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Biomedical subjects

B W Festoff

Publications and source records attributed to B W Festoff.

At least 109 records · Page 6Linked to original sources
PubMed1983

The identification of neurotrophic factor as a transferrin.

Partially purified neurotrophic factor (NTF) from chicken nerves comigrated with transferrin and a component in several preparations known to have neurotrophic effects on cultured skeletal muscle cells. One-dimensional gel electrophoretograms of proteolytic fragments of NTF and fragments obtained from transferrins purified from chicken eggs, serum and embryos were indistinguishable. These purified transferrins, like NTF, all stimulated the incorporation of [3H]thymidine and supported myotube formation to a similar degree as NTF. These studies suggest that NTF is a transferrin-like protein and that both transferrins and NTF act by initially promoting myoblast proliferation and subsequently supporting myogenesis in chick muscle cultures.

Animals↗
PubMed1983

Acetylcholine receptor turnover in clonal muscle cells: role of plasmin and effects of protease inhibitors.

Characteristics of acetylcholine receptors were evaluated in G8-1, a continuous skeletal muscle line. Peak binding of 125I-alpha-bungarotoxin was in 10-day-old contractile myotubes at 4-8 nm. Turnover was studied using two different methods; both indicated half-times as little as half as long as previously reported for primary cultures. The effects of a variety of protease inhibitors on receptor turnover were assessed to determine if G8-1 receptors were less stable or turned over faster because of increased neutral protease activity. Leupeptin, antipain, and chloroquine markedly slowed receptor degradation. Inhibitors of plasmin or plasminogen activator had definite but less dramatic effects on receptor turnover. Results from studies in which plasmin was increased in the tissue culture media indicated that a small but definite acceleration of receptor turnover occurred. In clonal G8-1 cells, total number of acetylcholine receptors is controlled by negative feedback and although the major pathway for receptor degradation is lysosomal, plasmin may play a role in initiating receptor internalization.

Animals↗
PubMed1983

Appearance of acetylcholinesterase molecular forms in noninnervated cultured primary chick muscle cells.

Asymmetric forms of AChE have generally not been detected in cultured chick skeletal muscle cells in the absence of cocultured neurons. To explore further neurotrophic effects of adult peripheral nerve extracts (NE) on muscle in vitro, we reexamined the appearance of various molecular forms of AChE in cultured chick muscle cells in the presence of NE. The various molecular forms of AChE were distinguished by sucrose gradient sedimentation and radioenzymatic techniques. In the presence of NE, cells proliferated during the first 48 hr of culture, then fused and formed spontaneously contracting myotubes by 6-8 days in culture. Total AChE, 5.4 S, and 11.5 S molecular forms reached activity plateaus by 8 days in culture which persisted until cultures were terminated at day 20. Between 1 and 6 days in culture, 19.5 S AChE (A12) was not detected. The A12 form was first observed at 7 days reaching a maximum of 11.3% of the total AChE at 14 days and then gradually declined to a level of 1% at day 20. Since the A12 form declined in older cultures but comprised 25% in embryonic muscle tissue, we examined the possible requirement of neurons in culture to attain higher levels of A12 AChE. Spinal cord neurons were plated onto 6-day muscle cultures and AChE activities were measured between 8 and 20 days. The results showed that 19.5 S AChE activity in the presence of both spinal cord neurons and NE was no greater than that found in the presence of NE alone. To suppress spontaneous contraction, 0.6 microM tetrodotoxin (TTX) or 15 microM d-tubocurarine (dTC) were added to 5-day-old muscle cultures at a time when myotubes were differentiated but contractile activity had not begun. TTX had cytotoxic effects and inhibited further development of myotubes. In contrast, dTC had no deleterious effect on morphological development, eliminated contraction, but did not interfere with the appearance of any forms of AChE including the A12 form. These studies show that primary chick muscle cells are capable of producing the A12 form of AChE if cultured in NE-supplemented medium. In this culture system, production of the A12 form does not require activity or innervation.

Acetylcholinesterase↗
PubMed1983

Occurrence of reduced alpha 2-macroglobulin and lowered protease inhibiting capacity in plasma of amyotrophic lateral sclerosis patients.

Lowered levels of plasma alpha 2-macroglobulin were found in 13 patients with amyotrophic lateral sclerosis using anti-alpha 2-macroglobulin-embedded agar plates. Levels of this major protease inhibitor in ALS patients were contrasted with those in disease controls, consisting of patients with a variety of neurologic disorders, as well as with those in normal individuals. The patients' levels, in milligrams per deciliter, were 191 +/- 39 (SD). The disease controls had levels of 269 +/- 33. Local normal values compared with the reported normal values in the literature (260 +/- 70). A correlation was not made for either the form of motor neuron disease, disease severity, or age. As a measure of functional protease inhibition, trypsin inhibitory capacity of sera was estimated using a radioactive fibrinolytic assay. An increase in the half-maximal inhibitory concentration of sera was found in the amyotrophic lateral sclerosis group. These findings likely relate to the known increases in proteolytic activity in denervated muscle and collagenolytic activity in skin of patients with amyotrophic lateral sclerosis. The determination of whether altered protease inhibitory potential in such patients is primary or secondary may help in understanding the pathogenesis of this enigmatic disorder and provide suggestions for future approaches to intervention.

Adult↗
PubMed1982

Extracellular matrix synthesis by skeletal muscle in culture. Proteins and effect of enzyme degradation.

Extracellular matrix proteins produced by a mouse skeletal muscle cell line, G8-1, were isolated and characterized. Cultures were incubated with [35S]methionine or [3H]glycine and [3H]proline, and the labeled, substrate-attached proteins were obtained after cellular proteins were extracted by deoxycholate in neutral salt. The labeled matrix was analyzed by gel electrophoresis and fluorography before and after enzymatic digestion. Of the nine major bands present in the matrix, four were identified. Fibronectin and collagen were detected on the bases of their relative mobilities, differential labeling with 3H-versus 35S-labeled amino acids and their solubilization by protease free collagenase. High molecular weight material which was present in the matrix was also sensitive to collagenase and probably included cross-linked collagen and laminin. Proteins co-migrating with actin and myosin were also present in the extracellular matrix. These results are novel in that they demonstrate that the skeletal muscle phenotype, not contaminated with fibroblastic elements, is able to synthesize basal lamina-type macromolecules and incorporate them into an insoluble, extracellular matrix. Since this cell line is able to form functional synaptic contacts with neuronal cells, the influence of nerve on basal lamina production by muscle in vitro is possible.

Animals↗
PubMed1982

Plasminogen activator: the major secreted neutral protease of cultured skeletal muscle cells.

Clonal mouse skeletal muscle cells which differentiate in culture and form synapses with neuronal cells were found to secrete high levels of protease activity as measured with an 125I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean trypsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occurred with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromuscular contacts in experimental and pathologic denervation.

Animals↗
PubMed1982

Dose-dependent initiation of myogenesis by neurotrophic factor.

The kinetics of primary chick muscle cells in response to partially purified neurotrophic factor (NTF) were investigated. Chick muscle cells are routinely plated in the absence of NTF but in the presence of horse serum to allow cell attachment to the substratum; a maximum number of cells attaches 5-8 hr after plating, but cells do not proliferate in the absence of NTF, or do so extremely slowly. Subsequent to a routine attachment period of 6 hr, the medium is aspirated and replaced with medium containing 2-20 micrograms/ml of NTF as well as horse serum. Dose responsiveness at elevated NTF concentrations is observed only if F12 medium, supplemented with serum, insulin, transferrin, and selenium is used. In the presence of commercial F10, although supplemented with serum, dose responsiveness is not apparent at more than 2 micrograms/ml of NTF. Upon exposure to NTF, in the presence of supplemented F12 and serum, an 8-hr lag ensues; then, thymidine is incorporated at NTF dose-dependent rates by growing cell populations. The rates of incorporation into cell populations depend on the initial number of cells plated and the amount of NTF supplied. Cell counting indicates that dose-dependent proliferation of muscle cells has occurred during the first 48 hr of exposure to NTF. The specific activity of NTF is repeatedly shown to be at least 10-fold greater than unfractionated nerve extract. Shortly after 48 hr of exposure to NTF, myoblasts characteristically begin to fuse with one another, and myogenesis is visible. These studies show that the initiation of in vitro myogenesis in primary chick muscle cells depends on the amount of NTF supplied. The work also indicates a requirement for optimum medium conditions in order to detect dose-responses to NTF.

Animals↗
PubMed1981

Posttetanic potentiation in a patient with myasthenia gravis.

We studied a patient with clinically typical myasthenia gravis (MG) and high serum titer of antibodies to acetylcholine receptor. Unlike the usual response in MG, there was an increment in the amplitude of the electrical response (308% of control) after 10 seconds of voluntary tetanus. Posttetanic facilitation is usually less than 200% in MG and over 200% in Eaton-Lambert syndrome. However, there have been several other cases of typical MG with increments over 200%. Facilitation of this magnitude has also been seen with curare administration in animals and man. However, in myasthenia, as opposed to curare poisoning, competitive blocking is not thought to exist.

Autoantibodies↗
PubMed1980

Bidirectional axonal transport of 16S acetylcholinesterase in rat sciatic nerve.

Axonal transport of the 16S Molecular form of acetylcholinesterase (16S-AChE) in doubly ligated rat sciatic nerves was studied by means of velocity sedimentation analysis on sucrose gradients. This form of AChE was selectively confined to motor, and not to sensory, fibers in the sciatic nerve, where it represented 3--4% of total AChE. Its activity increased linearly with time (4--20 hr) in nerve segments (7 mm) proximal to the central ligature (4.5 mU/24hr) and distal to the peripheral ligature (2.0 mU/24 hr). From the linear rates of accumulation of 16S-AChE, we conclude that the enzyme is conveyed by anterograde and retrograde axonal transport at velocities close to those previously defined for the movement of total AChE (410 mm/day, anterograde; 220 mm/day, retrograde). The transport of AChE molecular forms, other than the 16S form, could not be resolved presumably due to their presence in blood as well as at extraaxonal sites. The present findings are consistent with the view that in rat sciatic nerve most, if not all, of the small portion of total AChE (approximately 3%) which is transported may be accounted for by 16S-AChE.

Acetylcholinesterase↗
PubMed1980

Neuromuscular junction macromolecules in the pathogenesis of amyotrophic leteral sclerosis.

An hypothesis regarding the pathogenesis of amyotrophic lateral sclerosis is presented, which places emphasis on extraneural cells. Classical experimental denervation is compared and contrasted with motor neuron disease, both from information in the literature as well as concepts deriving from the hypothesis. Background information regarding neuromuscular junction-specific (16S) acetylcholinesterase and a basal lamina-enriched surface glycoprotein (fibronectin) are presented, which suggest not only their mutual interaction, but likely parallel regulation on muscle cell surfaces by the motor nerve. Since 16S acetylcholinesterase likely contains basal lamina-type collagen and fibronectin specifically associates with collagen, a model relating activation of latent collagenase enzyme in amyotrophic lateral sclerosis is described. It is suggested that continued degeneration, including transneuronal effects, of the motor system ensues from random, continuous loss of nerve-muscle adherence resulting from collagen resorption at the neuromuscular junction.

Acetylcholinesterase↗
PubMed1979

Evaluation of insulin receptor in myotonic dystrophy.

Only 2 of 6 patients with myotonic dystrophy had glucose intolerance and hyperinsulinemia. All, however, had markedly reduced insulin binding to specific receptors on circulating monocytes. A significant difference in receptor affinity for insulin was observed, but the difference in affinity was not as marked as that in the amount of 125I-insulin bound per cell, which was sixfold greater in control cells. No evidence for a circulating factor competing with insulin for binding sites could be demonstrated. These alterations in binding did not directly correlate with glucose intolerance or hyperinsulinemia and are consistent with a postulated generalized membrane defect. Study of the insulin receptor in this disorder may uncover regulatory mechanisms in normal and pathological conditions, including myotonic dystrophy.

Glucose↗
PubMed1979

Neurotrophic control of 16S acetylcholinesterase at the vertebrate neuromuscular junction.

Endplate 16S acetylcholinesterase (16S-AChE) from rat anterior gracilis muscle was assessed, 6 hr to 10 days after denervation, by velocity sedimentation analysis on linear sucrose gradients. The innervating obturator nerve was transected either close (1--2 mm, short stump) or far (35--40 mm, long stump) from the muscle. In both instances, the activity of 16S-AChE gradually decreased and reached approximately the same level (10%--20% of control) by 6 days after denervation. However, enzymatic decay started considerably earlier in short stump (12--24 hr) as compared to long stump (4--5 days preparations, i.e., the time of onset of 16S-AChE loss depended on the length of nerve that remained attached to the muscle. Whether this result extended to other AChE molecular forms (10S, 4S) in muscle endplates could not be determined because, in contrast to 16S-AChE, these forms were also detected in red blood cells (4S) and plasma (10S). Only small amounts of 16S-AChE were found in intact obturator nerves (1/100 of that in gracilis endplate regions). Thus a faster depletion of enzyme from shorter nerve stumps after axotomy could not entirely account for the substantial effect of nerve stump length on 16S-AChE. Since muscle contraction ceases immediately following nerve transection, regardless of nerve stump length, the results can be ascribed to the lack of some neural influence other than nerve-evoked muscle activity. The present findings are consistent with the view that maintenance of 16SAChE at neuromuscular junctions primarily depends on regulatory substances which are conveyed by axonal transport and released from nerve terminals.

Acetylcholinesterase↗
PubMed1979

Monovalent cation transport in myotonic dystrophy. Na-K pump ratio in erythrocytes.

Myotonic dystrophy is a dominantly-inherited disorder which affects skeletal muscle in combination with several other systems. Because of abnormalities in red blood cells, a universal membrane defect has been proposed as the primary disturbance. Erythrocyte cation pump ratios have also been reported to be abnormal. Hyperinsulinemia and glucose intolerance are present in a large number of patients. Since dramatic effects of insulin on membrane cation transport have been shown in several tissues, notably skeletal muscle, we wished first to confirm reports of altered pump ratio in these patients and then to evaluate the effects of insulin on cation fluxes. However, in our experiments myotonic dystrophy patients had normal pump ratios when compared with disease controls.

Carrier Proteins↗
PubMed1977

In vitro studies of skeletal muscle membranes. Adenylate cyclase of fast and slow twitch muscle and the effects of denervation.

Sarcolemmal membranes were prepared from slow-twitch (red) and fast-twitch (white) skeletal muscle of the rat. A sensitive adenylate cyclase assay was used and basal, fluoride- and catecholamine-stimulated activities measured. The greater in vivo sensitivity of red muscle to the effects of catecholamines correlates, in the present study, with approximately a twofold stimulation of its sarcolemmal adenylate cyclase with isoproterenol (10 micronm). The white muscle enzyme, on the other hand, is only minimally stimulated (20%) at the same concentration of beta-adrenergic agonist. Fast-twitch muscle is known to be physiologically insensitive to catecholamine in vivo. A course of sciatic nerve denervation was followed to further distinguish these two metabolic types of skeletal muscle and their respective adenylate cyclases. The slow-twitch muscle enzyme activities were completely and permanently lost on denervation. The white muscle enzyme, however, recovered almost completely after an initial reduction in specific activity the first week. Interestingly, the NaF-stimulated activity lagged behind both the basal and hormone-stimulated activities of the white muscle enzyme, in returning to control levels. The activities of cyclic nucleotide phosphodiesterase were evaluated in homogenates of the two muscle types in innervated rats and following denervation, in order to further define the neural influence on skeletal muscle cyclic nucleotide metabolism. The results suggest that the motor nerve may regulate some of the metabolic properties of slow-twitch muscle (which may involve cyclic AMP) by controlling the responsiveness of its sarcolemmal-bound adenylate cyclase system.

Adenylyl Cyclases↗