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Biomedical subjects

J B Harris

Publications and source records attributed to J B Harris.

At least 37 records · Page 2Linked to original sources
PubMed1997

Absence of calpain 3 in a form of limb-girdle muscular dystrophy (LGMD2A).

Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature alpha- and beta-dystroglycan proteins. The beta-dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processing of dystroglycan.

Animals↗
PubMed1997

The effect of palytoxin on neuromuscular junctions in the anococcygeus muscle of the rat.

Palytoxin, a highly toxic natural product isolated from zoanthids of the genus Palythoa, is accumulated by a wide range of fishes and marine invertebrates used as food in the Indo-Pacific. It is responsible for many incidents of human morbidity and mortality. The toxin is a potent smooth muscle spasmogen. The cause of the contraction of smooth muscle is unclear, but recent work strongly suggests that it is primarily initiated by the release of neurotransmitters from the motor innervation of the smooth muscle. We show here that palytoxin caused the swelling of the muscle cells and some internal organelles of the anococcygeus muscle of the rat, but no substantial structural damage to the tissue. Axons and Schwann cells were also swollen but the most dramatic feature was the depletion of synaptic vesicles from putative release sites in the axons. Some axons were physically damaged following exposure to the toxin, but this was relatively uncommon (< 10% of all axons studied). In the majority of axons there was no damage to nerve terminal membranes, but there was damage to mitochondria. The depletion of vesicles involved all types-clear, dense-cored, large and small. Our observations and pharmacological data gathered elsewhere, provide a neuropathological basis for the spasmogenic activity of palytoxin.

Acrylamides↗
PubMed1997

Determination of silicon in breast and capsular tissue from patients with breast implants performed by inductively coupled plasma emission spectroscopy. Comparison with tissue histology.

A method for analysis of silicon in tissue was developed to determine silicon content in breast parenchymal and periprosthetic capsular tissues of patients with silicone or saline implants and to compare levels in tissues from normal (nonaugmented) breasts. It is of interest to determine whether increased silicon content in tissues can be associated with morbidity in patients who have received silicone implants. This manuscript addresses the issues involved in analysis of breast tissue samples for silicon and compares silicon levels with tissue histologic findings and patient morbidity. One hundred sixty tissue samples were obtained for silicon analysis from 72 patients during augmentation, capsulectomy with or without replacement mammoplasty, mastectomy, or biopsy procedures and were frozen in acid-washed polystyrene tubes at 220 degrees C until analysis. Samples were thawed, sectioned to approximately 0.1 g (dry weight), and digested in nitric acid before analysis by inductively coupled plasma emission spectroscopy, monitoring emission intensity at 251.6 nm. Tissue silicon levels (breast parenchymal and periprosthetic capsular tissue) in patients with silicone gel implants were much higher (mean, 9,287 micrograms/g, n = 106) than in patients with saline implants (mean, 196 micrograms/g, n = 37) or nonaugmented breasts (mean, 64 micrograms/g, n = 17). Histologic examination was performed on 54 tissue samples stained with hematoxylin-eosin. Tissue samples were rated as to degree of inflammation and calcification, and amount of giant cells, foamy histiocytes, and vacuoles containing a colorless refractory material. Vacuolization and foamy histiocyte ratings correlated significantly with tissue silicon concentration. No correlations were found between tissue silicon concentration and inflammation, calcification, or giant cell rating. Implant age (number of years an implant was in place before sampling) correlated with capsular tissue silicon concentration in patients with intact implants but not in those with ruptured implants. No difference in tissue silicon concentration was found between patients with or without signs or symptoms of morbidity. Using 0.1 g of tissue, the method was linear to 1,000 micrograms/g, and sensitivity was 3.7 micrograms/g. Precision between runs (mean, 5.1 micrograms/g; coefficient of variance, 13.7%; n = 13) was calculated from multiple analyses of a bovine liver standard (National Bureau of Standards, reference material 1577a). Significant biologic variability (21.4% to 52.5%) was seen in tissues with high silicon levels. Paraffin-embedded, formalin-fixed tissues are not amenable to silicon analysis by this method, because of leaching of silicone from the tissues during preparation. Thus only fresh frozen tissue samples were used.

Age Factors↗
PubMed1997

Tuberculosis control in Asia and the western Pacific: a role for computer modelling.

Despite the availability of effective treatment, tuberculosis (TB) causes more deaths than any other infection. Most of the world's TB cases and deaths occur in Asia and the Western Pacific, and the growing prevalence of multiple drug-resistant TB and the spread of human immunodeficiency virus (HIV) present ever greater obstacles to effective TB control. The management of TB remains difficult, and epidemiologic studies to assess control programmes require significant time and expense and may not be generalizable to other regions. Computer models are powerful and relatively inexpensive tools for rapidly planning and evaluating TB control strategies. Models have demonstrated the value of targeted chemoprophylaxis strategies for the prevention of TB among HIV-infected individuals, and programmes to ensure that all HIV-infected individuals receive TB chemoprophylaxis should be considered in Asia and the Western Pacific. Though directly observed therapy (DOT) is effective when designed to be attractive to patients, modelling has shown that DOT, if poorly accepted by patients, may lead to fewer patients seeking care and thus to paradoxical rises in TB case rates. Models may be used to make accurate predictions of TB morbidity and programme costs using local epidemiologic and demographic inputs. The use of models in Asia and the Western Pacific offers a low-cost way to compare programmes, to improve the evaluation of programmes, to project future cases, and to examine programme needs while providing insights into TB control helpful to countries in and out of the region.

AIDS-Related Opportunistic Infections↗
PubMed1996

Oxidative damage to muscle protein in Duchenne muscular dystrophy.

The hypothesis that reactive free radical species (ROS) may contribute to the pathogenesis of Duchenne muscular dystrophy (DMD) has been suggested previously, but experimental data obtained in support of the above have to date proved inconclusive. The recent discovery that nitric oxide synthase (required for muscle relaxation) is associated with the sarcolemmal protein dystrophin normally and that both proteins are absent in DMD has heightened interest in the potential role of ROS in this disorder. We therefore investigated oxidative damage to proteins in the quadriceps femoris muscle by quantifying protein carbonyl levels in six patients with DMD and six normal controls. In DMD, the mean protein carbonyl level in the quadriceps femoris muscle was increased by 211% (p < 0.005) compared with the normal control subjects. The data thus support the hypothesis for the role of ROS induced protein oxidation of muscle cell damage in DMD.

Adolescent↗
PubMed1996

Mutation detection in the med and medJ alleles of the sodium channel Scn8a. Unusual splicing due to a minor class AT-AC intron.

Analysis of a transgene-induced mutation at the mouse med locus led to the identification of the novel voltage-gated sodium channel gene Scn8a (Burgess, D. L., Kohrman, D. C., Galt, J., Plummer, N. W., Jones, J. M., Spear, B., and Meisler, M. H.(1995) Nat. Genet. 10, 461-465). We now report the identification of splicing defects in two spontaneous mutations of Scn8a. The original med mutation was caused by insertion of a truncated LINE element into exon 2 of Scn8a. The med transcript is spliced from exon 1 to a cryptic acceptor site in intron 2. A 4-base pair deletion within the 5' donor site of exon 3 in the medJ allele results in splicing from exon 1 to exon 4. Both mutant transcripts have altered reading frames with premature stop codons close to the N terminus of the protein. Loss of Scn8a expression results in progressive paralysis and early death. Intron 2 of Scn8a is flanked by minor class AT-AC splice sites. The observed splicing patterns of the med and medJ mutant transcripts provide the first evidence for preferential in vivo splicing between donor and acceptor sites of the same class. The apparent functional incompatibility may be a consequence of the different composition of spliceosomes bound to major and minor splice sites.

Alleles↗
PubMed1996

Differential susceptibility of human skeletal muscle proteins to free radical induced oxidative damage: a histochemical, immunocytochemical and electron microscopical study in vitro.

In this study we describe a novel experimental approach to quantify the relative susceptibility of (membrane-associated, contractile and mitochondrial) proteins in normal human muscle tissue sections to oxidative damage by the reactive oxygen species (ROS), hydroxyl (OH.) or superoxide (O2.-) radicals. The latter species were generated under controlled experimental conditions in vitro using a 60Co gamma radiation source, with subsequent analysis of damage to target proteins (dystrophin, beta-dystroglycan, beta-spectrin, fast and slow myosin heavy chain, NADH tetrazolium reductase, succinate dehydrogenase and cytochrome oxidase) via standard histochemistry, immunocytochemistry and electron microscopy of muscle tissue sections. In general terms, each of the proteins listed above was more susceptible to oxidative damage by OH., compared to O2.-. Different proteins (differing in structure, function or intracellular localisation) showed different susceptibility to oxidative damage, with certain mitochondrial proteins (succinate dehydrogenase, cytochrome oxidase) showing particular susceptibility. In addition, the use of monoclonal antibodies to four different regions of dystrophin showed the latter to contain both resistant and susceptible regions to ROS induced oxidative damage. At the ultrastructural level of subcellular organelle damage, mitochondria were identified as being particularly susceptible to ROS induced oxidative damage. We therefore speculate that oxidative damage to mitochondria and/or mitochondrial proteins may represent the principal initial route of free radical-induced damage within skeletal muscle tissue.

Dystrophin↗
PubMed1996

Comparison of protease and related enzyme activities in snake venoms.

We identified the presence of a range of proteolytic enzyme types, normally associated with the process of general intracellular protein catabolism in mammalian tissues, in venom samples from several species of snake and from three subspecies of Russell's viper. Although levels of protease activity in venoms were in general substantially lower than corresponding levels in mammalian tissues, activity levels were comparable with several other classes of enzyme normally considered as significant venom components. Based on the protease types and relative levels of activity present in venom samples, we suggest that a possible function of these enzymes (in addition to their generally held function to increase target tissue permeability to other venom components) may be to interfere with the process of neurotransmission in target tissues, via degradation of neurotransmitter/neuromodulatory oligopeptides; this may be particularly the case for proteases such as leucyl aminopeptidase, the activity of which is greater in some venom types than in mammalian tissues. For the purposes of inter- and intra-species taxonomic classification of snakes, we would suggest that determination of a comprehensive venom protease profile may be of considerable value (particularly for subspecies differentiation) either in conjunction with or in place of more conventionally applied techniques such as analytical electrophoresis.

Animals↗
PubMed1996

Myotoxic activity of the toxic phospholipase, notexin, from the venom of the Australian tiger snake.

Notexin is a neurotoxic and myotoxic phospholipase A2 derived from the venom of the Australian tiger snake, Notechis scutatus. Though the phospholipase activity has been implicated in the toxicity of notexin, little is understood of its site and mode of action. In this study we investigated the myotoxicity of notexin on the skeletal muscle of the rat. Using immunogold labeling both in vivo and in vitro, we demonstrated that notexin bound exclusively to the sarcolemma. At the early stages when notexin was injected into the muscle there was no evidence of internalization, though at more advanced degeneration when muscle fibers were necrotic, the toxin was able to penetrate the interior of the fibers to exhibit nonspecific labeling. We also showed the toxin was able to bind to glycolytic muscle fibers, which are known to be resistant to the myotoxic effects of notexin. Electron microscopy confirmed the localization of the binding site to the sarcolemma. Scanning electron microscopy showed that the primary pathological changes associated with exposure to notexin were the appearance of areas of hypercontraction along the muscle fibers associated with small lesions in the sarcolemma. At more advanced stages large tears appeared in the sarcolemma between adjacent areas of hypercontraction, revealing apparently intact myofibrils below. We conclude that the sarcolemma is the binding site for the toxin. We propose that the hydrolytic activity causes the appearance of small lesions in the membrane, the loss of ion gradients, and hypercontraction. The weakened area between sites of hypercontraction is then ruptured, leading ultimately to the degeneration of the muscle fibers.

Animals↗
PubMed1996

Silicon analysis of breast and periprosthetic capsular tissue from patients with saline or silicone gel breast implants.

The ubiquitousness of silicon is well known. Recent work has demonstrated measurable baseline levels of silicon in nonaugmented cadavers, subsequent to numerous reports of significant elevations of such levels within patients with silicone breast implants and even more reports alleging a causal relation between silicone gel prostheses and connective-tissue diseases. Despite the lack of scientifically substantiated data that such a relation exists, the calamitous silicone breast implant controversy has ensued. Saline-filled breast implants are constructed with a silicone elastomer envelope that remains in direct contact with periprosthetic capsular tissue following implantation. Although there is no evidence to link saline implants with any disorders, it is important to know if saline breast implants contribute any silicon to human body baseline silicon levels. The present study measured tissue silicon levels in 28 breasts of 16 patients with saline-filled implants to determine if the silicone envelope of these prostheses can contribute to the elevation of such levels. These data were compared with data from 116 breasts of 65 patients with silicone gel-filled prostheses as well as breast tissue from 17 patients (controls) who had never been exposed to either type of implant. Samples of breast tissue and periprosthetic capsular tissue were obtained from patients with both intact and ruptured implants. Silicon levels of breast tissue specimens from patients with saline-filled implants were within the range of the controls if the implants were intact. Silicon levels in periprosthetic capsular tissue from patients with intact saline-filled implants were significantly higher than controls (p < 0.02); however, they were still 100-fold less than capsular tissue levels from patients with intact gel-filled implants. Silicon levels measured in both types of tissue were significantly elevated in patients with silicone gel-filled implants compared with controls (p < 0.01). In the case of ruptured gel implants, breast tissue demonstrated higher silicon levels than did similar specimens from patients with intact implants (p < 0.054); periprosthetic capsular tissue levels also were elevated, although the differences were not statistically significant (p = 0.54). These findings are independent of the implant brand or length of exposure to the particular prosthesis. The finding of elevated levels of silicon in both breast and periprosthetic capsular tissue in patients with silicone gel-filled implants in no way implies or substantiates any claim of a causal relationship between silicone and any reported illnesses.

Adult↗
PubMed1995

Effect of neurotoxic metal ions in vitro on proteolytic enzyme activities in human cerebral cortex.

In order to develop a clearer understanding of the role of aberrant protein turnover in the pathogenesis of neurodegenerative disorders, the effect of a series of potentially neurotoxic metal ions on a wide range of proteases (lysosomal and cytoplasmic proteinases and peptidases) from human cerebral cortex was determined in vitro. The response of lysosomal and cytoplasmic proteases to inhibition by metal ion species (0.05-5 mmol/l) was broadly similar; Sr2+, Mg2+, Ba2+ or Ca2+ showed little inhibitory effect at any concentration for most protease types, whilst Cu2+, Cd2+, Pb2+, Mg2+ or Zn2+ showed a substantial degree of inhibition, depending on metal ion concentration and enzyme type. Ca2+ activated neutral proteinases were no more susceptible to general metal ion inhibition than most other protease types. Some proteases showed marked activation of activity in the presence of several metal ion species. Both lysosomal and cytoplasmic proteases were relatively insensitive to inhibition by Al3+, compared with that obtained with other metal ion species. It is of note that cathepsin D was particularly resistant to inhibition by most metal ion species, whilst pyroglutamyl aminopeptidase was particularly susceptible to inhibition by low concentrations of many metal ions. The above data suggest that in considering the potential role of neurotoxic metal ions in the pathogenesis of neurodegenerative disorders of the CNS (via protease inhibition in the intracellular protein degradation pathway), attention should be focused on the interactions between a wide range of metal ion species and protease types, rather than be restricted to the Al3+/calpain system (as is presently the case in Alzheimer's disease research). In particular, the potential role of pyroglutamyl aminopeptidase in intracellular protein degradation (in addition to more specialized functions such as neurotransmitter processing) and the pathological consequences of the susceptibility of this enzyme to inhibition by neurotoxic metal ions requires further investigation.

Aluminum↗
PubMed1995

Neuromuscular effects of Papuan Taipan snake venom.

Snakebite is a cause of significant morbidity in Central Province, Papua New Guinea. Three adult patients with clinical evidence of neurotoxicity following envenomation by the Papuan taipan had serial neurophysiological examinations over the course of their subsequent hospitalization. All required artificial ventilation for 2.5 to 5 days. The compound muscle action potential (CMAP) amplitudes declined over the first 2 to 4 days after envenoming and then gradually increased in parallel with clinical recovery. Repetitive stimulation studies revealed a distinctive pattern of abnormality. Activation resulted in brief potentiation of the CMAP followed by significantly greater decrement than observed at rest. This effect lasted up to 30 minutes and was not altered after intravenous edrophonium. Single-fiber electromyographic recordings during the recovery phase of the illness were abnormal with marked blocking and increased jitter. All patients were able to return home.

Adult↗
PubMed1995

Comparison of structural protein and proteolytic enzyme levels in degenerating and regenerating rat muscle induced by Notechis scutatus venom.

To develop a clear understanding of the biochemical mechanism of muscle degeneration and regeneration induced by a single dose of Notechis scutatus scutatus venom, we have correlated changes in the levels of a series of muscle structural proteins and proteolytic enzymes. The degradation of structural proteins post-injection fell into two broad groups; those completely degraded within 3-6 hr (e.g. C- and M-proteins, skelemin), and within 1-2 days (e.g. myosin, actin, troponin), respectively. Similarly, activation of proteases followed two general patterns; those enzymes showing substantially increased activity after 12-24 hr (lysosomal cathepsins, leucyl aminopeptidase) and those enzymes showing decreased activity after 12-24 hr, with substantially increased activity after 3-4 days (mainly cytoplasmic proteases). The data suggest that activation of cathepsins B, L and D and in particular leucyl aminopeptidase, may be responsible for the early stages of structural protein catabolism, and are thus potential therapeutic targets to prevent myonecrosis following envenomation.

Actins↗
PubMed1995

The expression of dystrophin-associated glycoproteins during skeletal muscle degeneration and regeneration. An immunofluorescence study.

The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.

Animals↗
PubMed1994

Screening of snake venoms for neurotoxic and myotoxic effects using simple in vitro preparations from rodents and chicks.

Eight snake venoms designated by the WHO as International Reference Venoms, and one additional venom were assessed for neurotoxic and myotoxic effects in vitro using the chick biventer cervicis and the rat and mouse phrenic nerve-diaphragm preparations. The objective was to determine whether any of the preparations could be used to detect evidence of neurotoxic or myotoxic activity prior to a more detailed examination. Bungarus multicinctus venom at concentrations above 1 microgram ml-1 selectively blocked neuromuscular transmission, with no direct effect on muscle fibres. Naja naja kaouthia and Notechis scutatus venoms selectively blocked neuromuscular transmission at low concentrations, but at higher concentrations both venoms caused direct effects on skeletal muscle resulting in contractures, loss of tension following direct stimulation and a loss in sensitivity to elevated [K+]0. Vipera russelli (Thailand) venom also blocked neuromuscular transmission but it was less potent than the venoms of B. multicinctus, N. n. kaouthia and N. scutatus. It also caused contractures in the chick biventer cervicis muscle. The venoms of Echis carinatus (Iran and Mali), Crotalus atrox, Bothrops atrox asper and Trimeresurus flavoviridis had limited neuromuscular blocking activity, and most of these venoms blocked [K+]0 and cholinoceptor stimulation in the chick muscle. Although both chick and rodent muscles allowed the assessment of neurotoxic and myotoxic activity, the chick biventer cervicis was simpler and more robust in use than either of the rodent phrenic nerve-diaphragm preparations. We propose that the chick biventer cervicis muscle could be used as a standard preparation for the screening of snake venoms for neurotoxic and myotoxic effects, and that it may be possible to use this preparation as a means to check that antivenoms can neutralize neurotoxic and direct myotoxic actions of venoms.

Animals↗
PubMed1994

The effects of five phospholipases A2 from the venom of king brown snake, Pseudechis australis, on nerve and muscle.

The effects on vertebrate neuromuscular function of five homologous phospholipases A2 (PLA2) (Pa-3, Pa-8, Pa-9C, Pa-10F and Pa-12B) from the venom of the Australian king brown snake, Pseudechis australis, were determined. These isoenzymes (0.2-1.6 microM) reduced, with different potencies, responses of chick biventer cervicis preparations to nerve stimulation and to exogenously applied acetylcholine, carbachol and KCl in a time- and concentration-dependent way but with different potencies. They also blocked twitches of mouse hemidiaphragm preparations evoked by nerve and by direct muscle stimulation. Pa-8 was the most active and Pa-9C was the least potent. There was a strong correlation between the enzymatic activity and the effect of toxins on the responses of mouse hemidiaphragm to direct muscle stimulation, but weak correlation between the effects on indirect responses and enzymatic activity. Intracellular recording from endplate regions of mouse triangularis sterni nerve-muscle preparations showed that Pa-10F and Pa-12B at 0.2 microM significantly reduced quantal content after 10 min. Pa-8 (0.2 microM) reduced the amplitude of endplate potentials by about 25% and abolished miniature endplate potentials within 15 min. Pa-3 (0.2 microM) and Pa-9C (0.8 microM) also significantly reduced quantal content by about 30% of control after 30 min. Among these toxins, Pa-3 and Pa-8 at 0.2 microM depolarised mouse muscle fibres after 30 min. Extracellular recording of action potentials at motor nerve terminals of mouse triangularis sterni preparations indicated that these isoenzymes reduced the waveforms associated with both Na+ and K+ conductances. Since no facilitatory effect on the release process has been observed, the apparent blockade of K+ conductance by some of these toxins may not be a selective action on K+ channels, but may be secondary to membrane depolarisation. An in vivo study with Pa-8 and Pa-10F demonstrated myotoxic effects. Light microscopic examination showed a degeneration of mouse and rat skeletal muscle fibres caused by Pa-8 and Pa-10F. For the in vivo study, rats received 80 micrograms/kg of the toxins s.c. and mice were injected i.m. with the toxins (40 micrograms/kg). Myotoxicity appears to be the predominant effect of these five toxins.

Acetylcholine↗