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J B Harris

Publications and source records attributed to J B Harris.

At least 55 records · Page 3Linked to original sources
PubMed1994

Palytoxin-induced transmitter release in the autonomic nervous system of the rat.

A combination of pharmacological, histochemical, immunocytochemical, neurochemical and microscopic techniques has been used to test the hypothesis that smooth muscle contractions caused by palytoxin are primarily a response to toxin-induced release of transmitter from motor nerve terminals. In the anococcygeus, palytoxin caused a dose-dependent contraction; the dose-response curve was particularly steep in the dose range 0.3-100 nM. This part of the response was abolished by phentolamine and absent in the muscles of reserpinized animals. A single, large dose of palytoxin (100 nM) caused a biphasic contraction. Phentolamine blocked the first phase by 50% and the second by > 80%. Transmitter overflow studies showed that the toxin enhanced the release of 3H after loading with 3H-NA, and immunofluorescence showed the loss of adrenergic transmitters from the innervation. In the vas deferens, palytoxin caused a biphasic contraction of the muscle. Phentolamine blocked first phase by > 80% and the second by 47%. Immunofluorescence studies showed that stores of adrenergic transmitter were depleted but stores of NPY were not greatly affected. Indirect evidence suggested that palytoxin did not cause the release of purinergic transmitters. A direct component to the contraction was apparently present in most preparations, but though variable in extent it was usually slight. It is concluded that the contractions of smooth muscle caused by palytoxin are primarily the result of toxin-induced transmitter release.

Acrylamides↗
PubMed1994

The expression of vimentin in satellite cells of regenerating skeletal muscle in vivo.

The expression of the intermediate filament protein, vimentin, was studied in skeletal muscle during a cycle of degeneration and regeneration. Venom from the Australian tiger snake, Notechis scutatus scutatus, was used to initiate the breakdown of the soleus muscle of young, mature rats in vivo. Cryosections and Western blots of muscle samples were labelled using antibodies to vimentin, and examined at fixed time points after venom injection. Vimentin was absent in control adult muscle fibres, but was identified in activated satellite cells 12 h after venom assault. The amount of this protein rose during the early stages of regeneration, reaching its peak at 2-3 days. At this time, the expression of muscle-specific intermediate filament protein, desmin, began. As the abundance of desmin increased with the maturation of the regenerating myofibres, the abundance of vimentin declined until it was no longer detectable in mature regenerated fibres. It is suggested that vimentin plays an important role during satellite cell activation in the early stages of regeneration, and that the expression of vimentin may act as a stimulus for the expression of desmin at later stages of regeneration.

Animals↗
PubMed1993

Production of recombinant notechis 11'2L, an enzymatically active mutant of a phospholipase A2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli.

We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine-amino-acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11'2L, an isoform of notexin of Notechis scutatus scutatus venom. Notechis 11'2L is a mutant of the naturally occurring notechis 11'2 [Bouchier, C., Boyot, P., Tesson, F., Trémeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Ménez, A. (1991) Eur. J. Biochem. 202, 493-500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/l culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N-terminal sequence of notechis 11'2L. Notechis 11'2L and the wild notechis 11'2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11'2L was directly generated in a correctly folded form.

Base Sequence↗
PubMed1993

The clinical, genetic and dystrophin characteristics of Becker muscular dystrophy. II. Correlation of phenotype with genetic and protein abnormalities.

We have correlated a detailed clinical assessment of 67 patients with proven Becker muscular dystrophy with the results from genetic and protein analyses. There was an overall deletion frequency of 80%, rising to 92.6% in the large group of patients defined on clinical grounds as being of "typically" mild severity. The deletions in this group were all clustered in the region of the gene between exons 45 and 59; the most common deletion was of exons 45-47 and all but one started at exon 45. No similar deletions were seen in the patients with more severe disease, in whom the diverse genetic defects included a duplication and a very large deletion. Dystrophin patterns in the "typical" group were also very characteristic, and in both groups were as predicted from the genetic defect, the size of deletions being inversely proportional to the size of the protein produced.

Adolescent↗
PubMed1993

Effect of growth promoters on pig muscle structural protein and proteolytic enzyme levels in vivo and in vitro.

To elucidate the biochemical mechanism by which the growth hormone porcine somatotrophin (PST) promotes skeletal muscle growth, we have determined the activity of a comprehensive range of protein catabolizing proteolytic enzymes and structural proteins (determined by analytical electrophoresis, SDS-PAGE) in longissimus dorsi muscle from control and PST treated pigs. There was no significant difference in the levels of muscle structural proteins, or in the activity of muscle proteolytic enzymes at point of slaughter in control or PST treated animals. Similarly, in post-mortem muscle proteolysis time course experiments at pH 5.5 or pH 7.5, there was no significant difference in the rate of structural protein degradation by endogenous muscle proteases (determined via SDS-PAGE) using muscle from control or PST treated animals. In addition, investigation of a range of beta-agonist related drugs (clenbuterol, salbutamol, pirbuterol, fenoterol) showed no effect (10(-4)-10(-8) M) in vitro on the activity of individual protease types in control muscle, or on the degradation rate of muscle structural proteins by endogenous proteases in time course experiments at pH 5.5 or pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1992

The fate of desmin and titin during the degeneration and regeneration of the soleus muscle of the rat.

We studied the fate of desmin and titin in rat skeletal muscle during a cycle of degeneration and regeneration induced in vivo by the inoculation of a snake venom. Cryosections of muscle were labelled using antibodies to the two proteins, and examined at fixed time points after venom injection. Early pathological changes in the muscle, such as hypercontraction, preceded the loss of desmin. Immunolabelling using anti-desmin antibodies showed that desmin bridges were still intact when adjacent myofibrils were no longer aligned. The results suggested that although the hydrolysis of desmin is not necessary for the hypercontraction of muscle fibres, it probably contributes to complete fibre breakdown. Titin, or at least the part which lies close to the M-line, remained intact longer than desmin, but was also hydrolysed prior to complete disintegration of the fibres. Both desmin and titin were re-expressed in the regenerating myotubes by 2 days after venom inoculation, and became well organised even before the myofibrils became aligned. We conclude that desmin and titin are involved in both establishing and maintaining the structural integrity of the muscle fibres.

Animals↗
PubMed1992

The fate of dystrophin during the degeneration and regeneration of the soleus muscle of the rat.

Immunocytochemistry and Western blotting were used to monitor the fate of dystrophin in the soleus muscle of the rat during a cycle of degeneration and regeneration induced by inoculation of the muscle with the venom of Notechis scutatus scutatus (the Australian tiger snake). In control muscle dystrophin was localised close to the plasma membrane. Dystrophin began to break down 3-6 h after venom inoculation, giving a characteristic discontinuous labelling pattern. At 12 h dystrophin was absent from the plasma membrane, and by 1 day the architecture of the muscle fibers had completely broken down. By 2 days post inoculation regeneration had commenced. The regenerating myofibres possessed well-organised myofibrils and the plasma membrane was intact. Dystrophin was detected by Western blot at 3 days, but was not seen in sections until regeneration of the muscle was well advanced, at 4 days post inoculation. The results suggested that although dystrophin was present in the myofibres at 3 days, it was not incorporated into the plasma membrane until 4 days post inoculation. This may be due to the influence of the functional reinnervation of the regenerating fibres, which occurs at 4-5 days, or to the growing fibres reaching a critical diameter.

Animals↗
PubMed1992

The distribution of desmin and titin in normal and dystrophic human muscle.

We have used monoclonal antibodies to desmin and titin, and a combination of immunofluorescence and immunogold labelling to study the disposition of these two proteins in normal human muscle fibres and in fibres at various stages of degeneration in dystrophic muscle. The normal pattern of desmin labelling, in particular the subsarcolemmal labelling, became disrupted at an early stage of fibre breakdown. There was a change from a transverse to a longitudinal orientation of the labelled intermediate filaments as the myofibrils sheared relative to one another. Thus, while it is probable that the desmin filaments are able to play a role in the mechanical integration of the myofibrils in healthy muscle, our results suggest that they cannot withstand the excessive forces generated by the hypercontraction and stretching of dystrophic muscle. However, small accumulations of desmin persisted between the damaged myofibrils until necrosis reached an advanced stage. In general, the degradation of titin appeared to occur before the degradation of desmin, and at the ultrastructural level, labelling with antibodies to epitopes from parts of the titin molecule close to the A-I-band junction was lost before labelling with an antibody to an epitope in the A-band. This suggests that different regions of the titin molecule break down at different stages in the breakdown of the fibre. We propose that lysis of titin in the I-band may underlie 'slippage', an abnormality often seen in dystrophic muscle, in which the A-band slips to one pole of the sarcomere such that it abuts onto the Z-line. Breakdown of the A-band section of titin may facilitate the disassembly of the A-filaments.

Connectin↗
PubMed1992

Physiological and biochemical studies on the cerebellar cortex of the murine mutants "jolting" and "motor end-plate disease".

The activity of cerebellar Purkinje cells in the murine mutants "jolting" and "med" has been determined using extracellular electrical recordings in vivo and in isolated cerebellar slices. Most of the cells in the mutant brains failed to generate simple spontaneous action potentials, but they responded to climbing fibre inputs by generating complex potentials. The few mutant cells that were spontaneously active exhibited much lower firing frequencies than normal cells, and interval histograms of spontaneous activity were skewed towards longer intervals. The silent cells in mutant cerebellar slices could be activated by direct intracellular stimulation, by antidromic excitation and by the application of glutamate and high [K+]0. Activity was not restored by the application of bicuculline. It seems that the failure of the cerebellar Purkinje cells to generate simple spontaneous action potentials is not due to an inherent inexcitability of the soma or to the excessive activity of GABA-ergic inputs onto the cells. It is suggested that an abnormality in the behaviour of a Ca2+ channel is the most likely origin for the deficit in these mutant mice.

Aging↗
PubMed1992

The action of palytoxin on the isolated detrusor muscle of the rat.

1. The effects of a coelenterate toxin, palytoxin (PTX) have been studied in the isolated detrusor muscle. of the rat. 2. PTX (1-100 nM) initiated concentration-dependent contractions of the detrusor; the contraction led to an irreversible tachyphylaxis. Muscle desensitized to PTX continued to respond to acetylcholine (ACh) and excess K+ but the contractions were reduced compared to pre-PTX contractions. 3. Contractions evoked by PTX were not affected by the presence of atropine (10 microM), indomethacin (10 microM) or tetrodotoxin (0.5 microM) but were greatly reduced by nifedipine (3 microM) and by the absence of K+. PTX could not evoke contractions in the absence of Ca2+ or in tissues depolarized by exposure to excess K+. 4. PTX abolished the neurogenic contractile responses to electrical field stimulation (EFS). 5. Combined treatment with atropine (10 microM) plus nifedipine (3 microM) abolished contractile responses to EFS and greatly reduced the contractile response to PTX. 6. The contractile response to PTX (100 nM) was reduced following exposure of the muscle to alpha, beta-methylene ATP. 7. Exposure to PTX (100 nM) for 1-3 h reduced both the ACh content of the detrusor (by more than 80%), and the immunoreactivity of neuropeptide Y-containing nerve fibres compared to control. 8. It is concluded that the primary effect of PTX is to promote the release of endogenous motor transmitters, leading to their eventual depletion.

Acetylcholine↗
PubMed1992

Dystrophin or a "related protein" in Duchenne muscular dystrophy?

Previously we have shown low levels of dystrophin immunoreactivity in muscle from patients with DMD. According to the "frame-shift hypothesis" DMD muscle should not synthesize any dystrophin through to the C-terminus and it has been suggested that the protein detected is not dystrophin, but a related autosomal homologue. We have labelled serial sections of DMD muscle with specific monoclonal antibodies to the amino, rod and C-terminal domains of dystrophin and find labelling on the same individual fibres, allowing us to conclude that the protein detected is Xp21-encoded dystrophin. This has an impact on the interpretation of myoblast transfer experiments. The abundance (on blots) of "C-terminal dystrophin" appears lower than "rod dystrophin" in both BMD and DMD.

Antibodies, Monoclonal↗
PubMed1991

Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy.

We have identified 7 patients with Becker muscular dystrophy (BMD) in whom analysis of dystrophin by immunoblotting shows a full-sized molecule produced at reduced abundance compared with controls. They have no detectable deletion in their dystrophin cDNA. One patient presented atypically with unusually severe cramps as his only symptom for 25 years. These patients were investigated using the polymerase chain reaction (PCR) with 3 sets of primers within the promoter region of the dystrophin gene, followed by dot blot and restriction analysis. In the patient with the atypical history, one of the expected fragments on PCR failed to amplify. A large deletion was excluded by the finding of normally sized fragments on amplification with the other primer sets. The mutation was localised to the 3' end of the forward primer binding site by dot blot and restriction analysis. This result supports the hypothesis that, in patients with a full-sized dystrophin molecule produced at reduced abundance, the phenotype may result from a mutation in the promoter region of the dystrophin gene. The atypical history of the patient in whom this was detected adds to the variety of phenotypes now known to exist as BMD.

Adolescent↗
PubMed1991

Myosin isoform transitions and physiological properties of regenerated and re-innervated soleus muscles of the rat.

Soleus muscles in young female rats were destroyed by the local injection of the crude venom of the Australian tiger snake, Notechis scutatus and allowed to regenerate. The regenerated muscles consisted almost exclusively of type I muscle fibres, and histograms of fibre cross-sectional area were unimodal. In contrast the normal contralateral muscles consisted of a mixture of type I and IIa fibres, and histograms of fibre cross-sectional area were bimodal. There was no change in the ability of the regenerated muscles to generate tension in response to indirect stimulation, and muscle fibre number and motor unit index were similar to controls. The regenerated muscles often contained split muscle fibres, but if re-innervation of the regenerated muscles was delayed, splitting did not occur. The principal features of the regenerated muscles (fibre type homogeneity, unimodal histograms of muscle fibre area, normal numbers of muscle fibres and normal motor unit index) were reproduced in soleus muscles that were simply denervated by nerve crush and allowed to re-innervate. We conclude that the phenotypic homogeneity of the regenerated muscles is a consequence of the temporary disconnection of muscle from nerve, rather than of the cycle of degeneration/regeneration, and suggest that this disconnection results in the reprogramming of the soleus motor neurones.

Animals↗
PubMed1991

Immunogold labelling of dystrophin in human muscle, using an antibody to the last 17 amino acids of the C-terminus.

Immunolabelling with a 10 nm gold probe was used to localize dystrophin at the ultrastructural level in human skeletal muscle. The primary antibody was raised against a synthetic peptide containing the last 17 amino acids at the C-terminus of dystrophin. Using this antibody, labelling was almost entirely confined to a narrow band enclosing 40 nm either side of the plasma membrane and including the membrane itself. Histograms of the position of the gold probe relative to the plasma membrane showed modes lying over the membrane itself or the extracellular face of the membrane. One interpretation of these results is that the C-terminus of dystrophin is inserted in the plasma membrane alongside the glycoproteins with which it is tightly associated. Histograms of the distances between gold probes displayed modes at approximately 120 nm in both transverse and longitudinal sections suggesting that dystrophin forms a lattice-like network adjacent to the plasma membrane.

Amino Acid Sequence↗
PubMed1991

Long-term results of pectoralis major muscle transposition for infected sternotomy wounds.

During an 11.5-year period, 100 consecutive patients (79 male, 21 female) underwent repair of an infected sternotomy wound. Sixty-five patients had failed attempts at wound closure by other physicians. Median age was 61.5 years (range, 5 to 85 years). Reconstruction included muscle in 79 patients, omentum in 4, and both in 15. A total of 175 muscles were transposed, including 169 pectoralis major, 3 rectus abdominis, 2 external oblique, and 1 latissimus dorsi. Median number of operations was four (range, 1 to 11). Mechanical ventilation was required in 30 patients. Two perioperative deaths occurred, one related to sepsis. Median follow-up was 4.2 years (range, 1.3 to 13.5 years). Twenty-six patients had recurrent infection. Median time from our closure to recurrence was 5.5 months (range, 0.3 to 27.6 months). Cause of recurrence was inadequate removal of cartilage in 16 patients, bone in 6, and retained foreign body in 4. Eighteen patients had the wound reopened with further resection; 10 had another muscle or omentum transposition. There were 30 late deaths, only one related to recurrent infection. At the time of death or last follow-up, 92 patients had a healed chest wall. Transposition of the pectoralis major muscle remains an excellent method of management for infected sternotomy wounds. Failure is directly related to persistent infection of cartilage, bone, or retained foreign bodies.

Adolescent↗
PubMed1991

Neuromuscular transmission at newly formed neuromuscular junctions in the regenerating soleus muscle of the rat.

1. A study of neuromuscular function in regenerating skeletal muscle fibres in the rat soleus muscle has been made. The muscle fibres were damaged in vivo by the injection of the myotoxic venom component notexin, and then allowed to regenerate spontaneously. 2. Regenerating muscle fibres generated action potentials and contracted following direct intracellular stimulation as early as 4 days after the injection of notexin. 3. Miniature endplate potentials (MEPPs) were recorded at a minority of synapses at 3 days, and from all synapses by 5 days. The mean amplitude of MEPPs in a given fibre was directly proportional to muscle fibre input resistance. 4. Spontaneous transmitter release in the regenerating fibres was relatively insensitive to changes in [K+]0 but the effect of Ruthenium Red on spontaneous release was similar in the regenerating and control muscle fibres. 5. Functional innervation, defined as the ability to generate an indirect action potential, was restored in 97% of fibres by 10 days. The generation of an action potential was always associated with a twitch of the muscle fibre. Those fibres that were unable to generate an action potential usually exhibited a low membrane potential (ca -50 mV). These fibres could generate action potentials if they were hyperpolarized using an intracellular current-passing microelectrode. 6. The quantal content of endplate potentials (EPPs) was estimated from the mean EPP and mean MEPP amplitudes in cut muscle fibre preparations. These estimates suggested that quantal content was low at the earliest stages of regeneration, but increased as the muscle fibres matured and became normal at 10-21 days. 7. During repetitive stimulation at 30 Hz there was a fall in the amplitude of EPPs of 40-45%. The fall was similar in regenerating and control fibres. Conducting synapses never exhibited failure to generate an EPP during the period of high-frequency stimulation.

Action Potentials↗