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V Askanas

Publications and source records attributed to V Askanas.

At least 73 records · Page 4Linked to original sources
PubMed1993

Long-term treatment with glucocorticoids increases synthesis and stability of junctional acetylcholine receptors on innervated cultured human muscle.

We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose- and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.

Animals↗
PubMed1993

Expression of muscle-type phosphorylase in innervated and aneural cultured muscle of patients with myophosphorylase deficiency.

Patients with McArdle's myopathy lack muscle glycogen phosphorylase (M-GP) activity. Regenerating and cultured muscle of patients with McArdle's myopathy presents a glycogen phosphorylase (GP) activity, but it is not firmly established whether M-GP or non-M-GP isoforms are expressed. We have cultured myoblasts from biopsy specimen of five patients with McArdle's myopathy. Skeletal muscle was cultured aneurally or was innervated by coculture with fetal rat spinal cord explants. In the patients' muscle biopsies and in their cultured innervated and aneural muscle we studied total GP activity, isoenzymatic pattern, reactivity with anti-M-GP antiserum, and presence of M-GP mRNA. There was no detectable enzymatic activity, no immunoreactivity with anti-M-GP antiserum, and no M-GP mRNA in the muscle biopsy of all patients. GP activity, M-GP isozyme, and anti-M-GP antiserum reactivity were present in patients' aneural cultures, increased after innervation, and were undistinguishable from control. M-GP mRNA was demonstrated in both aneural and innervated cultures of patients and control by primer extension and PCR amplification of total RNA. Our studies indicate that the M-GP gene is normally transcribed and translated in cultured muscle of patients with myophosphorylase deficiency.

Adult↗
PubMed1992

Strong immunoreactivity of beta-amyloid precursor protein, including the beta-amyloid protein sequence, at human neuromuscular junctions.

At the postsynaptic domain of the human neuromuscular junction (NMJ), we have demonstrated strong concentrations of the N-terminus 45-62, C-terminus 676-695 and beta-amyloid protein sequences of beta-amyloid precursor protein (beta APP). We used well-characterized monoclonal and polyclonal antibodies for co-localization with three other postsynaptic proteins, applying double and triple fluorescence labeling. Strong immunoreactivity of all three beta APP sequences was found at all NMJs identified by bound alpha-bungarotoxin (alpha BT), where they co-localized with alpha BT and with immunoreactive desmin and dystrophin, which are postsynaptic proteins of human NMJs. This appears to be the first demonstration of beta APP sequences concentrated postsynaptically at human NMJs. beta APP may have a role in normal junction biology and possibly in some diseases affecting NMJs.

Amyloid beta-Protein Precursor↗
PubMed1992

The influence of muscle contractile activity versus neural factors on morphologic properties of innervated cultured human muscle.

In contrast to aneurally cultured human muscle, which is immature in regard to its morphologic phenotype and only rarely and weakly contracts spontaneously, innervated cultured human muscle fibres have: (1) nearly continuous, d-tubocurarine-inhibitable contractions; (2) well-developed cross-striations, basal lamina, t-tubules, and postsynaptic folds of the neuromuscular junctions; (3) the majority of their nuclei peripheralized; and (4) acetylcholinesterase-positive sites present only at the neuromuscular junctions. To see whether the expression of the muscle morphologic phenotype is induced only by neural factors generated from the spinal cord explants or also by their frequent contractile activity, we paralyzed innervated cultured human muscle fibres with 2 microM tetrodotoxin for four weeks, either from the first day of muscle contractions or following four weeks of muscle contractions. In both experimental designs, by light microscopy tetrodotoxin paralysis abolished cross-striations and caused prominent internalization of muscle nuclei; however, it did not influence the intensity of acetylcholinesterase staining at the neuromuscular junctions. By electron microscopy, there was no difference between paralyzed and contracting muscle fibres in development of t-tubules, basal lamina and postsynaptic folds. Our study demonstrates that in human muscle contractile activity: (1) regulates peripheral migration of nuclei and development of cross-striations; and (2) does not influence development of the neuromuscular junction, basal lamina, and t-tubules, which are mainly regulated by neural influences. This culture model may be useful for studying detailed mechanisms of human muscle fibre development and structural abnormalities in human neuromuscular diseases.

Acetylcholinesterase↗
PubMed1992

Glucocorticoid increases acetylcholinesterase and organization of the postsynaptic membrane in innervated cultured human muscle.

We have studied the influence of hydrocortisone (HC) on the neuromuscular junctions (NMJs) established on cultured human muscle fibers that had been innervated by fetal rat spinal cord neurons. Treatment with HC was begun 4 weeks after innervation and continued for 1-28 days. Four weeks of treatment significantly increased (a) size of acetylcholinesterase (AChE)-positive sites, indicative of NMJs; (b) intensity of AChE staining; (c) A12-AChE (junctional) molecular fraction; and (d) organization of junctional postsynaptic folds. The effect of HC depended on the dose and duration of treatment. These effects on the molecular properties of the postsynaptic component of the human neuromuscular junction could be through an action of HC directly on the muscle fiber or indirectly by affecting the motor neuron. Because the increased organization of the postsynaptic folds and the increased AChE seem to be salutory effects on the NMJ of prolonged HC treatment, these changes of the NMJ itself might contribute to the long-term beneficial effect of prednisone, another glucocorticoid, in myasthenia gravis patients.

Acetylcholinesterase↗
PubMed1992

Different degradation rates of junctional and extrajunctional acetylcholine receptors of human muscle cultured in monolayer and innervated by fetal rat spinal cord neurons.

It is well demonstrated that in intact animals the degradation rate of the junctional acetylcholine receptor (AChR) is significantly slower than that of the extrajunctional receptor. Such data, however, are not available for human AChRs because the required experimentation cannot be performed in humans. We have now studied the degradation rate of the junctional and extrajunctional AChRs, utilizing our tissue culture model, in which well-differentiated neuromuscular junctions (NMJs) form on human muscle cultured in monolayer and innervated long-term by fetal rat spinal cord neurons. Half-life of AChRs was studied by a method utilizing the autoradiography of 125I-alpha bungarotoxin and computerized video image analysis. Extrajunctional AChRs degraded with a half-life of 1.3 days whereas junctional AChRs degraded with a half-life of 3.5 days. Our studies demonstrate for the first time that in innervated cultured human muscle: (a) the life span of human junctional AChR, is approximately 3 times longer than that of the extrajunctional AChR and (b) the stability of human AChR is neuronally regulated. This system can now be applied to evaluate the influence of pharmacologic agents on the stability of human junctional AChR, which is of potential importance in the treatment of myasthenia gravis and other diseases of the NMJ.

Animals↗
PubMed1992

Immunocytochemical localization of ubiquitin at human neuromuscular junctions.

Using several specific monoclonal and polyclonal antibodies, we found that ubiquitin is highly concentrated in the post-synaptic domain of human neuromuscular junctions. The distribution and pattern of ubiquitin immunoreactivity is identical to the localization of bound alpha-bungarotoxin, suggesting that ubiquitin may be involved in post-translational modification or turnover of the acetylcholine receptor or other junctional proteins of the muscle fibre.

Antibodies, Monoclonal↗
PubMed1992

Light and electron microscopic localization of beta-amyloid protein in muscle biopsies of patients with inclusion-body myositis.

In 11 of 11 inclusion-body myositis (IBM) patients, including one hereditary case, vacuolated muscle fibers contained large and multiple small inclusions immunoreactive for beta-amyloid protein (beta AP). All IBM muscle biopsies had characteristic cytoplasmic tubulo-filaments (CTFs) by electron microscopy. None of 14 control muscle biopsies contained the beta AP immunoreactive (IR) inclusions characteristic of IBM. On the light microscopy level, beta AP-IR inclusions colocalized with ubiquitin immunoreactivity. By immunogold electronmicroscopy, beta AP immunoreactivity was localized to a) amorphous, poorly defined structures, b) dense floccular material, c) clusters of loosely packed amyloidlike fibrils 6-8 nm in diameter, and d) poorly defined loose fibrillar structures 6-8 nm in diameter. beta AP immunoreactive structures were often in proximity to CTFs, but CTFs themselves never contained beta AP-IR. Our study provides the first demonstration of beta AP accumulations in abnormal human muscle. This finding suggests that in addition to Alzheimer's disease, Down syndrome, and Dutch-type hereditary cerebrovascular amyloidosis, beta AP may play an important role in the pathogenesis of other diseases, including ones outside the central nervous system, for example, IBM.

Actin Cytoskeleton↗
PubMed1991

Immunolocalization of ubiquitin in muscle biopsies of patients with inclusion body myositis and oculopharyngeal muscular dystrophy.

In 10/10 inclusion body myositis (IBM) patients and 2/2 oculopharyngeal muscular dystrophy (OPMD) patients, vacuolated muscle fibers contained darkly stained ubiquitin (Ub)-immunoreactive cytoplasmic inclusions. By electronmicroscopy, Ub-immunoreactive material was strictly localized to the 15-21 nm pathologic cytoplasmic tubulofilaments (CTFs). None of 18 control muscle biopsies contained the Ub-immunoreactive inclusions that are typical for IBM and OPMD. Thus, (a) finding that CTFs are ubiquitinated places their protein in the Ub-mediated turnover pathway and provides their first molecular marker; (b) easy accessibility, as compared to the central nervous system, of muscle tissue containing ubiquitinated inclusions should be advantageous for biochemical and molecular studies and may provide information important to both systems.

Adolescent↗
PubMed1990

Abnormalities of aneural and innervated cultured muscle fibers from patients with myotonic atrophy (dystrophy).

Innervation of human muscle cocultured in monolayer with explants of fetal rat spinal cord plus dorsal-root ganglia produces more mature fibers, which show spontaneous, neurogenic (d-tubocurarine-blocked) contractions. On the innervated myotonic atrophy (MA) muscle fibers, 96% of acetylcholinesterase-stained patches were simple, and only 4% appeared as complicated, pretzel-like, more mature-looking structures; on control innervated fibers, 37% of the acetylcholinesterase patches had the mature appearance. The normal trend from multifocal innervation toward unifocal innervation was decreased in innervated MA muscle fibers. Microelectrode studies compared parameters of cultured aneural muscle fibers and cultured innervated-contracting muscle fibers from 7 patients with MA and 10 control patients. The mean resting membrane potentials of the two groups (aneurally cultured MA muscle fibers and innervated-contracting cultured MA muscle fibers) were 8 and 9 mV lower, respectively, than those of their counterpart controls. The mean amplitude of action potentials, the maximum rate of rise of action potentials in innervated MA muscle fibers, and the action potential amplitude in aneural MA muscle fibers were significantly smaller than in corresponding control fibers.

Acetylcholinesterase↗
PubMed1990

Expression of muscle-specific isozymes of phosphorylase and creatine kinase in human muscle fibers cultured aneurally in serum-free, hormonally/chemically enriched medium.

Primary cultures of muscle cells derived from a biopsied adult human skeletal muscle were grown up to 6 weeks in a hormonally/chemically enriched serum-free medium. The expression of muscle-specific isozymes of creatine kinase, glycogen phosphorylase, and phosphoglycerate mutase, indicative of muscle cell maturation, was studied after 1, 4 and 6 weeks of growth. The maturation of muscle fibers cultured in serum-free medium was comparable to that achieved by muscle fibers cultured in medium containing 10% serum and supplemented with growth factors (insulin, epidermal growth factor, and fibroblastic growth factor) and was greater than that achieved in medium containing 10% serum only. Our study demonstrates that adult human muscle can be cultured aneurally for a long period of time in a serum-free medium, and that it can achieve a high degree of maturation. This study provides an important basis for investigations related to: (1) assessment of the influence of individual components of the medium on human muscle maturation in culture; (2) studies of regulation of abnormal gene expression in diseased human muscle cultured in serum-fre medium.

Adult↗
PubMed1990

Paralysis of innervated cultured human muscle fibers affects enzymes differentially.

Increased accumulation of muscle-specific isozyme (MSI) of creatine kinase (CK), lactate dehydrogenase (LDH), glycogen phosphorylase (GP), and phosphoglycerate mutase (PGAM) occurs with development and indicates muscle fiber maturation. The expression of MSIs of those four enzymes is greatly enhanced in innervated-contracting as compared to noninnervated and noncontracting cultured human muscle fibers. We have now studied the effect of contractile activity on developmental accumulation of MSIs in innervated-contracting, innervated-paralyzed (2 microM tetrodotoxin for 30 days), and noninnervated-noncontracting cultured human muscle fibers. Muscle acetylcholinesterase (AChE) and total enzyme activities were also studied under the same conditions. We observed a different dependency on contractile activity between total enzymatic activities of CK, LDH, and AChE, which were substantially reduced after paralysis, and GP and PGAM, which were unchanged. The expression of MSIs of CK, GP, PGAM, and LDH was always significantly increased in innervated as compared to noninnervated fibers. While the expression of MSIs of GP and PGAM was the same in contracting-innervated and paralyzed-innervated muscle fibers, the expression of MSIs of CK and LDH in paralyzed-innervated muscle fibers was very slightly decreased as compared to their contracting-innervated controls. Our studies demonstrate that in human muscle: (1) total enzymatic activities and the expression of MSIs of GP and PGAM are regulated by neuronal effect(s); (2) total enzymatic activities of CK, LDH, and AChE depend mainly on muscle contractile activity; and (3) MSIs of CK and LDH are regulated predominantly by neuronal factors and to a much lesser degree by muscle contractile activity.

Acetylcholinesterase↗
PubMed1990

Immunocytochemical localization of desmin at human neuromuscular junctions.

We localized desmin at human neuromuscular junctions (NMJs) using specific anti-desmin monoclonal and polyclonal antibodies. It was highly concentrated in the postsynaptic domain. It was found in all the NMJs examined. Compared with the area of acetylcholine receptors identified by bound alpha-bungarotoxin, the strong desmin immunoreactivity occupied a slightly larger area and was localized slightly deeper in the muscle fiber, suggesting that desmin might be accumulated within the postsynaptic folds and possibly below the folds. This appears to be the first identification of a protein unrelated to cholinergic transmission accumulated at human NMJs.

Animals↗