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Biomedical subjects

G Goldspink

Publications and source records attributed to G Goldspink.

At least 55 records · Page 3Linked to original sources
PubMed1998

The morphological basis of increased stiffness of rabbit tibialis anterior muscles during surgical limb-lengthening.

When the tibialis anterior muscle of the rabbit is progressively stretched during surgical limb distraction, the muscle fibres lengthen by addition of new serial sarcomeres, provided that stretch is carried out at an appropriate rate. However, in spite of the apparent adaptation to the new functional length, range of joint movement is greatly decreased. In this study we have first, made measurements of the passive tension developed by distracted muscles over the range of joint movement and secondly made quantitative measurements of endomysial and perimysial connective tissue content. It was found that at all ankle joint angles greater than 90 degrees, the passive tension developed by the distracted muscles was greater than both contralateral and sham-operated controls. Image analysis showed that the ratio of collagen to contractile material was increased in distracted muscles compared with muscles from sham-operated controls, due to increased deposition of collagen type III. Scanning electron microscopy showed the presence of a dense perimysial weave surrounding the distracted muscle fibres. These quantitative and qualitative changes in the connective tissue component could account for the increased stiffness demonstrated by the physiological measurements. It would seem that in distracted muscle the connective tissue element adapts less readily than the contractile component, with prolonged stretch leading to damage to the perimysial and endomysial network, with subsequent fibrosis and loss of muscle compliance. Such changes could help explain the loss of range of movement noted in the distracted limbs of patients undergoing surgical limb-lengthening and in other conditions that result in muscle contractures.

Adaptation, Physiological↗
PubMed1998

Cellular and molecular aspects of muscle growth, adaptation and ageing.

Although major advances have been made over the past few decades in prosthetic dentistry, deterioration in oral function and altered facial appearance are still common accompaniments of ageing. Molecular biology methods now allow us to understand these age-related changes at the level of gene expression. Muscle loss as well as bone loss still present major problems, the magnitude of which increases as the age profile of our society changes. Both muscle and bone tissue respond to mechanical signals for which bone depends on muscle and for muscle, stretch has been shown to be important as it induces protein synthesis and an increase in girth as well as length of the muscle fibres. The latter involves the production of more sarcomeres in series so that the jaw muscles adapt to a new functional length following changes in vertical dimension of occlusion. It also determines the postural position of the lower jaw. In our investigations into the control of muscle mass we have recently cloned a growth factor which is expressed in exercised and/or overloaded muscles. This comes in two forms: an autocrine or local form and a paracrine or systemic form. Both growth factors influence muscle growth markedly and it is probable that the systemic type is also involved in maintenance of bone. The discovery of these growth factors provides the mechanisms by which mechanical signals are transduced into chemical signals that in turn regulate gene expression and protein synthesis.

Adaptation, Physiological↗
PubMed1998

Intercostal muscles in the rabbit: surgical anatomy and flap construction.

Demos and colleagues (1967) obtained good antireflux results from transposing an intercostal myoneurovascular pedicle around the gastro-oesophageal junction in dogs. An intact neurovascular supply is essential for the viability of a muscle flap. The aim of this study was to delineate the nerve and arterial supply to the left 11th intercostal muscle in the rabbit and to assess whether this muscle could be mobilized as a viable flap. The innervation of the muscle was studied using the methods of gross dissection in cadaveric specimens, and histological staining techniques. The arterial supply was studied using gross dissection, and aortography. In three non-recovery experiments, intercostal muscle was transposed around the gastro-oesophageal junction. The distal motor latency was recorded after electrical stimulation of the intercostal wraps. Gross dissection, histological staining techniques, and aortography showed that the left 11th intercostal muscle group in the rabbit is supplied by segmental vein, artery and nerve, running between external and internal intercostal muscles. Aortography and electrical stimulation demonstrated that the muscle group could be mobilized with an intact neurovascular supply. The left 11th intercostal muscle group has potential as a viable muscle flap for use in surgical procedures within the upper abdomen.

Animals↗
PubMed1997

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.

Actins↗
PubMed1997

Comparison of the molecular, antigenic and ATPase determinants of fast myosin heavy chains in rat and human: a single-fibre study.

Combined methodologies of histochemistry, immunohistochemistry, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), reverse transcriptase polymerase chain reaction (RT-PCR) and a histochemical method specific for myofibrillar ATPase (mATPase) of the type IIX myosin heavy chain (MyHC) isoform were used to study human and rat single fibres to examine the homology between type II MyHC isoform-based fibres of both species. We demonstrate that human type II fibres exhibit antigenic mATPase and 3'-untranslated region (3'-UTR) sequence determinants homologous to the IIA and IIX but not the IIB MyHC isoforms of the rat. Both immunolabelling with anti-MyHC monoclonal antibodies and the mATPase method used with frozen sections confirmed that all human type II fibres express type IIA and/or type IIX MyHC. Quantitative immunohistochemistry failed to recognize human fibres with antigenic characteristics corresponding to hybrid IIXB MyHC-based fibres. Ca2+-stimulated maximum myosin ATPase activity, determined by quantitative histochemistry, revealed that human IIX fibres (with an optical density or OD = 0.707) display enzyme activity which is comparable to that of the rat type IIX (OD = 0.687) but lower than that of the rat type IIB fibres (OD = 0.836). The results do not support the notion that MyHC IIB is expressed in human limb muscles, even in hybrid fibres. We conclude that human type II fibres have been misclassified in numerous previous publications and that this has important implications in attempts to compare the physiological characteristics of fibre types, particularly when animal models are used.

Animals↗
PubMed1997

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Animals↗
PubMed1997

Changes in muscle fibre type, muscle mass and IGF-I gene expression in rabbit skeletal muscle subjected to stretch.

The relationship between IGF-1 and changes in muscle fibre phenotype in response to 6 d of stretch or disuse of the lower limb muscles of the rabbit was studied by combining in situ hybridisation and immunohistochemistry procedures. Passive stretch by plaster cast immobilisation of the muscle in its lengthened position not only induced an increase in IGF-I mRNA expression within the individual muscle fibres but also an increase in the percentage of fibres expressing neonatal and slow myosin. This change in phenotype was also found to be accompanied by a rapid and marked increase of muscle mass, total RNA content as well as IGF-I gene expression. In contrast, IGF-I appears not to be involved in muscle atrophy induced by immobilisation in the shortened position and the inactivity which results from this procedure. The level of increase in expression of IGF-I mRNA varied from fibre to fibre. By using adjacent serial sections, the fibres which expressed IGF-I mRNA at the highest levels were identified as expressing neonatal and the slow type 1 myosin. These data suggest that the expression of IGF-I within individual muscle fibres is correlated not only with hypertrophy but also with the muscle phenotypic adaptation that results from stretch and overload.

Adaptation, Physiological↗
PubMed1997

Evaluation of an intercostal myoneurovascular transposition as a lower esophageal neosphincter.

Previous work has shown promising results for an intercostal myoneurovascular transposition in the prevention of gastroesophageal reflux following esophagectomy. A first study evaluated the intercostal transposition procedure and compared it with the Nissen fundoplication using a rabbit model of gastroesophageal reflux. Group A underwent partial cardiomyectomy to produce gastroesophageal reflux. Group B underwent cardiomyectomy, and intercostal transposition around the gastric cardia. Group C underwent Nissen fundoplication and cardiomyectomy. All animals had preoperative and 1-week and 4-week postoperative intraesophageal manometry and pH studies. At the 4-week interval, macroscopic and microscopic esophageal histopathology was assessed. The mean change in values from preoperative to 4 weeks postoperative were compared. Group B showed significantly lower reflux time (P < 0.001) and grade of esophagitis (P < 0.005), and significantly greater average lower esophageal sphincter basal pressure (P < 0.001) and abdominal length of sphincter (P < 0.01) when compared with Group A. There was no statistical significance between the results of Group B and Group C. A second study assessed whether reflux was prevented by an anatomical structure, or a muscle flap acting in a physiological manner. At autopsy, the ten rabbits from Group B underwent removal of the intercostal wrap, and the right 11th intercostal muscle as a control. There was a significant difference in the quantity of viable muscle tissue between muscle flaps and controls (P < 0.001), the muscle flaps having generally little viable muscle left 4 weeks after surgery. A further experiment to evaluate this result found that loss of muscle tissue was due to excessive stretch and not due to damage of the intercostal neurovascular bundle during mobilization. Two groups of animals underwent electromyographic studies. The first group underwent recordings of all intercostal muscles. The second group underwent intercostal transposition around the gastric cardia, and insertion of recording electrodes into the muscle flap. The electromyographic activity of the muscle flap was recorded at 0, 2, and 4 weeks after surgery. The second group demonstrated activity in the muscle flaps simultaneous with diaphragmatic contractions. This activity, although much reduced, was still present 4 weeks after surgery. These studies showed that the intercostal transposition and Nissen fundoplication procedures are equally effective in preventing experimental gastroesophageal reflux. The antireflux properties of the intercostal transposition were possibly the result of anatomical buttressing of the gastroesophageal junction, and not due to a fully viable contracting muscle flap.

Animals↗
PubMed1996

The characterisation of the 5' regulatory region of a temperature-induced myosin-heavy-chain gene associated with myotomal muscle growth in the carp.

We have isolated and characterised the 5' region of a member of the carp myosin heavy chain gene family. Expression of this gene has previously been shown to be induced by an increase in environmental temperature and is restricted to the small-diameter white myotomal muscle fibres which are associated with growth. The whole isoform gene, including potential regulatory sequence 5' to the transcription start site and the 3' untranslated region was cloned in a lambda2001 bacteriophage vector. Studies of the structure of the 5'-end of the gene revealed high amino acid sequence similarity with translated exons 3-7 of mammalian myosin heavy chain genes indicating identical exon/intron boundaries. The overall length of the gene was however only about one half of that in mammals and birds due to shorter introns. The region 5' to the transcription unit was sequenced and revealed the presence of putative TATA and CCAAAT boxes. In order to study the regulation of expression, a series of endonuclease-generated fragments from the 5' flanking sequence were spliced to chloramphenicol acetyltransferase reporter vectors and used in cell transfection assays or direct gene injection into carp skeletal muscle. The 5' flanking region, which contains a consensus sequence known as an E-box (CANNTG) and a MEF2 binding site, was shown to improve the expression of the reporter gene in fish acclimated at 18 degrees C or 28 degrees C. Unlike the coding region, there was little similarity between the 5'-upstream sequence (promoter region) when compared with sequences flanking the 5'-end of the other myosin heavy chain genes in mammals or chicken.

Acclimatization↗
PubMed1996

Cloning and characterization of an IGF-1 isoform expressed in skeletal muscle subjected to stretch.

To ascertain if IGF-1 is a regulator of local muscle growth, total RNA was extracted from rabbit muscle induced to undergo rapid hypertrophy using active stretch and from control muscles. This was analysed by Northern hybridization with a 280 base pair probe containing sequences derived from exons 3 and 4 of the insulin-like growth factor 1 gene. Two types of insulin-like growth factor 1 mRNA were shown to be strong expressed in the stretched muscles. In situ hybridization using the same probe (280 base pair) showed that IGF-1 is strongly expressed in muscle that is induced to grow rapidly and is expressed in the muscle fibres themselves. Using RT-PCR a single insulin-like growth factor 1 isoform cDNA (IGF-1Ea) could be cloned from the normal resting muscles. However, an additional isoform of insulin-like growth factor 1 (insulin-like growth factor 1Eb) was found to be expressed in stretched muscle undergoing hypertrophy. The E domain sequence of the additional isoform differs from the liver insulin-like growth factor 1Ea by the presence a 52 base pair insert. This changes the reading frame of the derived carboxyl-terminal resulting in a different precursor insulin-like growth factor 1 isoform. This insulin-like growth factor 1 mRNA probably encodes the precursor insulin-like growth factor 1 isoform that is responsible for local muscle growth regulation in response to mechanical stimulation. To confirm that alternative splicing of the insulin-like growth factor 1 gene occurs in muscle in response to physical activity, oligonucleotide primers were made which specifically amplify the cDNAs of two isoforms (insulin-like growth factors 1Ea and Eb) in the human as well as the rabbit. Following altered physical activity for 2 h to 6 days, appreciable levels of insulin-like growth factor 1Eb (in human the Ec) isoform were detected in skeletal muscle by using RT-PCR. In contrast very little if any of this splice variant could be detected in control muscle not subjected to stretch or extra physical activity.

Animals↗
PubMed1996

Muscle growth and muscle function: a molecular biological perspective.

Molecular biological methods are pervading all biomedical fields and it is likely that they will soon introduce new techniques to veterinary diagnostics and have a major impact on food and fibre production in animal agriculture. The ability to manipulate muscle growth and phenotype will present new ethical problems, particularly if the techniques are used to manipulate muscle development in greyhounds and racehorses where the financial rewards could be very substantial. Muscle has been a useful tissue for the study of the molecular control of tissue development because terminal differentiation results in the production of large quantities of highly specialised proteins. Now that the functional anatomy of structural genes in muscle is being elucidated, a coherent picture is beginning to emerge of the way in which post-natal muscle growth and phenotype are regulated at the gene level. The hormones and growth factors involved in regulating the quantitative and qualitative changes in gene expression are now better understood, together with the ability of the tissue to adapt to physical signals and hence new activity patterns. The myosin heavy chain isoform genes which encode the myosin cross-bridges (the force generators for muscular contraction) exist as a large multigene family. The contractility and other characteristics of muscle depend to a large extent on the differential expression of members of this and other gene families. Muscle fibres adapt for increased power output by expressing a subset of "fast' genes and for increased economy of action by expressing a slow subset of genes and producing more mitochondria. With the increasing understanding of gene expression in muscle, there are prospects for manipulating the mass, contractility and other characteristics of muscle and also to change its phenotype and understand certain disease states.

Agriculture↗
PubMed1995

Editing of human alpha-galactosidase RNA resulting in a pyrimidine to purine conversion.

During a study of the gene coding for alpha-galactosidase (EC 3.2.1.22), the lysosomal enzyme deficient in Fabry's disease, RT-PCR amplification of alpha-galactosidase mRNAs obtained from three different tissues isolated from males revealed a substantial number of clones with a U to A conversion at the nucleotide position 1187. Such a modification of the coding sequence would result in an amino acid substitution in the C-terminal region (Phe396Tyr) of the enzyme. Neither PCR analysis of the genomic sequence nor the RT-PCR amplification of RNA obtained by in vitro transcription of the wild-type cDNA showed this change in the sequence. Multiple genes, pseudogenes are allelic variants were excluded. Hence, we propose RNA editing as a mechanism responsible for this base change in the alpha-galactosidase RNA.

Base Sequence↗
PubMed1995

Characterization of human skeletal muscle fibres according to the myosin heavy chains they express.

Using a method of single muscle fibre analysis, we investigated the presence of RNA transcripts for various isoforms of the myosin heavy chain (MyoHC) gene in histochemically, immunohistochemically and electrophoretically characterized individual muscle fibres (n = 65) from adult human vastus lateralis muscle. A cDNA clone isolated in this study was shown to contain the 3' end of a previously uncharacterized human MyoHC gene which is expressed specifically in human fast IIA muscle fibres and we conclude that this clone contains part of the human fast IIA MyoHC gene. In all the fibres histochemically, immunohistochemically and electrophoretically characterized as containing the previously classified IIB MyoHC (n = 23), it was shown that the human equivalent to the rat type IIX MyoHC gene is expressed. This observation was taken to suggest that the previously classified IIB muscles fibres in human muscle express a MyoHC isoform equivalent to the rat IIX, not the IIB, and would therefore be more accurately classified as IIX fibres.

Adult↗
PubMed1995

Small-diameter white myotomal muscle fibres associated with growth hyperplasia in the carp (Cyprinus carpio) express a distinct myosin heavy chain gene.

A carp myosin heavy chain gene isoform was isolated from a genomic clone, restriction mapped and partially sequenced to reveal the location of various exons. The clone contains a complete gene of approximately 12.0 kb which is half the size of the corresponding mammalian and avian myosin heavy chain genes. The mRNA transcript of this gene, however, is the same size as mammalian and avian striated muscle myosin heavy chain genes (about 6000 nucleotides), illustrating that the difference in size at the genomic level is due to shorter introns. A 169 bp NsiI restriction fragment containing only the 3' untranslated region of this gene was subcloned and used as an isoform-specific probe to study the expression of this particular isoform. Hybridisation analysis could only detect expression of this myosin heavy chain gene in the white muscle of adult carp that had been subjected to an increased environmental temperature. No expression of this gene was detected in carp under 1 year of age. In situ hybridisation demonstrated that expression of this gene is limited to small-diameter white muscle fibres of adult carp, which are thought to be responsible for muscle growth by fibre hyperplasia.

Adaptation, Physiological↗
PubMed1995

The response of muscle to leg lengthening.

We used an experimental rabbit model of leg lengthening to study the morphology and function of muscle after different distraction rates. Lengthening was in twice-daily increments from 0.4 to 4 mm per day. New contractile tissue formed during lengthening, but some damage to the muscle fibres was seen even at rates of less than 1 mm per day; abnormalities increased with larger rates of lengthening. There was proliferation of fibrous tissue between the muscle fibres at distraction rates of over 1 mm per day. Active muscle function showed adaptation when the rate was 1.0 mm per day or less, but muscle compliance was normal only after rates of 0.4 mm per day. Muscle responded more favourably at rates of distraction slower than those shown to lead to the most prolific bone formation. At present the rate of distraction in clinical practice is determined mainly by factors which enhance osteogenesis. Our study suggests that it may be advisable to use a slower rate of elongation in patients with poor muscle compliance associated with the underlying pathology; this will allow better accommodation by the contractile and connective tissues of the muscles.

Animals↗
PubMed1995

Expression of factor VII by muscle cells in vitro and in vivo following direct gene transfer: modelling gene therapy for haemophilia.

Direct injection of plasmid DNA into skeletal muscle has been proposed as a method of effecting somatic gene therapy. This article describes the construction and testing of a plasmid derived expression cassette believed to confer skeletal muscle specific expression. Expression constructs were designed containing the full-length cDNAs for both coagulation factor VIII and factor VII. The engineered genes were flanked by two muscle specific regulatory elements from different myosin isoforms and by an artificial polyadenylation signal sequence. In vitro transfection of C2-myoblasts led to expression of the factor VIII gene, shown by reverse transcription and polymerase chain reaction, upon differentiation of the myoblasts. The expression of the FVII construct was tested in a C2 cell culture system and also when injected directly into mouse muscle. It was found that in cell culture the level of factor VII antigen outside the cell, ie in the cell culture medium was two- to three-fold higher than inside the cell, ie in the cell lysate. This level of expression was found to continue for the duration of cell culture maintenance and a fully functional protein was produced. In vivo transfection experiments in mice showed a substantial increase in factor VII antigen compared with the background level 4-5 days after injection. An anti-human factor VII antibody was detected 7-10 days after injection. We conclude that muscle cells in vitro secrete and efficiently carry out post-translational modifications of the engineered gene product and in vivo secrete the gene product resulting in elevation of systemic levels. The data provide the basis for the use of muscle cells as an in vivo expression system for coagulation proteins in the treatment of inherited haemostatic and thrombotic disorders.

Animals↗
PubMed1994

Age-related changes in collagen gene expression in the muscles of mdx dystrophic and normal mice.

It has been shown that there is a marked accumulation of collagen (notably type III) in the muscles of Duchenne muscular dystrophy patients. Although not as marked, there is also an accumulation of collagen in normal skeletal muscle with age. To attempt to determine whether dystrophy-related and/or age-related collagen accumulation are the result of increased collagen gene expression, sections from muscles of mdx and control mice at different ages were subjected to in situ hybridization with cRNA probes for procollagen I and III to estimate changes in specific mRNA levels. Greater amounts of collagen I and III mRNA were noted in skeletal muscles and the diaphragm of the mdx mice than in the same muscles of age matched controls. The over-expression of these collagen genes was particularly noticeable in the mdx diaphragm and may possibly be explained by the greater activity of this muscle as compared to the limb muscles of these dystrophic mice. There was, however, an age-related decline of collagen expression in both normal and dystrophic muscle and this strongly suggests that the increased fibrosis of skeletal muscle with age is not the result of increased collagen gene expression but is most likely due to an impairment of degradation. In contrast, the excess accumulation of collagen in dystrophic muscle compared to normal muscle is related to increased gene expression, probably triggered by an injured tissue response induced by muscle fibre damage due to the lack of dystrophin.

Aging↗