Orwik

Consensus statement on standard of care for congenital muscular dystrophies.

Congenital muscular dystrophies are a group of rare neuromuscular disorders with a wide spectrum of clinical phenotypes. Recent advances in understanding the molecular pathogenesis of congenital muscular dystrophy have enabled better diagnosis. Howev... expand abstracter, medical care for patients with congenital muscular dystrophy remains very diverse. Advances in many areas of medical technology have not been adopted in clinical practice. The International Standard of Care Committee for Congenital Muscular Dystrophy was established to identify current care issues, review literature for evidence-based practice, and achieve consensus on care recommendations in 7 areas: diagnosis, neurology, pulmonology, orthopedics/rehabilitation, gastroenterology/ nutrition/speech/oral care, cardiology, and palliative care. To achieve consensus on the care recommendations, 2 separate online surveys were conducted to poll opinions from experts in the field and from congenital muscular dystrophy families. The final consensus was achieved in a 3-day workshop conducted in Brussels, Belgium, in November 2009. This consensus statement describes the care recommendations from this committee. collapse abstract

Best practice guidelines on molecular diagnostics in DuchenneBecker muscular dystrophies.

Severe phenotype of a patient with autosomal recessive centronuclear myopathy due to a BIN1 mutation.

Centronuclear myopathy (CNM) is a rare hereditary congenital myopathy characterized by muscular hypotonia and abnormal centralization of nuclei in muscle fibers. The autosomal recessive (AR) form presents from birth to childhood, followed by a mild p... expand abstractrogression of muscle weakness. Despite recently identified genetic loci in the AR form, genotype-phenotype correlations are poorly established. Our index case is a 17 year old boy with recessive CNM causing loss of ambulation at 13 years of age and requiring ventilatory assistance nightly. Recent genetic testing revealed a c.1723A > T mutation in the BIN1 gene. The phenotype of the index case contrasts to previously published cases, where recessive CNM patients have lost ambulation in their 20s and have not required ventilatory assistance. The disease severity of our index case, carrying a c.1723A > T mutation, widens the phenotypic spectrum of AR CNM to include earlier loss of ambulation and respiratory failure. collapse abstract

Mice expressing L345P mutant desmin exhibit morphological and functional changes of skeletal and cardiac mitochondria.

Desmin mutations underlie inherited myopathies/cardiomyopathies with varying severity and involvement of the skeletal and cardiac muscles. We developed a transgenic mouse model expressing low level of the L345P desmin mutation (DESMUT mice) in order ... expand abstractto uncover changes in skeletal and cardiac muscles caused by this mutation. The most striking ultrastructural changes in muscle from DESMUT mice were mitochondrial swelling and vacuolization. The mitochondrial Ca(2+) level was significantly increased in skeletal and cardiac myocytes from DESMUT mice compared to wild type cells during and after contractions. In isolated DESMUT soleus muscles, contractile function and recovery from fatigue were impaired. A SHIRPA screening test for neuromuscular performance demonstrated decreased motor function in DESMUT compared to WT mice. Echocardiographic changes in DESMUT mice included left ventricular wall hypertrophy and a decreased left ventricular chamber dimension. The results imply that low levels of L345P desmin acts, at least partially, by a dominant negative effect on mitochondria. collapse abstract

Deletion in TNNI3 gene is associated with restrictive cardiomyopathy.

In dilated and hypertrophic cardiomyopathies, over ten disease-causing genes have been identified in each entity. In contrast, mutations in only desmin and cardiac troponin T and I (TNNI3) have been shown to cause restrictive cardiomyopathy (RCM). We... expand abstract applied a candidate gene approach and identified a novel one nucleotide deletion, resulting in frame shift and predicted formation of a premature stop codon, deletion of part of exon 7 and all exon 8, and truncation of significant C-terminal portion of TNNI3. Western blot analysis showed approximately 50% reduction of total troponin I content in myocardial tissue. The clinical hallmark was a restrictive type of cardiac hemodynamics, and congestive heart failure, leading to the death of the patient at the age of 28. collapse abstract

Sustained favorable effects of cognitive training in children with acquired brain injuries.

The overall aim of the present study was to assess in greater detail the sustained effects of a broad-based cognitive training programme on the neuropsychological performance of children with acquired brain injury. In particular, the long term (6 mon... expand abstractths) effects on cognitive functions, as well as how various moderators (gender, age at the time of injury/diagnosis, time since injury/diagnosis, age at the training) might influence outcome were investigated. A group of 38 children, 9-16 years of age, with various types of acquired brain injury had earlier been randomly assigned into treatment and control groups. These two groups had first been assessed directly after completion of the training and were now reassessed 6 months later. The treatment group exhibited significantly more persistent improvements with respect to complex tasks of attention and memory in comparison to the control group. In contrast there were no differences on simple reaction time tests. We conclude that the long term effects on cognitive functions of this broad-based neuro-cognitive training is encouraging. These positive results should be further investigated in larger more specific diagnostic groups and in different settings. collapse abstract

Long-term follow-up of children with obstetric brachial plexus palsy I: functional aspects.

The aims of this study were to describe the development of sequelae in obstetric brachial plexus palsy (OBPP) and to identify possible differences in functional outcome from 5 years of age to follow-up, 2 to 15 years later. A cohort of 70 participant... expand abstracts (35 males, 35 females; age range 7-20y, mean 13y 6mo [SD 4y 3mo], median 13y) with OBPP of varying degrees of severity were monitored. Differences in status between 5 years of age and follow-up were studied. Active joint motion in the shoulder and hand function, especially grip strength, generally remained unchanged or improved, whereas a slight but significant deterioration occurred in elbow function. Shoulder surgery resulted in considerable improvement of shoulder function. Participants with nerve reconstruction had a similar profile of change as the non-operated group. It was concluded that ongoing follow-up of children with OBPP, beyond the preschool years, is required due to decreases in elbow function, a commonly occurring restriction in external rotation of the shoulder, together with individual variations in long-term outcomes. In a related article (part II: neurophysiological aspects) long-term neurophysiological and sensory aspects of OBPP are reported. collapse abstract

Long-term follow-up of children with obstetric brachial plexus palsy II: neurophysiological aspects.

The aim of this study was to examine long-term neurophysiological outcomes and sensory function in patients with obstetric brachial plexus palsy (OBPP). The same 70 children/adolescents examined in part I: functional aspects (35 males, 35 females; ag... expand abstracte range 7-20y, mean 13y 6mo [SD 4y 3mo], median 13y) were examined with neurophysiological methods at 3 to 7 months and at 7 to 20 years of age. Thirteen of the 70 participants underwent nerve reconstruction before 1 year of age. Electromyography (EMG) was performed on deltoid and first interossei muscles; Quantitative Sensory Test was used for C6 and C8 dermatomes. Tests for functional sensibility and 2-point discrimination for C6 and C8 were performed. This study shows that considerable EMG changes can be observed in OBBP, even in those fully recovered. EMG changes in the deltoid were shown to deteriorate over time, and sensibility is considerably less affected than motor function. collapse abstract

Desmin mutations in a St. Petersburg cohort of cardiomyopathies.

Several desmin mutations have been described over the past few years in patients with dilated and restrictive cardiomyopathy, often in association with distal myopathy. However, the role of desmin mutations as a cause of various types of cardiomyopat... expand abstracthy is still undetermined. The aim of this study was to analyse the frequency of desmin mutations in patients with cardiomyopathy identified and diagnosed in the St. Petersburg area of Russia. We screened 98 patients with dilated, 40 with hypertrophic and 4 with restrictive cardiomyopathy. All exons of the desmin gene were amplified by PCR and studied by sequencing. Two out of 98 patients showed the presence of desmin gene mutations, not previously described in dilated cardiomyopathy. A novel IVS2-2A-->G splice site mutation, presumably causing skipping of exon 3, was detected in a case of familial right ventricular dilated cardiomyopathy. An A213V mutation was associated with a case of late onset dilated cardiomyopathy. No desmin mutations were found in patients with hypertrophic or restrictive cardiomyopathy. Desmin mutations should be considered a relatively rare cause of dilated cardiomyopathy in this specific geographic area. collapse abstract

Variable pathogenic potentials of mutations located in the desmin alpha-helical domain.

Mutations in the desmin gene have been recognized as a cause of desminopathy, a familial or sporadic disorder characterized by skeletal muscle weakness, often associated with cardiomyopathy or respiratory insufficiency. Distinctive histopathologic fe... expand abstractatures include aberrant intracytoplasmic accumulation of desmin (DES). We present here comparative phenotypic, molecular, and functional characteristics of four novel and three previously reported, but not fully characterized, desmin mutations localized in desmin alpha-helical domain. The results indicate that the c.638C>T (p.A213V), c.1178A>T (p.N393I), and to some extent the c.1078G>C (p.A360P) mutations exhibit pathogenic potentials only if combined with other mutations in desmin or other genes and should therefore be considered conditionally pathogenic. The c.1009G>C (p.A337P), c.1013T>G (p.L338R), c.1195G>T (p.D399Y), and c.1201G>A (p.E401K) mutations make desmin filaments dysfunctional and are capable of causing disease. The pathogenic potentials of desmin mutations correlate with the type and location of the disease-associated mutations in the relatively large and structurally and functionally complex desmin molecule. Mutations within the highly conserved alpha-helical structures are especially damaging since the integrity of the alpha-helix is critical for desmin filament assembly and stability. collapse abstract

Forced expression of desmin and desmin mutants in cultured cells: impact of myopathic missense mutations in the central coiled-coil domain on network formation.

We recently demonstrated that inherited disease-causing mutations clustered in the alpha-helical coiled-coil "rod" domain of the muscle-specific intermediate filament (IF) protein desmin display a wide range of inhibitory effects on regular in vitro ... expand abstractassembly. In these studies, we showed that individual mutations exhibited phenotypes that were not, with respect to the severity of interference, predictable by our current knowledge of the structural design of IF proteins. Moreover, the behavior of some mutated proteins in a standard tissue culture cell expression system was found to be even more complex. Here, we systematically investigate the behavior of these disease mutants in four different cell types: three not containing desmin or the related IF protein vimentin and the standard fibroblast line 3T3, which has an extensive vimentin system. The ability of the mutants to form filaments in the vimentin-free cells varies considerably, and only the mutants forming IFs in vitro generate extended filamentous networks. Furthermore, these latter mutants integrate into the 3T3 vimentin network but all the others do not. Instead, they cause the endogenous network of 3T3 vimentin to reorganize into perinuclear bundles. In addition, most of these assembly-deficient mutant desmins completely segregate from the vimentin system. Instead, the small round to fibrillar particles formed distribute independently throughout the cytoplasm as well as between the collapsed vimentin filament arrays in the perinuclear area. collapse abstract

Beneficial effect from a cognitive training programme on children with acquired brain injuries demonstrated in a controlled study.

PRIMARY OBJECTIVE: To test the effectiveness of a cognitive training programme in children and adolescents with attention and memory deficits after acquired brain injury (ABI). RESEARCH DESIGN: Randomized controlled study. PARTICIPANTS: Thirty-eight ... expand abstractchildren with ABI, 9-16 years of age. METHODS AND PROCEDURES: The treatment group trained with the cognitive programme for 30 minutes per day interactively with a teacher or parent for a period of 17 weeks. Children in the control group had a freely chosen interactive activity 30 minutes daily for 17 weeks. Pre- and post-training assessments were made using a neuropsychological test battery. MAIN OUTCOME AND RESULTS: Significant improvements in the majority of neuropsychological tests of sustained and selective attention as well as in memory performance were shown in the treatment group as compared to controls. CONCLUSIONS: The immediate effect of the training programme improved complex attention and memory functions, indicating that this method may be a valuable treatment option for improving cognitive efficiency in children after ABI. On the basis of these results, the next step will be to evaluate long-term effects and further ecological validity. collapse abstract

Hereditary cardiomyopathies: a review. Mutation of structural proteins a common cause of hereditary cardiomyopathy

Cardiomyopathy is a disorder of the cardiac muscle and can be either primary or secondary. The primary disorders have been classified by WHO into 4 groups based on structure and function; hypertrophic, dilated and restricted cardiomyopathies and arry... expand abstractthmogenic right ventricle dysplasia. During the last decade the familial nature of many of these cardiomyopathies has been elucidated and different genes have been found to be mutated and causative of disease. Certain patterns can be distinguished in the mutated genes, e.g. in general the genes causing hypertrophic cardiomyopathies code for proteins involved in the contractile apparatus, the sarcomere, and the genes causing dilated cardiomyopathy code for proteins that anchor the sarcomere to the cell membrane and extracellular matrix. This article reviews these recent genetic findings and discusses their potential clinical applicability. collapse abstract

Attention and memory training in children with acquired brain injuries.

AIM: To test the feasibility of the Amsterdam memory and attention training for children (Amat-c) in Swedish children with acquired brain damage. METHODS: Amat-c consists of structured exercises in specific attention and memory techniques. Three Swed... expand abstractish children aged 9-16 y with acquired brain injuries and related memory and attention deficits trained with the Amat-c method for half an hour a day in school or at home interactively with a teacher or parent for a period of 20 wk. RESULTS: All children and their coaches completed the training without interruption. The results showed an improvement in several neuropsychological tests of sustained and selective attention as well as in memory performance. Questionnaires filled in by parents and teachers indicate that, using the Amat-c method, the children learned strategies that improved their school achievement and self-image. CONCLUSIONS: The Amat-c is a valuable treatment option for improving cognitive efficiency in children with acquired brain injuries. The results indicate improved performance in several psychometric measurements. On the basis of these results, the second step will be to modify the complexity and duration of the method, as well as to integrate a reward system before further evaluating the efficacy in a larger controlled study. collapse abstract

Cytoskeletal derangements in hereditary myopathy with a desmin L345P mutation.

Patients with abnormal accumulations of desmin have been described in myopathies with or without cardiac involvement. Desmin deposits were sometimes associated with abnormal aggregates of other cytoskeletal proteins. In the present study we present h... expand abstractow the cytoskeletal organisation of desmin, nestin, synemin, paranemin, plectin and alphaB-crystallin is altered in skeletal muscles from a patient with a L345P mutation in the desmin gene. In general, accumulations of desmin together with synemin, nestin, plectin and alphaB-crystallin were present between myofibrils and beneath the sarcolemma. However, as the biopsy samples were very myopathic, large variability in fibre size and fibre maturation was seen, thus the myofibrillar content and the cytoskeletal organisation varied considerably. In cultured satellite cells from the patient, desmin aggregates were not observed in initial passages, but occurred over time in culture in the form of perinuclear, peripheral or cytoplasmic deposits. Nestin colocalised to the abnormal desmin deposits to a larger extent than did vimentin. alphaB-Crystallin was only present in cells with a disrupted desmin network. Plectin was altered in a subset of cells with a disrupted desmin network, whereas synemin and paranemin were not detected. We conclude that the L345P desmin mutation has a profound influence on the cytoskeletal organisation both in vivo and in vitro, which reflects the pathogenesis of the desmin myopathy. collapse abstract

Lung clearance in children with Duchenne muscular dystrophy or spinal muscular atrophy with and without CPAP (continuous positive airway pressure).

Bronchiolar clearance was studied in 7 boys in the age range of 8 to 17 years, 6 with Duchenne muscular dystrophy (DMD) and 1 with spinal muscular atrophy type II (SMA-II). These boys had healthy lungs but a severely reduced muscular strength (wheelc... expand abstracthair dependent). In 6 of the boys, clearance was studied twice, at one occasion as a control and at the other occasion following treatment with continuous positive airway pressure (CPAP). A control group of healthy adults was used. In the clearance examinations, 6-microm Teflon particles, labeled with III In was inhaled extremely slowly, 0.05 L/s. This gives a deposition mainly in the bronchioles. Lung retention was measured after 0,24,48, and 72 hours. A model for deposition of particles in the adult lung was scaled down to represent the children in this study. Deposition in various airway generations was calculated to be similar in children and adults. Also the measured retentions were similar in the boys and the adults. In the clearance experiments during CPAP treatment, there was a significantly lower retention after 72 hours (but not after 24 and 48 hours) than in the control experiments. Theresults indicate that a severe reduction of muscular strength, and thereby a reduction of mechanical movement of the lung, does not affect clearance from large and small airways. However, some effect of clearance from small airways cannot be excluded due to the short measuring period. The small but significant effect of the CPAP treatment might have potential clinical importance and suggest that bronchiolar clearance can be affected by some form of mechanical force. collapse abstract

A missense mutation in the desmin rod domain is associated with autosomal dominant distal myopathy, and exerts a dominant negative effect on filament formation.

In some myopathies of distal onset, the intermediate filament desmin is abnormally accumulated in skeletal and cardiac muscle. We report the first point mutation in desmin cosegregating with an autosomal dominant form of desmin-related myopathy. The ... expand abstractL345P desmin missense mutation occurs in a large, six generation Ashkenazi Jewish family. The mutation is located in an evolutionarily highly conserved position of the desmin coiled-coil rod domain important for dimer formation. L345P desmin is incapable of forming filamentous networks in transfected HeLa and SW13 cells. We conclude that the L345P desmin missense mutation causes myopathy by interfering in a dominant-negative manner with the dimerization-polymerization process of intermediate filament assembly. collapse abstract

cDNA cloning and chromosomal localization of human alpha(11) integrin. A collagen-binding, I domain-containing, beta(1)-associated integrin alpha-chain present in muscle tissues.

We previously identified a novel integrin alpha-chain in human fetal muscle cells (Gullberg, D., Velling, T., Sjöberg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57-65). We have now isolated the full-length cDNA for this integrin subunit, alpha(11). ... expand abstractThe open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains seven conserved FG-GAP repeats, an I domain with a metal ion-dependent adhesion site motif, a short transmembrane region, and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. alpha(11), like other I domain integrins, lacks a dibasic cleavage site for generation of a heavy chain and a light chain, and it contains three potential divalent cation binding sites in repeats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the alpha(11) integrin sequence from other integrin alpha-chains. Amino acid sequence comparisons reveal the highest identity of 42% with the alpha(10) integrin chain. Immunoprecipitation with antibodies to alpha(11) integrin captures a 145-kDa protein distinctly larger than the 140-kDa alpha(2) integrin chain when analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Fluorescence in situ hybridization maps the integrin alpha(11) gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting, integrin alpha(11) mRNA levels are high in the adult human uterus and in the heart and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, alpha(11) mRNA and protein are up-regulated. Studies of ligand binding properties show that alpha(11)beta(1) binds collagen type I-Sepharose, and cultured muscle cells localize alpha(11)beta(1) into focal contacts on collagen type I. Future studies will reveal the importance of alpha(11)beta(1) for muscle development and integrity in adult muscle and other tissues. collapse abstract

Pediatric rhabdomyosarcomas express the intermediate filament nestin.

Previous findings that the intermediate filament nestin is expressed in immature skeletal muscle cells prompted us to compare the staining patterns of nestin and desmin in rhabdomyosarcomas (RMSs) and in other small cell tumors of infancy. We found t... expand abstracthat nestin immunoreactivity was present in all of 29 examined typical RMSs, which also expressed desmin. Two undifferentiated tumors, primarily suspected to be RMSs, expressed nestin, but not desmin. One of these nestin-positive, desmin-negative tumors was positive for the expression of the myogenic regulatory gene MyoD and is considered to represent an undifferentiated RMS. The other, a paratesticular tumor, did not contain transcripts for MyoD, and most likely does not represent a RMS. In several RMSs and nonmuscle tumors, a z-disc-associated nestin immunoreactivity occurred as a paramalignant phenomenon in cross-striated muscle fibers adjacent to the tumor cells. Our findings indicate that nestin, although present also in tumors of the central and peripheral nervous systems, as well as in endothelial cells and in some muscle cells adjacent to tumors, is a useful complementary marker for RMS, particularly in very undifferentiated desmin-negative tumors. collapse abstract

Distribution of nestin in the developing mouse limb bud in vivo and in micro-mass cultures of cells isolated from limb buds.

Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isol... expand abstractated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development. collapse abstract

Tenascin-C expression correlates with macrophage invasion in Duchenne muscular dystrophy and in myositis.

Tenascin-C (TN-C) is an extracellular matrix protein expressed during development in several tissues, but restricted to only a few areas in normal adult tissues. By immunizing mice with human fetal myoblasts we generated a monoclonal antibody to TN-C... expand abstract and mapped the epitope to the aminoterminal end containing EGF-like repeats. Using this antibody we detected by immunohistochemistry TN-C in the epimysium and perimysium of human fetal muscles, as well as in nonfibrillar deposits in myoblast cultures. In situ hybridization did not reveal any signal within human fetal muscle groups, suggesting that non-muscle cells synthesize the majority of the tenascin that localizes in and around human fetal muscle. Immunohistochemical analysis of muscle biopsies from Duchenne/Becker muscular dystrophy and myositis patients revealed that TN-C is expressed in skeletal muscle. Although the patterns of TN-C immunoreactivity were quite different in the two disease entities, the endomysial TN-C reactivity in both DMD/BMD and in myositis invariably correlated with the presence of macrophages. collapse abstract

Nestin distribution in the developing limb bud in vivo and in vitro.

Children with poliomyelitis in Sweden 1996

Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation.

The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the ext... expand abstractracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells. collapse abstract

Colocalization of nestin and vimentindesmin in skeletal muscle cells demonstrated by three-dimensional fluorescence digital imaging microscopy.

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more di... expand abstractstantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggest that charged amino acid residues in the alpha-helical rod domain may play a role in the interaction. collapse abstract