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Biomedical subjects

G Butler-Browne

Publications and source records attributed to G Butler-Browne.

At least 19 recordsLinked to original sources
PubMed2001

Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.

Animals↗
PubMed2001

Alpha-motoneurons of the injured cervical spinal cord of the adult rat can reinnervate the biceps brachii muscle by regenerating axons through peripheral nerve bridges: combined ultrastructural and retrograde axonal tracing study.

Following our previous studies related to brachial plexus injury and repair, the present experimentation was designed to examine the ultrastructural features of those motoneurons of the locally injured cervical spinal cord of adult rats that were seen to regenerate into peripheral nerve (PN) bridges and to reinnervate nearby skeletal muscles. Here, the peripheral connection of the PN bridge was made with the biceps brachii (BB) muscle. Three months postsurgery, the spinal motoneurons labelled by retrograde axonal transport of horseradish peroxidase (HRP), after its injection into the BB, were selected on thick sections, using light microscopy, for the presence of dark amorphous granules of the HRP reaction product. Serial ultrathin sections were then made from the selected material. For the 10 labelled neurons studied, we examined the synaptic boutons present on the membrane of the neuronal soma. For five of them, we could observe three of the six types of synaptic boutons described for the alpha-motoneurons of the cat (S-type with spherical vesicles, F-types with flattened vesicles, and C-type with subsynaptic cistern). The largest boutons (type C) are specific to alpha-motoneurons. In comparison to normal material, we noticed a decrease in the number of boutons and an increase in the number of glial processes. After a transient phase of trophic changes, the reinnervated BB muscles showed a return of their fibers to nearly normal diameters as well as evidence of fiber type grouping. Simultaneous staining with silver and cholinesterase also revealed the presence of new motor endplates frequently contacted by several motoneurons. The present study indicates that, after a local spinal injury, typical alpha-motoneurons can reinnervate a skeletal muscle by regenerating axons into the permissive microenvironment provided by a PN graft. These data offer prospects for clinical reconstruction of the brachial plexus after avulsion of one or several nerve roots.

Acetylcholinesterase↗
PubMed2001

Lack of desmin results in abortive muscle regeneration and modifications in synaptic structure.

Desmin, a muscle-specific intermediate filament protein, is expressed in all muscle tissues. Its absence leads to a multisystemic disorder involving cardiac, skeletal, and smooth muscles. In skeletal muscle, structural abnormalities include lack of alignment of myofibrils, Z disk streaming, and focal muscle degeneration. In this study, we have examined the consequences of an absence of desmin on the mechanisms of regeneration and the integrity of the neuromuscular junction. The muscles of desmin knock-out and wild-type mice were made to regenerate by injecting cardiotoxin and were examined 7 to 42 days following the injection. The absence of desmin resulted in a delayed and modified regeneration and an accumulation of adipocytes. This was associated with a persistence of small diameter muscle fibers containing both N-CAM and developmental myosin isoforms. The amount of the slow myosin was increased, whereas there was a decrease in the fast isoform in the regenerated muscles of desmin knock-out mice. Both regeneration and aging led to the appearance of elongated neuromuscular junctions with diffuse acetylcholinesterase staining and a decrease in the overall acetylcholinesterase activity in the muscles of these mice. The neuromuscular junctions were markedly disorganised and in some cases postjunctional folds were absent. We conclude that desmin is essential for terminal muscle regeneration, maturation of muscle fibers, and maintaining the complex folded structure of the postsynaptic apparatus of the neuromuscular junctions.

Animals↗
PubMed2001

A discrepancy resolved: human satellite cells are not preprogrammed to fast and slow lineages.

Satellite cells from chicken and mouse muscle when differentiated in vitro have been shown to display a myosin heavy chain phenotype that corresponds to the fibre from which they originated. Indirect evidence has suggested that this might not be the case for human satellite cells. In the present study we have compared the myosin heavy chain (MHC) profile expressed by differentiated cultures of satellite cells isolated from single fast or slow muscle fibres. The MHC composition of the isolated fibres was determined by sodium dodecyl sulfate glycerol gel electrophoresis and Western blotting. The MHC profile expressed by the differentiated myotubes was identified by immunostaining using specific antibodies. Our results show that all human satellite cells isolated from either fast or slow fibres form myotubes in vitro which co-express both fast and slow MHCs independently of the fibre type from which they originated. These results confirm that human satellite cells, in contrast to those of birds and rodents, are not confined to distinct fast and slow lineages.

Adult↗
PubMed2001

The muscle-specific enolase is an early marker of human myogenesis.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) is active as a dimer formed from three different subunits, alpha, beta and gamma, encoded by separate genes. The expression of these genes is developmentally regulated in a tissue-specific manner. A shift occurs during development, from the unique embryonic isoform alphaalpha, towards specific isoforms in two tissues with high energy demands: alphagamma and gammagamma in the nervous system, alphabeta and betabeta in striated muscles. The alphaalpha remains widely distributed in adult tissues. Here we report the results of the first extensive study of beta enolase expression during human development. Indeed, the beta subunit is specifically expressed at early stages of human myogenesis. Immunocytochemical analyses demonstrated that it is first detected in the heart of 3-week-old embryos and in the myotomal compartment of somites from 4-week-old embryos. At this stage, the muscle-specific sarcomeric protein titin is expressed in this structure, which will give rise to all body skeletal muscles, but embryonic myosin heavy chain is not yet present. Analyses at the protein level show that, during human ontogenesis, myogenesis is accompanied by an increase in beta enolase expression and by a decrease in the expression of the two other alpha and gamma subunits. Furthermore, beta enolase subunit is expressed in proliferating myoblasts from both embryonic and post-natal muscles. In addition, clonal analysis of primary cell cultures, obtained from the leg muscle of a 7-week-old human embryo, revealed that the beta subunit is present in the dividing myoblasts of all four types, according to the classification of Edom-Vovard et al. [(1999) J Cell Sci 112: 191-199], but not in cells of the non-myogenic lineage. Myoblast fusion is accompanied by a large increase in beta enolase expression. Our results demonstrate that this muscle-specific isoform of a glycolytic enzyme (beta enolase) is among the earliest markers of myogenic differentiation in humans.

Adult↗
PubMed2000

Age-related appearance of tubular aggregates in the skeletal muscle of almost all male inbred mice.

Tubular aggregates (TAs) which have been recently observed in a few mouse myopathies are identical to those described in human diseases. In this study we show that TAs are also found in the skeletal muscle of almost all normal inbred mice strains. In these inbred strains of mice the presence of TAs is shown to be related to both age and sex. Nine different muscles were stained with the modified Gomori trichrome method to reveal the general morphology of the muscles. Anti-SERCA1 ATPase was used to confirm that the TAs were in fact accumulations of sarcoplasmic reticulum and anti-MyHC IIB to demonstrate that these accumulations were found exclusively in the type IIB muscle fibers. An ultrastructural study confirmed the observations revealed by light microscopy that the TAs were derived from the sarcoplasmic reticulum. TAs were never observed in female inbred mice and were only found in type IIB glycolytic muscle fibers of male inbred mice. Therefore when analyzing the effect of genetic knock out and knock in experiments on the muscle phenotype of transgenic mice one should be aware that the presence of these aggregates is a non-specific phenomenon induced by inbreeding.

Aging↗
PubMed1999

The beta enolase subunit displays three different patterns of microheterogeneity in human striated muscle.

In higher vertebrates, the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolyase; EC 4.2.1.11) is active as a dimeric protein formed from three subunits--alpha: ubiquitous, beta: muscle specific, and gamma: neuron specific--encoded by different genes. In the present study, we have shown that an antiserum previously produced against the mouse beta beta enolase is also a specific reagent for the muscle specific human enolase. Using this antiserum to study human muscles, we demonstrated novel patterns of the beta subunit microheterogeneity which are distinctive from those observed previously in rodents and which appear to be independent of age, gender and muscular activity. Two variants of the beta subunit differing by their size have been detected: one heavy form of 46 kDa (beta H) and one light form of 45 kDa (beta L). Muscle biopsies expressed either beta H or beta L or beta H + beta L, and all muscles of an individual expressed the same variants. The products of in vitro translation of RNA prepared from human muscle displayed beta subunit variants identical to those of the protein present in the biopsy. Therefore the differences observed between individuals reveal a difference already present at the level of the RNA transcripts. These observations suggest the existence of an yet undescribed polymorphism of the human beta enolase gene which could affect the coding sequence. Comparative immunocytochemical and histochemical analyses of biopsies demonstrated that the beta subunit was expressed in all fast fibres (type II), but not in slow fibres (type I). No difference was observed in the intensity of beta enolase immunolabelling between the various types (IIA, IIAB, IIB) of fast fibres. No significant difference in fibre type composition and histological appearance was visible between muscles presenting either one of the three patterns of microheterogeneity.

Adult↗
PubMed1998

Dynamic left/right regionalisation of endogenous myosin light chain 3F transcripts in the developing mouse heart.

It has recently emerged that transcriptional differences exist between left and right cardiac chambers. An example is provided by transgenic mice with an nlacZ reporter gene under transcriptional control of the fast skeletal muscle alkali myosin light chain (MLC) 3 promoter and 3' enhancer, which express beta-galactosidase in a left ventricular-right atrial dominant pattern in the developing and adult heart. Here, we demonstrate that endogenous MLC3F transcripts are also left/right regionalised in the mouse heart during embryonic development. Regionalisation is observed as early as embryonic day (E) 8.5, and by E10.5 MLC3F transcripts are present predominantly in the future left ventricle and right atrium, and to a lesser extent in the left atrium. Subsequently, MLC3F transcripts are down-regulated in the left ventricle, and by E12.5 expression is restricted to both atria and left-ventricular trabeculae. No MLC3F protein can be detected in the adult or embryonic mouse heart, suggesting that post-transcriptional regulation prevents this fast myosin isoform contributing to myocardial contraction. Left ventricular-right atrial dominant MLC3F transgenes therefore reflect transitory left/right regionalisation of the endogenous gene, unlike other reported cases of transgene regionalisation. MLC3F transgenes, however, maintain an embryonic-like distribution throughout development suggesting that myocardial gene expression is controlled by distinct temporal, as well as spatial, regulatory modules.

Age Factors↗
PubMed1997

Acetylcholine receptor formation in mouse-chick chimera.

This study investigated possible interactions between motoneurons and somitic-derived muscle cells in the formation of neuromuscular synapses in the myotome. The peculiarities of the neuromuscular synaptic pattern in chick and mouse embryos provided a model for studying the achievement of synaptogenesis between chick motoneurons and mouse muscle cells. In chick embryo, initial AChR clustering occurs well before innervation of the myotome, whereas in mouse embryo nerve axons invade the myotome extensively before the appearance of AChR clusters. Our approach was to replace somites from a chick host embryo with those derived from mouse donor embryos. We show that muscle cells from mouse myotome can differentiate in the chick embryo environment and form neuromuscular contacts with chick motor axons. Host axons invaded in ovo differentiating mouse myotome at a time when they had not yet reached the host myotome. This particular ingrowth of motor nerves was attributable to the mouse transplant since use of a quail somite did not produce the same effect as the mouse somite, which suggests that developing mouse muscles specifically modify the time course of chick axogenesis. The synaptic areas formed between chick motor axons and mouse myotubes developed according to the mouse pattern. Both the timing of their appearance and their morphology correlated perfectly with events in mouse synaptogenesis. These results indicate the important role played by postsynaptic membrane in controlling the first steps of AChR formation.

Animals↗
PubMed1997

Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle.

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.

Adenosine Triphosphatases↗
PubMed1997

Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts.

We and others have previously cloned several cDNAs of human cardiac troponin T (cTnT), demonstrating the multiplicity of cTnT isoforms in the human heart. Four of them named cTnT1, 2, 3 and 4 result from a combinatorial alternative inclusion of 30- and 15-nucleotides in the 5' coding region of the cDNAs. In failing human ventricles, increased expression of cTnT4 has been reported at the protein level. More recent RT-PCR experiments showed increased expression of fetal-type splicing products in the 5' region, one of them corresponding to cTnT1. To clarify this issue, we examined the accumulation of the 4 cTnT mRNA and protein species in left ventricular specimens at the time of heart transplantation, and in control left ventricular samples using RNase protection and Western blotting. In all samples, cTnT3 was the major mRNA isoform, cTnT4 a minor isoform while cTnT1 and cTnT2 mRNAs were present but barely detectable. At the protein level, cTnT3, 4 and 1 were detected with the same relative abundance as that seen at the mRNA level. In addition, we detected a fourth TnT species of very low abundance corresponding either to a skeletal or to a "short" cardiac TNT isoform. Compared to controls, increased levels of cTnT4 mRNA and protein were detected in only half the failing ventricles independently of the cause of failure, suggesting that this increase may not be a general characteristic of left ventricular failure but instead could be related to stress. Unexpectedly, we found a decrease in cTnT1 protein expression in all failing ventricular samples studied, compared to controls.

Adult↗
PubMed1996

Intraspinal injection of embryonic neurons maintains muscle phenotype in adult chronic spinal rats.

A suspension of monoaminergic embryonic neurons was transplanted into the spinal cord of paraplegic rats. Enzyme histochemical, morphometric, and biochemical analyses of the hindlimb musculature were carried out 2-5 months later to determine the consequences on muscle atrophy and muscle phenotypes which were compared in three groups of rats: intact, spinalized, and spinalized and transplanted with embryonic cells. Our results indicate that this transplantation does not prevent muscular atrophy, which appears highly dependent on the level of muscular activity, but partially maintains the slow phenotype, especially in the soleus muscle. We conclude that fiber phenotypes are not determined by the level of muscular activity alone but are also dependent on putative trophic factors synthesized by motoneurones.

Animals↗
PubMed1994

The emergence of an adult muscle phenotype in urodelan amphibians: an immunohistochemical study.

Electrophoretic techniques adapted for the analysis of muscles of lower invertebrates reveal four myosin heavy chain isoforms in the dorsalis trunci of Pleurodeles waltlii: two fast (MHC-IIA, MHC-IIB), and one slow (MHC-I) in the adult and one isoform (MHC-La) in the larvae. Polyclonal antibodies were prepared against the larval (anti-MHC-La) and one of the fast myosin (MHC-IIA) isoforms and their specificity was confirmed by western blot analysis. An immunohistochemical analysis was then carried out on frozen sections of the dorsalis trunci of P. waltlii at different stages of development. From stage 44 it was possible to demonstrate the presence of MHC-IIA in the small diameter fibers at the periphery of the muscle; the number and diameter of these fibers increased from stage 44 to stage 56 when anatomical metamorphosis had finished. By stage 56 these fibers could also be readily identified using standard histochemical techniques as type IIA fibers. We conclude that fast IIA myosin is expressed well before the final adult muscle phenotype has been established and its expression is therefore independent of thyroid hormone.

Animals↗
PubMed1992

Modification of the dystrophic phenotype after transient neonatal denervation: role of MHC isoforms.

While it recently has been demonstrated that it is possible to modify the phenotypic expression of murine dystrophy (dy/dy) (i.e., prevent myofiber loss) by subjecting the extensor digitorum longus (EDL) muscle of 14-day-old dy/dy mice to transient neonatal denervation (Moschella and Ontell, 1987), the mechanism responsible for this phenomenon has not been determined. Since it has been suggested that the effects of dystrophy vary according to fiber type, the fiber type frequency in 100-day-old normal (+/+) and dy/dy EDL muscles subjected to transient neonatal denervation has been determined by immunohistochemical analysis of their myosin heavy chain (MHC) composition. This frequency has been compared with that found in the EDL muscles of 14- and 100-day-old unoperated +/+ and dy/dy mice, in order to determine whether the reinnervation of transiently denervated neonatal muscle results in a preponderance of fibers of the type that might be spared dystrophic deterioration. In unoperated dy/dy muscle there is a progressive decrease in the frequency and in the absolute number of fibers that express MHC2B, with 100-day-old dy/dy muscles having approximately 32% of the number of myofibers fibers containing MHC2B as is found in age-matched +/+ muscles. The number of fibers containing the other fast isoforms (MHC2A and MHC2X) is similar in +/+ and dy/dy muscles at this age, indicating that fibers with MHC2B are most affected by the dystrophic process. Reinnervation following transient neonatal denervation of both the +/+ and the dy/dy EDL muscles results in a similar decrease (approximately 62%) in the number of myofibers containing MHC2B and an increase in myofibers containing the other fast MHC isoforms (MHC2A and MHC2X). The selective effect of dy/dy on fibers containing MHC2B and the sparing of myofibers in transiently denervated dy/dy muscle (which contains a reduced frequency of fibers containing MHC2B) are consistent with, although not direct proof of, the hypothesis that alterations in the fiber type may play a role in the failure of myofibers in transiently denervated dy/dy muscles to undergo dystrophic deterioration. Evidence is presented suggesting that neurons that supply myofibers containing MHC2B may be at a selective disadvantage in their ability to reinnervate neonatally denervated muscles.

Animals↗
PubMed1990

Molecular cloning of human cardiac troponin I using polymerase chain reaction.

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.

Amino Acid Sequence↗
PubMed1989

Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development.

A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined. The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb. Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons. There are eight introns in both the human and the hamster des genes. Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved. A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns. These sequences all belong to the human AluI family. When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al. [Cell 43 (1985) 327-338] for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes [Minty and Kedes, Mol. Cell. Biol. 6 (1986) 2125-2136] in the 5'-flanking region. The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene. Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.

Amino Acid Sequence↗