[The distally based sural flap at the ankle and foot: complications analysis in a 27 cases study]. / Le lambeau sural à pédicule distal à la cheville et au pied : analyse des complications à propos d'une série de 27 lambeaux.

The distally based sural flap is widely used in foot and ankle skin and soft tissue repairs. It is described as an easy and reliable procedure. But in our experience, the flap necrosis was observed rather frequently. The analysis of this complication was the main goal of this retrospective study. The distally based sural flap has been used 27 times for skin repair at the foot and ankle. Twenty-six cases were post-traumatic. The success rate was 70%. Eight (mostly partial) necrosis occurred, one total necrosis lead to mid-leg amputation. In post-trauma reconstructions, this flap was not found so reliable. The rate of flap necrosis increases with age and comorbidities. No relationship could be established between the necrosis of the flap and its width. Heel and lateral localizations were found more risky. Technical modifications are discussed with a special focus on the two staged procedure.

Calcium-dependent proteolytic system and muscle dysfunctions: a possible role of calpains in sarcopenia.

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.

Iatrogenic peritonitis following an incident during ablation of a pedicle screw.

Immediate complications can arise due to faulty implantation of material during fusion procedures, but none have been reported in connection with ablation of material in the spine. We report a case of intraperitoneal migration of a pedicle screw during attempted removal. It crossed the psoas muscle and perforated a small-intestine loop, triggering hemorrhagic shock and peritonitis by perforation. We analyze the causes and mechanisms underlying this exceptional case of migration, with a view to sharing preventive measures. Initial extra-pedicular screw positioning and the pressure exerted to remove it were responsible for this serious incident.

Free calcium and calpain I activity.

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor.

Two muscle thiol proteinases causing partial degradation of myofibrillar constituents were isolated and purified from skeletal muscle. The two proteinases that differ significantly in calcium requirements were designated respectively high- and low-Ca2+-requiring proteinase. Both are inhibited, in vitro, by a specific inhibitor which is a protein also isolated from skeletal muscle. Experiments using carboxymethylated monomeric proteinases and inhibitor-conjugated Sepharose were carried out in order to understand the mechanism of control of the proteinases by the inhibitor. The results using increasing inhibitor concentrations show a non-competitive inhibition for both enzymes. The Ki value for the low-Ca2+-requiring form was 0.3 microM, while the Ki value for the high-Ca2+-requiring form was 0.9 microM. Likewise, the low-Ca2+-requiring form needs about 3-fold more inhibitor than the high-Ca2+-requiring form for the same per cent inhibition.

Hydrostatic pressure and calcium-induced dissociation of calpains.

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.

Ca(2+)-dependent proteinases (calpains) and muscle cell differentiation.

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.

Degradation of protein kinase Malpha by mu-calpain in a mu-calpain-protein kinase Calpha complex.

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

M-calpain levels increase during fusion of myoblasts in the mutant muscular dysgenesis (mdg) mouse.

Previous studies have led to the hypothesis of a possible role for the calcium-dependent neutral protease m-calpain in myoblast fusion in culture. To evaluate this hypothesis, we chose as our model, the "muscular dysgenesis" mouse (mdg), which presents in vivo and in vitro characteristics of an elevated process of fusion (Yao and Essien, 1975; Dussartre, 1993; Ashby et al., 1993, Joffroy et al., 1999). The aim of this study was to demonstrate using myoblast cell lines and muscle biopsies from this mdg mutant, that the amount of m-calpain increases significantly as multinucleated myotubes are formed. Using immunoblot analysis, it was shown that the m-calpain concentration in a dysgenic cell line (GLT) increased 3-fold compared to what it was upon the introduction of the differentiation medium. On the other hand, in a normal cell line (NLT), the concentration of m-calpain did not vary significantly. Thus, when the transition from myoblasts to myotubes was slow, and the absolute level of fusion was reduced, as in the NLT cell line, the level of m-calpain was stable. In contrast, when the process of fusion was precocious and fast, and the level of fusion was elevated, such as in the GLT cell line, the concentration of m-calpain increased during fusion. Moreover, when myoblast fusion was prevented by the addition of calpain inhibitor II, the process was reduced by approximately 93%. Taking into account these observations, it is clear from our data that the muscular dysgenesis mouse provides a relevant model to study myoblast fusion and that m-calpain is involved in this process.

Management of upper cervical spine fractures in elderly patients: current trends and outcomes.

Upper cervical spine fractures in the elderly represent serious injuries. Their frequency is on the rise. Their early accurate diagnosis might be compromised by the existence of extensive degenerative changes and deformities. Adequate stabilisation allowing fracture healing is of paramount importance. However, the debate is ongoing as to the best protocol that can be applied taking into consideration the presence of comorbidities and the increase risk of mortality in this frail patient population. A literature review, based on PubMed, related to protocols reporting on fracture fixation of the upper cervical spine, fractures (C1-C2) was carried out. Papers including information about type of fracture, treatment carried out, complication rates, mortality and morbidities were eligible to be included in this study. Fourteen papers met the inclusion criteria. Six reported on all types of injuries of the upper cervical spine, and eight only odontoid fractures (C2). Overall mortality rate ranged between 0 to 31.4%. Overall morbidity rate was from 10.3 to 90.9%. No significant difference was identified between three types of treatment (rigid collar cuff without fracture reduction, halo cast with reduction of fracture displacement, and surgical treatment). Halo-cast got the highest rate of complications. Surgical treatment got a mortality rate from 0 to 40.0%, and a morbidity rate from 10.3 to 62.5%. Non-union rate ranged between 8.9 to 62.5%. Elderly patients with upper cervical spine fractures must be notified that these injuries are associated with high incidence of non-union, morbidity and mortality.

Calpastatin-modulation of m-calpain activity is required for myoblast fusion.

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.

Rat myoblast fusion requires exteriorized m-calpain activity.

Our previous studies demonstrated that fibronectin could be proteolyzed by m-calpain during muscle cell differentiation. Recent results indicated also that m-calpain could be exteriorized and more particularly associated to extracellular matrix components. To clarify one of the possible physiological functions of this proteinase during myogenesis, we have analyzed the incidence of added purified m-calpain and calpain inhibitors on the fusion kinetics of cultured myoblasts. Our results provided evidence that at low concentration (0.01 microgram/ml), added m-calpain induces precocious fusion and increases myoblast fusion by 78%. At high concentrations (10 micrograms/ml), the viability of the cells was not affected but the myoblasts were unable to fuse. Leupeptin and calpastatin--potent m-calpain inhibitors--added to the culture medium reduced myoblast fusion by 70%. On the other hand, the addition of monospecific m-calpain polyclonal antibodies to the culture medium induced a 76% decrease of myoblast fusion. In order to trap exteriorized m-calpain, myoblasts were incubated for 24 h with m-calpain antibodies. Following this treatment, nonpermeabilized myoblasts exposed to labeled secondary antibodies showed fluorescent spots scattered at the cell surface. These results strongly support that m-calpain which was involved in myoblast fusion was exteriorized and suggest therefore that this enzyme may play an important role extracellularly.

Evidence for a MARCKS-PKCalpha complex in skeletal muscle.

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.

Evidence for a Ca2+-independent association between calpain II and phospholipid vesicles.

Possible interactions between calpain II and phospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylcholine were studied using fluorescence and gel filtration techniques. Changes in fluorescence intensity of purified calpain II show that the enzyme strongly interacts with phosphatidylinositol and phosphatidylserine and to a lesser extent with phosphatidylcholine. These results are corroborated by the gel filtration technique which permits the isolation of the enzyme phospholipid complex. Association between calpain II and various phospholipid vesicles can occur in the absence of calcium. Such binding occurs without any observable change of the molecular mass of the two subunits on SDS-polyacrylamide gel electrophoresis.

Evidence for fibronectin degradation by calpain II.

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.

Quantitative measurement of calpain I and II mRNAs in differentiating rat muscle cells using a competitive polymerase chain reaction method.

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-beta, 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.

In vitro digestion of dystrophin by calcium-dependent proteases, calpains I and II.

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 Ca2+ 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.

[Neutral calcium dependent proteinase from rabbit skeletal muscle: activity on myofibrillar proteins]. / Protéinase neutre calcium dépendante de muscle squelettique de lapin : activité sur les protéines myofibrillaires.

Experiments have been carried out to explore the proteolytic cleavage of rabbit skeletal myofibrils by a calcium dependent neutral proteinase (CaANP). Polyacrylamide gel elctrophoresis on great slabs showed the ability of CaANP to degrade myofibrils more readily than supposed. Besides the hydrolysis of troponin T and the apparition of degradation product of 30,000 molecular weight, the activity of this enzyme is obvious too on some components of the M-line and on heavy subunits of tropomyosin as well as on three unidentified proteic fractions. The variety of the degradation products which appear suggest that the specificity of CaANP is not as selective as presumed. The participation of this proteinase in the postmorten evolution of muscle and its intervention in the turnover of myofibrillar proteins is discussed.

[66% glucose, a safe sclerosant. Experimental study]. / Le glucose à 66%, sclérosant de sécurité. Etude expérimentale.

Although accidents due to the intra-arterial injection of detergent sclerosant are very rarely observed, they are dramatic in their effects and often result in amputations, a risk accepted with difficulty for a treatment with a functional aim. To avoid these incidents, which may occur even when treatment is applied by the most experienced surgeons, the authors have used 66% glucose solution without accident since 1948. To confirm efficacy of the method, an experimental study compared 66% glucose (66 G) with a very commonly used product, 1% sodium tetradecyl sulfate (STD), in the rabbit. Except when enormous doses of 66 G are employed, the only effect noted was eosinophilic necrosis of the vessel wall without clinical symptoms, whereas doses eight times lower of STD produced an irreversible ischemia from obliterating endarteritis of the branches of the vascular tree injected. The 66% glucose solution appears to be a very safe, gently acting sclerosant, and the product of choice for peri- and post-operative sclerosis, particularly in regions where accidental arterial puncture is anatomically possible.

[Giant cell chondroma of soft tissues. Apropos of a case and review of the literature]. / Chondrome à cellules géantes des tissus mous. A propos d'un cas avec revue de la littérature.

Soft tissue chondroma is a benign cartilaginous tumour most often located at the extremity of the upper limbs. We report a very unusual case of soft tissue chondroma due to its location in the sole of the foot resulting in delayed diagnosis. Diagnosis of soft tissue chondroma was confirmed by microscopic examination and by immunohistochemistry showing S 100 protein in the cells of mature and immature areas. The presence of numerous giant cells is exceptional. Labelling of the giant cells by KP 1 protein in the present case suggest that they are derived from macrophages.