH by two steps: DEAE-Sephacel and Phenyl-Sepharose. The molecular mass of the enzyme subunit size was 67 kDa. The purified enzyme is recognized by anti rat liver mitochondrial BDH antibodies. Furthermore, BDH activity was absolutely dependent upon phospholipids. BDH is also characterized by specific enzymatic parameters: an optimum pH of approximately 8 for the oxidation reaction, and approximately 7 for the reduction reaction and kinetic constant (Michaelis and dissociation constants) values of 1.07±0.13 mM for K MBOH , 0.21±0.01 mM for K MNAD + , 1.04±0.20 mM for K DNAD + , 0.29±0.01 mM for K MAcAc , 0.27±0.03 mM K MNADH and 1.12±0.18 mM for K DNADH .

MARCKS (Myristoylated Alanine Rich C Kinase Substrate) is a protein known to cross-link actin filament and consequently, is very important in the stabilization of the cytoskeletal structure. In addition, it has been recently demonstrated that the phosphorylation rate of this protein changes during myogenesis and that this protein is implicated in fusion events. For a better understanding of the biological function of MARCKS during myogenesis, we have undertaken to identify and purify this protein from rabbit skeletal muscle. Three chromatographic steps including an affinity calmodulin-agarose column were performed. The existence of a complex between the two proteins was confirmed by non-denaturing gel electrophoresis and immunoprecipitation. Two complexes were isolated which present an apparent molecular weight of about 600 kDa. Such interactions suggest that MARCKS is either a very good PKCalpha substrate and/or a regulator of PKC activity. These results are supported by previous studies showing preferential interactions and co-localization of PKC isozyme and MARCKS at focal adhesion sites. This is the first time that MARCKS has been purified from skeletal muscle and our data are consistent with a major role of this actin- and calmodulin-binding protein in cytoskeletal rearrangement or other functions mediated by PKalpha. Our results provide evidence for a tight and specific association of MARCKS and PKCalpha (a major conventional PKC isozyme in skeletal muscle) as indicated by the co-purification of the two proteins.

In previous studies, we isolated and identified a μ-calpain/PKCα complex from rabbit skeletal muscle. Here, we have used specific purification procedures in order to study the interactions between μ-calpain and PKC in mouse hippocampus, a brain structure implicated in memory processes. We observed that μ-calpain and conventional PKCs (α, βII and γ) are co-eluted after anion exchange chromatography. In contrast to our previous results obtained on skeletal muscle, μ-calpain and PKC isoenzymes were dissociated after gel filtration chromatography. Furthermore, μ-calpain induced the proteolytic conversion of PKCα, βII, and γ into PKMα, βII, and γ with a preferential hydrolysis of PKCγ, a specific isoenzyme of the nervous system. Although the μ-calpain/PKC interactions in the hippocampus are quite different from skeletal muscle, our results however, point out the functional importance of these inter-relations. Moreover, as PKCγ has been involved in the biochemical events underlying learning and memory, the preferential relationship between μ-calpain and PKCγ promotes the importance of the role that μ-calpain could play in the cellular mechanisms of memory formation.

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.

In previous studies, we isolated and identified a μ-calpain-PKCα complex from rabbit skeletal muscle. At the same time we pointed out that an association between μ-calpain and PKCα could occur at the level of the plasma membrane of muscle cells, and that PKCα could thus be considered as a potential μ-calpain substrate. In the present study, using the μ-calpain-PKCα complex as a model, we report that μ-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 μM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed μ-calpain forms and PKCα hydrolysis which leads to the formation of PKMα; (2) in certain experimental conditions, autolyzed μ-calpain forms are able to hydrolyze PKMα independently of the presence of diacylglycerol.

The effects of pressure on mu and m-calpain stability and specific activity have been examined. Activity and stability of these neutral calcium-dependent heterodimeric proteinases were studied using an in-house built bioreactor allowing on-line spectrophotometric monitoring with retention of pressure. Both isozymes were founded to be rather baro-sensitive with t1/2 at 1500 bar of 6 min and 11 min for mu and m-calpain respectively. Activity measurements under pressure showed a biphasic behavior for both proteinases with a slight activation for pressure up to 500 bar and 750 bar for m and mu-calpain respectively. Activation volume changes indicated that the proteolytic reaction was alternatively favored and disfavored by pressure due to catalytic step activation associated with enzyme-substrate binding step being continuously inhibited by pressure. Furthermore, autoproteolysis of calpain, a calcium dependent phenomenon was inhibited by application of pressure indicating that pressure inhibition of proteolytic activity could also be due to Ca2(+)-binding decrease under pressure. Implication of these results with catalytic mechanism of these heterodimeric proteinases is also discussed.

Summary Previous studies have demonstrated a role for m-calpain in myoblast fusion. Moreover, the presence, in differentiated cells, of a highly specific endogenous inhibitor of calpain, calpastatin, has led to the hypothesis that a regulation of or a protection against m-calpain activity by calpastatin could also occur during the earlier stages of muscle cell differentiation. In order to verify this hypothesis, we have investigated, in myoblast culture, the appearance of calpastatin-mRNA and its corresponding protein. Our results provide evidence that calpastatin is already present at the earlier stages of myoblast differentiation and that a significant decrease of the levels of calpastatin mRNA and its protein precedes myoblast fusion. In addition, the induction of an artificial decrease in calpastatin level, via an appropriate antisense oligodeoxyribonucleotide methodology, leads to earlier and faster myoblast fusion. Together with previous studies, these results indicate that m-calpain and calpastatin are functionally involved in myoblast fusion. Our findings also demonstrate that an acute "hyperactivity" of m-calpain resulting from the decrease of calpastatin synthesis is necessary during the early stages of this step of differentiation.

Previous studies have led to the hypothesis of a possible role for m-calpain (EC 3.4.22.17) in myoblast fusion in culture in vitro. To support this hypothesis, an antisense strategy has been used with cultured primary rat myoblasts. Using an appropriate antisense oligodeoxyribonucleotide to m-calpain mRNA, an inhibition of myoblast fusion has been observed, the maximum being obtained when the cell culture was treated with 30 microM of oligomer. Synthesis of m-calpain was decreased by 48% while high concentrations of antisense oligonucleotide do not significantly affect myoblast proliferation. The specificity of m-calpain intervention during fusion has also been confirmed using antisense oligonucleotides to mu-calpain and p94 mRNAs, respectively.

A novel extracellular β-fructosidase (EC 3.2.1.26) produced by Bacillus stearothermophilus has been identified and purified. The purified enzyme, obtained by using successive QEAE Sepharose fast flow and Sephacryl S300 HR columns, has a 600 kDa relative molecular weight (M r ) and is composed of 60 kDa subunits indicating a multimeric structure. The pH and temperature for optimal activity are 6-5 and 65°C respectively, the enzyme being thermostable at this temperature. The apparent K m values for sucrose and inulin are 3.56 mmol −1 and 1 mmol 1 −1 respectively, the total invertase (EC 3.2.1.26)/total inulinase (EC 3.2.1.7)ratio being 4

Levels of calpain I and calpain II mRNAs were analyzed at different stages of rat skeletal myoblast differentiation using a competitive polymerase chain reaction method. The results provide evidence that only calpain II mRNAs were present in significant quantities on the second day while calpain I mRNAs were identified on the fourth day of differentiation. If there is no compelling reason to believe that synthesis of calpains I and II is regulated at the level of mRNA, our results suggest that calpain II will be more particularly involved in Ca 2+ -mediated events accompanying myoblast fusion. On the other hand, calpain I, because of its later appearance may probably act on specific substrates such as myofibrillar proteins, associated myofibrillar proteins or the control of enzyme metabolism. Added factors such as insulin, which is known to induce enhancement of myoblast growth or myoblast fusion, had a significant effect on the amounts of calpain I and II mRNAs. In the presence of TGF-β, a potent inhibitor of myoblast fusion, calpain I and II mRNAs were decreased. These results confirm first that a Ca 2+ - dependent proteolytic system is positively correlated with myoblast fusion ( via calpain II) and second, that transcriptional regulation of calpains I and II may be negatively modulated during myoblast differentiation.

Peroxiredoxin IV (Prx IV), a member of the peroxiredoxin family, has been shown to be involved in cell protection against radiation. Peroxiredoxins are also overexpressed and involved in the progression of several tumours. Calpains have been shown to be over-activated in alveolar rhabdomyosarcoma (ARMS). The present study focused on the possible cross-regulations between Prx IV and calpains in ARMS cells. Prx IV abundance was quantified by Western blot analysis in ARMS cells and compared with non-malignant LHCN-M2 cells. Its abundance is quantified in ARMS cells treated or untreated with calpain inhibitors moreover its mRNA expression is also quantified by real-time RT-PCR in these cells. The study showed that Prx IV is overexpressed by five times in ARMS cells when compared to non-malignant myoblasts. Moreover, the inhibition of calpains using chemical inhibitors led to a decrease in Prx IV abundance (64.32 ± 8.25 and 76.79 ± 4.60 for the precursor and secretable forms, respectively, with calpain inhibitor III treatment). It is the first time that a Prx IV calpain-dependent up-regulation is revealed. In summary, calpains may be implied in the tumour phenotype of ARMS cells especially through Prx IV regulation and may, thus, represent a potential therapeutic target to stop progression of ARMS tumour.

International audience; Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern. Although sarcopenia is well documented, the molecular mechanisms of this condition still remain unclear. The calcium-dependent proteolytic system is composed of calcium-dependent cysteine proteases named calpains. Calpains are involved in a large number of physiological processes such as muscle growth and differentiation, and pathological conditions such as muscular dystrophies. The aim of this study was to determine the involvement of this proteolytic system in the phenotype associated with sarcopenia by identifying key proteins (substrates or regulators) interacting with calpains during muscle aging. Immunoprecipitations coupled with proteomic analyses and protein identification by mass spectrometry have been undertaken. Reverse co-immunoprecipitation, cellular colocalisation by confocal microscopy and calpain-dependent in vitro proteolysis of several of the identified proteins have been also carried out. We identified ATP synthase subunit alpha and alpha actinin 3 as key partners of calpains during muscle aging. Such interactions would suggest that calpains are implicated in many processes altered during aging including cytoskeletal disorganisation and mitochondrial dysfunction.

International audience; Rhabdomyosarcoma (RMS) are soft-tissue sarcoma commonly encountered in childhood. RMS cells can acquire invasive behavior and form metastases. The metastatic dissemination implicates many proteases among which are mu-calpain and m-calpain. Study of calpain expression and activity underline the deregulation of calpain activity in RMS. Analysis of kinetic characteristics of RMS cells, compared to human myoblasts LHCN-M2 cells, shows an important migration velocity in RMS cells. One of the major results of this study is the positive linear correlation between calpain activity and migration velocity presenting calpains as a marker of tumor aggressiveness. The RMS cytoskeleton is disorganized. Specifying the role of mu- and m-calpain using antisense oligonucleotides led to show that both calpains up-regulate alpha- and beta-actin in ARMS cells. Moreover, the invasive behavior of these cells is higher than that of LHCN-M2 cells. However, it is similar to that of non-treated LHCN-M2 cells, when calpains are inhibited. In summary, calpains may be involved in the anarchic adhesion, migration and invasion of RMS. The direct relationship between calpain activity and migration velocities or invasive behavior indicates that calpains could be considered as markers of tumor aggressiveness and as potential targets for limiting development of RMS tumor as well as their metastatic behavior.

International audience; Calpain 3 is a calcium-dependent cysteine protease that is primarily expressed in skeletal muscle and is implicated in limb girdle muscular dystrophy type 2A. To date, its best characterized function is located within the sarcomere, but this protease is found in other cellular compartments, which suggests that it exerts multiple roles. Here, we present evidence that calpain 3 is involved in the myogenic differentiation process. In the course of in vitro culture of myoblasts to fully differentiated myotubes, a population of quiescent undifferentiated "reserve cells" are maintained. These reserve cells are closely related to satellite cells responsible for adult muscle regeneration. In the present work, we observe that reserve cells express higher levels of endogenous Capn3 mRNA than proliferating myoblasts. We show that calpain 3 participates in the establishment of the pool of reserve cells by decreasing the transcriptional activity of the key myogenic regulator MyoD via proteolysis independently of the ubiquitin-proteasome degradation pathway. Our results identify calpain 3 as a potential new player in the muscular regeneration process by promoting renewal of the satellite cell compartment.

Dystrophin is a cytoskeletal protein which is thought to play an important role in membrane physiology since its absence (due to gene deficiency) leads to the symptoms of Duchenne muscular dystrophy (DMD). Some disruption in the regulation of intracellular free Ca 2+ levels could lead to DMD-like symptoms. In this study, calpains, which are very active calcium-dependent proteases, were examined for their capacity to hydrolyse dystrophin in vitro . The results show that calpains are able to split dystrophin and produce breakdown products of different sizes (the degree of cleavage being dependent on the incubation time with proteases). The time-course of protease degradation was examined by Western immunoblot using three polyclonal sera which were characterized as being specific to the central (residues 1173–1728) and two distal parts of the molecule ie specific to the N-terminal (residues 43– 760) or the C-terminal (residues 3357–3660) extremities of the dystrophin molecule. The cleavage patterns of dystrophin showed an accumulation of some major protease-resistant fragments of high relative molecular mass (250–370 kDa). These observations demonstrate that calpains digest dystrophin very rapidly when the calcium concentration is compatible with their activation. For instance, it is clear that calpains first give rise to large dystrophin products in which the C-terminal region is lacking. These observations suggest that dystrophin antibodies specific to the central domain of the molecule should be used to detect dystrophin for diagnostic purposes and before any conclusion as to the presence or absence of dystrophin can be deduced from results obtained using immunoanalyses of muscle biopsies.

TMAO-ase is an enzyme of great economic significance in the fish industry since, even below the freezing point, it can produce large amounts of formaldehyde. During frozen storage the resulting formaldehyde-protein interactions induce deleterious effects on the functional properties of frozen fish minces. Knowledge of the structural and enzyme properties is incomplete at present and produces certain ambiguities. A new procedure for extracting TMAO-ase from saithe kidney (Pollachius virens (L.)), using a non-denaturing detergent, has been developed. Subsequent anion exchange chromatography separated three independent enzyme fractions, differing mainly in their isoelectric points (5.0, 4.5 and 4.1). The three fractions revealed identical pH optima for activity (pH = 5.10), affinity constants (12 mM) and activation energies (4 kcal mol−1). However, differences were observed concerning some of their physicochemical properties such as T1/2 denaturation and spectrophotometric characteristics (unusual absorption at λ = 258 nm, related to the presence of DNA fragments). From a structural point of view, as evidenced by the elution profiles, TMAO-ase activity seems to be constituted of high MW protein groups (20 × 106 and 2 × 105) closely associated with mixed micelles of phospholipids.

International audience; The reduced regenerative potential of muscle fibres, most likely due to a decreased number and/or function of satellite cells, could play a significant role in the progression of muscle ageing. Accumulation of reactive oxygen species has been clearly correlated to sarcopenia and could contribute to the impairment of satellite cell function. In this work we have investigated the effect of oxidative stress generated by hydrogen peroxide in cultured human skeletal muscle satellite cells. We specifically focused on the activity and regulation of calpains. These calcium-dependent proteases are known to regulate many transduction pathways including apoptosis and play a critical role in satellite cell function. In our experimental conditions, which induce an increase in calcium concentration, protein oxidation and apoptotic cell death, a significant up-regulation of calpain expression and activity were observed and ATP synthase, a major component of the respiratory chain, was identified as a calpain target. Interestingly we were able to protect the cells from these H2O2-induced effects and prevent calpain up-regulation with a natural antioxidant extracted from pine bark (Oligopin®). These data strongly suggest that oxidative stress could impair satellite cell functionality via calpain-dependent pathways and that an antioxidant such as Oligopin® could prevent apoptosis and calpain activation.

Le facteur de croissance myostatine initialement identifié chez la souris, est un régulateur négatif de la masse musculaire. Des mutations naturelles ou expérimentales dans le gène codant ce facteur sont à l’origine d’un phénotype d’hypermuscularité, notamment chez les bovins culards. La myostatine régule la myogenèse et la balance atrophie/hypertrophie musculaire. Elle intervient dès la vie foetale en contrôlant la prolifération des cellules musculaires et donc le nombre total de fibres musculaires. Pendant la vie postnatale elle participe au contrôle de la taille des fibres musculaires en régulant l’activité des cellules satellites et la synthèse protéique. Elle est galement impliquée dans la fonte musculaire. Il semblerait qu’elle intervienne aussi dans le contrôle de l’adipogenèse et de l’ostéogenèse. Cette revue fait le point sur l’état des connaissances actuelles concernant l’expression et l’activité de ce facteur et leur régulation moléculaire. Ces données permettront à terme d’envisager une utilisation raisonnée de ces connaissances en agronomie pour la production de viande., In livestock the double-muscled phenotype is well known and has been selected in certain breeds, in order to increase carcass yield . Even though the chromosomal location of the gene responsible for the double-muscled phenotype in cattle has been known for a long time, the gene was identified more recently with studies in mice. In fact, mutations present in the growth factor myostatin gene cause the doublemuscled phenotype. Myostatin is a negative regulator of muscle mass and plays a key role in muscle homeostasis. Myostatin also appears to be involved in the control of adipogenesis and osteogenesis. This review provides an update on the current state of the knowledge of myostatin and on the rational use of such knowledge in agronomy for meat production.

Caveolae are specialised RAFTs (detergent-resistant membrane microdomains enriched in cholesterol and glycosphingolipids). Caveolin, the main caveolae protein, is essential to the organisation of proteins and lipids, and interacts with numerous mediating proteins through a 'Caveolin Scalfolding Domain'. Consequently, caveolae play a major role in signal transduction and appear to be veritable signalling platforms. In muscle cells, caveolae are essential for fusion and differentiation, and are also implicated in a type of muscular dystrophy (LGMD1C). In a preceding work, we demonstrated the presence of active milli-calpain (m-calpain) in myotube caveolae. Calpains are calcium-dependent proteases involved in several cellular processes, including myoblast fusion and migration, PKC-mediated intracellular signalling and remodelling of the cytoskeleton. For the first time, we have proved the cholesterol-dependent localisation of m-calpain in the caveolae of C 2 C 12 myotubes. Calpain-dependent caveolae involvement in myoblast fusion was also strongly suggested. Furthermore, eight differentially expressed caveolae associated proteins were identified by 2-DE and LC-MS/MS analyses using an m-calpain antisense strategy. This proteomic study also demonstrates the action of m-calpain on vimentin, desmin and vinculin in myotube caveolae and suggests m-calpain's role in several mitochondrial pathways.

International audience; The calcium-dependent proteolytic system is composed of cysteine proteases named calpains. They are ubiquitous or tissue-specific enzymes. The two best characterised isoforms are the ubiquitously expressed mu- and m-calpains. Besides its regulation by calcium, calpain activity is tightly controlled by calpastatin, the specific endogenous inhibitor, binding to phospholipids, autoproteolysis and phosphorylation. Calpains are responsible for limited proteolytic events. Among the multitude of substrates identified so far are cytoskeletal and membrane proteins, enzymes and transcription factors. Calpain activity is involved in a large number of physiological and pathological processes. In this review, we will particularly focus on the implication of the calcium-dependent proteolytic system in relation to muscle physiology. Because of their ability to remodel cytoskeletal anchorage complexes, calpains play a major role in the regulation of cell adhesion, migration and fusion, three key steps of myogenesis. Calcium-dependent proteolysis is also involved in the control of cell cycle. In muscle tissue, in particular, calpains intervene in the regeneration process. Another important class of calpain substrates belongs to apoptosis regulating factors. The proteases may thus play a role in muscle cell death, and as a consequence in muscle atrophy. The relationships between calcium-dependent proteolysis and muscle dysfunctions are being further developed in this review with a particular emphasis on sarcopenia.

Aging is associated with a progressive and involuntary loss of muscle mass also known as sarcopenia. This condition represents a major public health concern with high socio-economics implications. Although sarcopenia is well documented, the aetiology of this condition still remains poorly understood. Calpains are ubiquitous proteases regulated in part by a specific inhibitor, calpastatin. They are well known to have major implications in muscle growth and differentiation. The aim of the present study was to determine if this proteolytic system could be involved in the phenotype associated with sarcopenia. Calpains and calpastatin levels, subcellular distributions and activities were compared between muscles from 3 and 24 months old rats. Altogether, the results we obtained showed an overall increase in calpain activities associated with muscle aging. These findings suggest that the calcium-dependent proteolytic system is indeed involved in sarcopenia.

International audience; Recent research carried out in our laboratory has shown that IGF-1, TGF-beta1, and insulin were able to strongly stimulate myoblast migration by increasing milli-calpain expression and activity. However, the signalling pathways involved in these phenomena remain unknown. The aim of this study was to identify the signalling pathway(s) responsible for the effects of IGF-1, TGF-beta1, and insulin on myoblast migration and on milli-calpain expression and activity. For this purpose, wound healing assays were carried out in the presence of growth factors with or without specific inhibitors of ERK/MAP kinase and PI3K/Akt pathways. The results clearly showed that the inhibition of the ERK/MAP kinase pathway prevents the effects of growth factors on myoblast migration. Secondly, the expression and the activity of milli-calpain were studied in cells treated with growth factor, alone or with ERK/MAP kinase inhibitor. The results demonstrated that the up-regulation of milli-calpain expression and activity was mediated by the ERK/MAP kinase pathway. Finally, the possible implication of MyoD and myogenin, myogenic regulatory factors able to regulate milli-calpain expression, was studied. Taken together our results clearly showed that the ERK/MAP kinase signalling pathway is responsible for the effects of the three growth factors on myoblast migration and on milli-calpain expression and activity. On the opposite, the PI3K/Akt signalling pathway, MyoD and myogenin seem to be not implicated in these phenomena.

The calcium-dependent proteolytic system is a large family of well-conserved ubiquitous and tissue-specific proteases, known as calpains, and an endogenous inhibitor, calpastatin. Ubiquitous calpains are involved in many physiological phenomena, such as the cell cycle, muscle cell differentiation, and cell migration. This study investigates the regulation of crucial steps of cell motility, myoblast adhesion and spreading, by calpains. Inhibition of each ubiquitous calpain isoform by antisense strategy pinpointed the involvement of each of these proteases in myoblast adhesion and spreading. Moreover, the actin cytoskeleton and microtubules were observed in transfected cells, demonstrating that each ubiquitous calpain could be involved in the actin fiber organization. C2C12 cells with reduced μ- or m-calpain levels have a rounded morphology and disorganized stress fibers, but no modification in the microtubule cytoskeleton. Antisense strategy directed against MARCKS, a calpain substrate during C2C12 migration, showed that this protein could play a role in stress fiber polymerization. A complementary proteomic analysis using C2C12 cells over-expressing calpastatin indicated that two proteins were under-expressed, while six, which are involved in the studied phenomena, were overexpressed after calpain inhibition. The possible role of these proteins in adhesion, spreading, and migration was discussed. Cell Motil. Cytoskeleton 63: 2006. © 2006 Wiley-Liss, Inc.

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.

MARCKS (myristoylated alanine-rich C kinase substrate) is a major cytoskeletal protein substrate of PKC (protein kinase C) whose cellular functions are still unclear. However numerous studies have implicated MARCKS in the stabilization of cytoskeletal structures during cell differentiation. The present study was performed to investigate the potential role of Ca2+-dependent proteinases (calpains) during myogenesis via proteolysis of MARCKS. It was first demonstrated that MARCKS is a calpain substrate in vitro. Then, the subcellular expression of MARCKS was examined during the myogenesis process. Under such conditions, there was a significant decrease in MARCKS expression associated with the appearance of a 55 kDa proteolytic fragment at the time of intense fusion. The addition of calpastatin peptide, a specific calpain inhibitor, induced a significant decrease in the appearance of this fragment. Interestingly, MARCKS proteolysis was dependent of its phosphorylation by the conventional PKCα. Finally, ectopic expression of MARCKS significantly decreased the myoblast fusion process, while reduced expression of the protein with antisense oligonucleotides increased the fusion. Altogether, these data demonstrate that MARCKS proteolysis is necessary for the fusion of myoblasts and that cleavage of the protein by calpains is involved in this regulation.

Calpains, also called calcium activated neutral cysteine proteases are presently known to play pivotal roles in physiological and biological phenomena such as signal transduction, cell spreading and motility, apoptosis, regulation of cell cycle and regulation of muscle cell differentiation. Concerning this last point, calpains have been shown to play a crucial role during the earlier myogenesis. In this study we have analyzed the involvement of calpains during an important step of myogenesis: myoblast migration. Our findings show that myoblast migration was drastically reduced when the expression of micro- and m-calpain was decreased. We have also observed that MARCKS (myristoylated alanine rich C kinase substrate), a protein localized at focal adhesion sites, was significantly accumulated when the expression levels of calpains were decreased. Also, using phorbol myristate acetate, (an activator of PKC) and plasmids carrying the full-length cDNA of MARCKS or a cDNA fragment lacking the phosphorylation site domain, we demonstrated that normal myoblast migration is dependent on MARCKS phosphorylation and localization.

Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.

The calcium-activated cysteine protease m-calpain plays a pivotal role during the earlier stages of myogenesis, particularly during fusion. The enzyme is a heterodimer, encoded by the genes capn2, for the large subunit, and capn4, for the small subunit. To study the regulation of m-calpain, the DNA sequence upstream of capn2 was analyzed for promoter elements, revealing the existence of five consensus-binding sites (E-box) for several myogenic regulatory factors and one binding site for myocyte enhancer factor-2 (MEF-2). Transient transfections with reporter gene constructs containing the E-box revealed that MyoD presents a high level of transactivation of reporter constructs containing this region, in particular the sequences including the MEF-2/E4-box. In addition, over-expression of various myogenic factors demonstrated that MyoD and myogenin with much less efficiency, can up-regulate capn2, both singly and synergistically, while Myf5 has no effect on synthesis of the protease. Experiments with antisense oligonucleotides directed against each myogenic factor revealed that MyoD plays a specific and pivotal role during capn2 regulation, and cannot be replaced wholly by myogenin and Myf5.

We investigated the status and the regulation of the cyclin-dependent kinases (CDK) inhibitor p27(Kip1) in a choroidal melanoma tumor-derived cell line (OCM-1). By contrast to normal choroidal melanocytes, the expression level of p27(Kip1) was low in these cells and the mitogen-activated protein (MAP) kinase pathway was constitutively activated. Genetic or chemical inhibition of this pathway induced p27(Kip1) accumulation, whereas MAP kinase reactivation triggered a down-regulation of p27(Kip1) that could be partially reversed by calpain inhibitors. In good accordance, ectopic expression of the cellular calpain inhibitor calpastatin led to an increase of endogenous p27(Kip1) expression. In vitro, p27(Kip1) was degraded by calpains, and OCM-1 cell extracts contained a calcium-dependent p27(Kip1) degradation activity. MAP kinase inhibition partially inhibited both calpain activity and calcium-dependent p27(Kip1) degradation by cellular extracts. Immunofluorescence labeling and subcellular fractionation revealed that p27(Kip1) was in part localized in the cytoplasmic compartment of OCM-1 cells but not of melanocytes, and accumulated into the nucleus upon MAP kinase inhibition. MAP kinase activation triggered a cytoplasmic translocation of the protein, as well as a change in its phosphorylation status. This CRM-1-dependent cytoplasmic translocation was necessary for MAP kinase- and calpain-dependent degradation. Taken together, these data suggest that in tumor-derived cells, p27(Kip1) could be degraded by calpains through a MAP kinase-dependent process, and that abnormal cytoplasmic localization of the protein, probably linked to modifications of its phosphorylation state, could be involved in this alternative mechanism of degradation.

p94 belongs to the calpain family of enzymes, also called calcium-activated neutral proteases and is mainly expressed in the skeletal muscle. Mutations affecting the gene coding for p94 are responsible for a myopathy syndrome called Limb Girdle Muscular Dystrophy type 2A (LGMD2A). Although the activity of p94 seems necessary for muscle function, the biological role of the enzyme is still unknown. The goal of this study was to develop a muscle cell line in which the expression level of p94 can be regulated, by an inducible way. In this study, a biological system was developed which allowed mimicking, in vitro, of part of the events occurring in patients (i.e. a decrease of p94 activity). The first results indicate that the decrease in p94 activity results in a significant increase of myogenin level, a high specific transcription factor involved in myoblast fusion. This muscle specific inducible system is an interesting biological tool to assess specifically p94 function(s) in cultured muscle cells. According to the present results, p94 seems at least to be involved in a myogenesis regulation pathway via its action on certain proteins belonging to the myogenic regulator factor family.

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C2C12) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.

We recently reported that when myoblasts fuse, m-calpain could be exteriorized. Indeed, at present a number of works support this hypothesis because this enzyme was localized intercellularly and more particularly associated to extracellular matrix components. Knowing that the cell surface of the fusing myoblast is supposed to undergo many changes, we addressed the question whether m-calpain could be involved in the phenomenon of fusion via fibronectin cleavage or degradation. Using different digestion experiments, we demonstrated that soluble purified fibronectin and highly insoluble fibronectin fibrils represent very good substrates for this proteinase; moreover, at the burst of fusion, fibronectin proteolytic fragments could be identified. On the other hand, we have conducted biological assays on cultured myoblasts using a defined medium supplemented by exogenous factors capable of stimulating or inhibiting m-calpain activity. The effects of such factors on rat myoblast fusion and concomitantly on the targeted glycoprotein were analyzed and quantified. When m-calpain activity and the phenomenon of fusion were reduced (defined medium without insulin), the amount of the 220-kDa fibronectin band was increased by 43%. When m-calpain activity and myoblast fusion were prevented by addition of antibodies to m-calpain or calpain inhibitor II, the fibronectin concentration was higher since it was increased by approximately 67 and approximately 71%, respectively. In addition, when observed at the ultrastructural level, m-calpain seems to be localized at the potential fusion site of myoblasts and more particularly associated to the extracellular matrix when muscle cells were initially treated by anti-m-calpain IgG. Taken together, these results support the hypothesis that exteriorized m-calpain could be, in part, involved in myoblast fusion via fibronectin alteration or degradation.

Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B+L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.

Summary It has already been reported that, in vitro , intermediate filaments such as desmin and vimentin are very susceptible to proteolysis by calpains (Ca 2+ -activated cysteine proteinases). On the other hand, desmin and m-calpain are both present at the onset of myoblast fusion and throughout this phenomenon. Based on these observations, the aim of this study was to demonstrate, with cultured rat myoblasts, that the amount of desmin decreased significantly as multinucleated myotubes were formed. Using immunoblot analysis, it has been shown that the desmin concentration decreased 41% as myoblasts fuse. Moreover, under conditions which stimulate myoblast fusion, desmin concentration was reduced by 21% compared to the control culture. Under our experimental conditions, which lead to a reduced desmin level, the amount of m-calpain was increased about three-fold. These results suggested that m-calpain could be involved in myoblast fusion via desmin cleavage. This hypothesis was confirmed by the results obtained after calpeptin treatment. In the presence of this cell-penetrating inhibitor of calpains, desmin seems not to be degraded. Taking into account the observations obtained after different hydrolysis assays and as compared to those observed on cultured cells, it seems conceivable that m-calpain would be able to initiate desmin cleavage leading to the formation of proteolytic fragments which should be immediately degraded.

The chronology of appearance of calpain I and calpain II was analyzed during myogenesis of embryonic myoblasts in culture. The influence of the hormones insulin and corticosterone, and insulin growth factor-1 (IGF-1) and transforming growth factor-beta (TGF-beta) on the modulation of calpain-calpastatin levels during myogenesis was also analyzed. Immunodetection assays using specific antibodies and enzymic activities showed that during muscle cell differentiation in vitro, calpain II is present from the beginning of myoblast fusion (2nd day) increasing until the 6th day and then reaching a plateau. These observations were confirmed by an analysis of the expression of total calpain mRNAs which followed the same time profile, thereby providing evidence for a transcriptional regulation in the expression of calpains. Even if an increase in calpain II activity occurs at approximately the same time as an increase of fusion, calpain II activity and rate of fusion are not closely correlated. The involvement of calpain II in some event that follows myoblast fusion is suggested. On the other hand, calpain I and calpastatin were detected only on the 6th day of cell culture growth; these results enable us to argue that if calpain I has any biological role (which remains to be established), this role occurs during the final stages of muscle cell differentiation. The presence of exogenous factors which are known to affect muscle cell differentiation by altering either the rate of protein synthesis, or degradation or both, significantly affects the modulation of calpain-calpastatin levels. Such a regulation at the transcriptional level suggests that calpains do not act as housekeeping enzymes during myogenesis.

Recent work supports the hypothesis that calpain II can be exteriorized. Indeed, this cysteine calcium-dependent proteinase was shown to be intercellularly, and, more particularly, associated to extracellular matrix components. Thereby, calpain II could be involved in hydrolysis of pericellular matrix components such as fibronectin, which is known to play an important role in cellular differentiation. Our in vitro studies provide evidence that fibronectin is a potential substrate for calpain II. On cultured cells, our findings show that calpain II is able, on the one hand, to cleave the fibrillar network of fibronectin secreted by fibroblasts, and, on the other, to decrease dramatically the fibronectin amount secreted by myoblasts just before fusion. Moreover, following this treatment, myoblasts become spherical due to the cleavage of this attachment factor. However, these cells, plated on an appropriate substrate are still able to differentiate. Our results suggest that calpain II is indeed involved in myoblast fusion via the fibronectin cleavage since it is well established that myogenic lineages lose this glycoprotein at the time of fusion.

En tant que reseau departemental charge de mieux articuler entre elles les propositions faites aux jeunes, la Maison des adolescents est un lieu d’observation legitime de la sante individuelle et communautaire. Le travail en reseau aupres des professionnels et des jeunes des differents territoires d’un departement, les rapports des uns avec les autres, les enjeux qui s’y font jour questionnent tout particulierement les criteres de la cooperation et son corollaire, la coordination. A partir de la MDA44, nous discutons la notion de « clinique du partenariat » visant a prendre soin de l’ensemble des protagonistes d’une aventure interdisciplinaire et interinstitutionnelle.