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Biomedical subjects

J B Harris

Publications and source records attributed to J B Harris.

At least 73 records · Page 4Linked to original sources
PubMed1991

Rat lung alveolar type I epithelial cell injury and response to hyperoxia.

Hyperoxia has been shown to cause extensive lung injury, which involves all components of the alveolar septum, although the type I epithelium has generally been reported to be resistant to significant injury. Electron microscopic morphometry was performed to define changes in volumes of subcellular components of alveolar epithelial cells in rats exposed to 85% O2 for 0, 7, and 14 d. Because of their large size, type I cells in control animals actually contain a greater volume of most of the organelles involved in cell metabolism than do type II cells. Hyperoxic exposure causes a dramatic change in the subcellular composition of the average type I cell, suggesting significant injury and/or response. Injury was suggested by the finding that lysosomes plus peroxisomes increased 1,250% after 7 d in hyperoxia and remained elevated by 200% after 14 d of exposure. Volumes of mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum, and Golgi apparatus increased by 100%, 51%, 91%, and 500%, respectively, after hyperoxia. Qualitative analysis showed an altered, ruffled air border with focal areas of cytoplasmic translucency (suggesting injury) and focal areas of subcellular hypertrophy. Exposure to hyperoxia was associated with more organelles being found in peripheral or attenuated portions of type I alveolar cells. Since the increase in type I organelles exceeds the volume of these organelles in its progenitor, the type II cell, it is likely that hyperoxia causes hypertrophy of the type I alveolar epithelium itself, independent of simple type II cell differentiation. Because of the large size and wide distribution of the type I cell, dramatic shifts in cell substructure caused by hyperoxia are more difficult to detect and require quantitative analysis to fully ascertain the extent of cell alterations.

Animals↗
PubMed1990

Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling.

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

Cell Membrane↗
PubMed1990

Very mild muscular dystrophy associated with the deletion of 46% of dystrophin.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), a much milder form of the disease where the age of onset can sometimes be as late as the third or fourth decade of life, are caused by mutations in the same X-linked gene, a 14 kilobase (kb) transcript which is spread over more than 2 megabases of the human X chromosome. The corresponding protein, dystrophin, has a relative molecular mass of 400,000. Most mutations causing DMD and BMD are deletions and deletions associated with both phenotypes are observed throughout the gene sequence. This observation led to the suggestion that DMD patients possess deletions that disrupt the reading frame of the protein, whereas BMD patients have deletions that retain the translational reading frame and enable the muscle cells to produce altered dystrophin products. This theory is supported by immunoblotting studies, which show that DMD patients lack dystrophin in their muscle cells or that dystrophin is present at very low levels, whereas BMD patients produce a protein with reduced abundance or abnormal size. Here we describe a deletion of the dystrophin gene in a family segregating for very mild BMD, one member of which was still ambulant at age 61 years, which removes a central part of the dystrophin gene encompassing 5,106 base pairs of coding sequence, almost half the coding information. Immunological analysis of muscle from one of the patients demonstrates that this mutation results in the production of a truncated polypeptide localized correctly in the muscle cell. These results are particularly significant in the context of gene therapy which, if it is ever envisaged, would be facilitated by the replacement of the very large dystrophin gene with a more manipulatable mini-gene construct.

Blotting, Western↗
PubMed1990

Radioimmunotherapy of human colon cancer in nude mice.

Nude mice bearing subcutaneous human colon cancer xenografts (LS174T) were treated with 120 microCi of yttrium 90-labeled anti-carcinoembryonic antigen monoclonal antibodies (specific therapy), 120 microCi of 90Y-labeled anti-melanoma monoclonal antibodies (nonspecific therapy), or phosphate-buffered saline solution (no treatment control). Mean (+/- SD) tumor growth rates (percent increase per day) over the first 30 days of the study were as follows: 0.6% +/- 0.2% per day (specific therapy); 17.7% +/- 5.7% per day (nonspecific therapy); and 30.5% +/- 4.2% per day (control). In all three groups, tumors over 1 g had similar doubling times (5.74 +/- 0.71 d). Specific therapy caused a lag in tumor growth corresponding to a 3-logarithm cell kill. Estimated tumor dose of radiation obtained by tissue analysis was 34 and 14 Gy for specific and nonspecific therapy, respectively. In conclusion, 120 microCi of 90Y-labeled anti-carcinoembryonic antigen monoclonal antibodies was effective in suppressing growth of human colon cancer xenografts. Clinical studies with this preparation are recommended.

Animals↗
PubMed1990

Heterogeneity of dystrophin expression in patients with Duchenne and Becker muscular dystrophy.

This report documents the results of an integrated biochemical and immunocytochemical investigation into the expression of dystrophin (the protein product of the Duchenne muscular dystrophy gene) in muscle biopsies from 226 patients. It is the first study in which dystrophin has been analysed on blots and on tissue sections in such a large number of patients using the same (monoclonal) antibody. The 140 patients with Xp21 muscular dystrophy who were included in this study represent a continuous spectrum of disease severity and this range was reflected in the heterogeneity of dystrophin expression which was observed with respect to abundance, size and the pattern of tissue localisation. Approximately 40% of biopsies obtained from patients diagnosed as having Duchenne muscular dystrophy (DMD) contained isolated clearly positive fibres and a further 20% had very weak labelling on a large number of fibres. Biopsies from patients with Becker muscular dystrophy (BMD) showed labelling patterns which varied from weak labelling on the majority of fibres to clear labelling on all fibres. Typically, however, there was inter- and intra-fibre variation in labelling intensity. Approximately 85% of the 52 BMD and 54 DMD patients who had unequivocal labelling on blots demonstrated a protein of abnormal size. The remaining 15% had a protein of normal size but reduced abundance. Overall, the estimated abundance of dystrophin correlated well with clinical assessments of the disease severity expressed in patients. We conclude that dystrophin analysis is an essential and dependable technique for the differential diagnosis of patients with Xp21 muscular dystrophy.

Adolescent↗
PubMed1990

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles.

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.

Animals↗
PubMed1990

Effects of notexin on neuromuscular transmission in the guinea pig vas deferens.

The effects of notexin on neuromuscular transmission in the isolated vas deferens of the guinea pig have been investigated using intracellular recording techniques. The toxin had no effect on the resting membrane potential, the amplitude or frequency of spontaneous excitatory junctional potentials or on the time constant of the decay of the evoked excitatory junctional potential in the smooth muscle cells. The amplitude of the evoked excitatory junctional potential was selectively reduced in the presence of notexin. The reduction in amplitude lasted for 20-30 min. Recovery occurred despite the continued presence of the toxin. Once recovery had been achieved the preparation remained insensitive to further exposure to the toxin. Although the amplitude of excitatory junctional potentials recovered in the continued presence of notexin, facilitation remained depressed. Action potentials, when generated, were indistinguishable from those in control tissues. It is concluded that notexin is a presynaptic inhibitor of transmission in the guinea pig vas deferens.

Action Potentials↗
PubMed1990

The assessment of muscle fibre loss after the injection of the venom of Notechis scutatus (Australian tiger snake).

We have used the W.H.O. International Reference Venom from the Australian tiger snake, Notechis scutatus, to study possible methods for the assessment of local myonecrosis caused by this venom. We made subcutaneous injections of various doses (0.25-20.0 micrograms) of venom into the antero-lateral aspect of the rat hind limb. The soleus muscle was removed after 24 hr and muscle fibre loss calculated from photo-montages of histological sections. Muscle tissue which had been either frozen or wax-embedded was preferable to resin-embedded tissue for making muscle fibre counts. There was a dose-dependent relationship between muscle fibre loss and the amount of venom inoculated. One microgramme of crude venom caused the loss of 50% of muscle fibres from the soleus muscle. This dose of venom neutralized by 1.5 microliters of the W.H.O. International Standard Antivenom for Notechis scutatus. Muscle wet weight increased following the inoculation of venom, to reach a peak of 42% at a dose of 0.5 microgram. There was no correlation between fibre loss and increase in wet weight. Biochemical analysis of both the venom-damaged muscle and the plasma showed that there was a strong linear correlation (r = 0.95) between loss of muscle aspartate aminotransferase and muscle fibre loss. There was a non-linear relationship between muscle fibre loss and the increase of plasma aspartate aminotransferase (EC 2.6.1.1). There was no correlation between either the loss of muscle creatine kinase or the increase of plasma creatine kinase and muscle fibre loss. We conclude that direct measurements are required to calculate muscle fibre loss with precision, but that the loss of muscle aspartate aminotransferase AST and its release into the plasma may also be important criteria to be used when studying local necrosis.

Animals↗
PubMed1990

Comparison of the intensity and duration of effects of inhaled bitolterol and albuterol on airway caliber and airway responsiveness to histamine.

Inhaled beta-agonists can produce bronchodilatation and reduce airway hyperreactivity in patients with asthma. Using these two measures, we compared inhaled bitolterol (three puffs, 1110 micrograms), albuterol (two puffs, 180 micrograms), and placebo administered by metered-dose inhaler in a blinded, crossover study of 40 subjects with chronic asthma. On each study day, subjects underwent histamine challenges at 1 1/2 hours before, and 1/2, 2, 4, 6, and 8 hours after inhaling one of the three test-drug treatments. Both drugs produced significant bronchodilatation at 30 minutes through 4 hours and significant effects on airway reactivity at 30 minutes through 2 hours (p less than 0.05). Bitolterol also produced small but significant bronchodilator effects at 6 hours and effects on airway reactivity at 4 hours (p less than 0.05). Effects of bitolterol on airway reactivity diminished significantly more slowly than effects of albuterol in subjects with baseline provocative concentration causing a 20% fall in FEV1 greater than or equal to 1.0 mg/ml of histamine (half-life of biologic effect 1.37 versus 0.92 hours; p less than 0.05) but not in subjects with baseline provocative concentration causing a 20% fall in FEV1 less than or equal to 1.0 mg/ml (half-life of biologic effect of 1.01 versus 1.00 hours; p greater than 0.05).

Administration, Inhalation↗
PubMed1990

Muscle necrosis caused by snake venoms and toxins.

Snake bite is often associated with localised soft tissue necrosis. Less frequently victims may suffer extensive muscle damage leading to rhabdomyolysis and the loss of muscle-specific protein. This review describes the organisation and structure of mammalian skeletal muscle, and its response to myotoxic venoms and to isolated pure myotoxic venom fractions. The clinical reports of muscle damage in man following snake bite are discussed, and the various classes of myotoxic toxins are introduced. Muscle damage caused by the toxins is next described, particular emphasis being placed on the correlation between muscle pathology seen at the light level and the morphological changes seen at the level of the electron microscope. Where known, those subcellular components of the muscle fibre that are especially sensitive to assault, and those components that appear to be spared, are identified. The relevance of the selective sparing of some components to the regenerative capacity of the skeletal muscle is considered.

Animals↗
PubMed1989

Dystrophin in skeletal muscle. I. Western blot analysis using a monoclonal antibody.

The value of analysing dystrophin on Western blots of skeletal muscle for the differential diagnosis of Xp21 muscular dystrophies is now fairly well established. Here we describe a sensitive system based on monoclonal antibodies to dystrophin. The specificity of the antibodies was established and experiments were undertaken to identify the source of dystrophin-related protein bands which were detected on blots of normal skeletal muscle. These investigations formed a necessary preliminary study to the application of the assay to samples of muscle obtained at biopsy from patients with Duchenne and Becker muscular dystrophy.

Antibodies, Monoclonal↗
PubMed1989

Dystrophin in skeletal muscle. II. Immunoreactivity in patients with Xp21 muscular dystrophy.

In the preceding paper a sensitive Western blotting analysis system based on the use of a monoclonal antibody to dystrophin was described. Here we report the immunoreactivity on blots and on unfixed frozen sections of muscle from patients with Duchenne (DMD) and Becker (BMD) muscular dystrophy. Muscle from 3 BMD patients showed variation both in the band pattern observed on blots and in the immunocytochemical labelling of dystrophin on frozen sections. In contrast to previous reports, we were able to detect some minor dystrophin bands on blots from 6 of 9 DMD biopsy samples. Tissue sections from 8 of the 9 contained isolated fibres with dystrophin-positive labelling. We conclude that the majority of DMD patients have muscle fibres which can synthesize dystrophin in a limited manner.

Adolescent↗
PubMed1989

Dye injection confirms the electrophysiological identification of regenerating muscle fibres in the rat.

Microelectrodes were inserted at random into superficial fibres in rat regenerating soleus muscles. The fibres from which recordings of electrical properties were made were marked using the dye Lucifer Yellow CH and then processed for histological studies. It was shown that the microelectrodes had sampled small regenerating muscle fibres, and that random penetration does not select for any undamaged fibres that may be present.

Animals↗
PubMed1989

Improved diagnosis of Becker muscular dystrophy by dystrophin testing.

We assessed the quantity (relative cellular abundance) and quality (approximate molecular weight) of dystrophin in muscle biopsies from 97 patients with a diagnosis of possible Becker muscular dystrophy. Fifty-four (all male) had dystrophin abnormalities and were deemed to have true Becker muscular dystrophy. The other 43 patients (14 female, 29 male) had no detectable dystrophin abnormalities. Of the dystrophin-verified Becker dystrophy patients, 35% (19/54) had a family history consistent with X-linked recessive inheritance. On the other hand, none of the 43 patients with apparently normal dystrophin had a clear X-linked family history, suggesting that few of these 43 actually had a form of Becker dystrophy. The data suggest that of all patients with a clinical picture consistent with Becker dystrophy but no family history, about 60% will be true Becker patients. The correlation of both the biochemical and clinical data suggests that Duchenne/Becker dystrophy can be divided into 4 clinically useful categories: Duchenne dystrophy (wheelchair at about age 11 years; dystrophin quantity less than 3% of normal); severe Becker dystrophy (wheelchair age 13 to 20 years; dystrophin 3% to 10%); and moderate/mild Becker dystrophy (wheelchair greater than 20 years; dystrophin quantity greater than or equal to 20%). Given the observed clinical variability of Becker dystrophy, it appears that dystrophin analysis is required for accurately distinguishing between Becker dystrophy and clinically similar autosomal recessive myopathies.

Adolescent↗
PubMed1988

Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.

A deficiency of the protein dystrophin has recently been shown to be the probable cause of Duchenne's muscular dystrophy. We sought to determine the relation between the clinical phenotype and the status of dystrophin in muscle-biopsy specimens from 103 patients with various neuromuscular disorders. We found very low levels (less than 3 percent of normal levels) or no dystrophin in the severe Duchenne phenotype (35 of 38 patients), low concentrations of dystrophin in the intermediate (outlier) phenotype (4 of 7), and dystrophin of abnormal molecular weight in the mild Becker phenotype (12 of 18). Normal levels of dystrophin of normal molecular weight were found in nearly all the patients (38 of 40) with 20 other neuromuscular disorders we studied. These data show the clinical consequences of both quantitative alterations (in Duchenne's and intermediate dystrophy) in a single protein. The biochemical assay for dystrophin should prove helpful in delineating myopathies that overlap clinically with Duchenne's and Becker's dystrophies, and it shows promise as an accurate diagnostic tool.

Adolescent↗
PubMed1988

Neuronal anti-nuclear antibody in paraneoplastic sensory neuronopathy.

Sera from 3 patients with a paraneoplastic sensory neuronopathy and small cell carcinoma of the lung were found to contain an IgG directed against neuronal nuclei and the nuclei of cells in adrenal medulla. Plasma from one of these patients was injected into laboratory mice for 100 days but passive transfer of the syndrome could not be effected. Plasma did not affect the viability of the dorsal root ganglia cells in tissue culture but did appear to bind to their nuclei. The role of this antibody remains uncertain.

Aged↗