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Biomedical subjects

W K Engel

Publications and source records attributed to W K Engel.

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

Strong immunoreactivity of alpha 1-antichymotrypsin co-localizes with beta-amyloid protein and ubiquitin in vacuolated muscle fibers of inclusion-body myositis.

In 10 of 10 inclusion-body myositis (IBM) patients, including 1 hereditary case, vacuolated muscle fibers contained large or small cytoplasmic inclusions immunoreactive for alpha 1-antichymotrypsin (alpha 1-ACT). All IBM muscle biopsies had characteristic cytoplasmic tubulo-filaments by electron microscopy. None of 17 control muscle biopsies contained the alpha 1-ACT immunoreactive inclusions characteristic of IBM. In vacuolated muscle fibers, alpha 1-ACT immunoreactive inclusions colocalized with beta-amyloid protein and ubiquitin immunoreactivities. Our study provides the first demonstration of alpha 1-ACT accumulations in abnormal human muscle, and it suggest that, as in Alzheimer's disease and Down's syndrome, alpha 1-ACT may be involved in the pathogenesis of IBM.

Adolescent↗
PubMed1993

New organotypic model to culture the entire fetal rat spinal cord.

We have developed a system for culturing separately the entire longitudinally-cut ventral and dorsal parts of the whole spinal cord of 12-14 day rat embryos, either on polylysine-collagen mixture or co-cultured with human muscle. At the time of explanation and throughout the period of culturing (up to 21 days) choline acetyltransferase (ChAT) activity in dorsal macroexplants (D-MEs) was negligible (2% of that in the ventral macroexplants (V-MEs)). During culturing neuronal survival was evaluated by measurements of ChAT and enolase. The densities and lengths of neuronal processes outgrowing from the MEs were measured in living and fixed cultures. In the latter, immunocytochemical and cytochemical stainings, allowing visualization of neurites, were evaluated by computerized video image analysis. The ChAT activity in the MEs cultured under our two experimental conditions differed only during the first few days of culturing, being significantly higher in the V-MEs co-cultured with muscle as compared to those cultured on a polylysine-collagen mixture. After 3 weeks of culturing, ChAT activity was not statistically different. However, the co-cultured muscle fibers definitely influenced: (a) neurite outgrowth, (b) the expression of neurofilament proteins and (c) the ability to maintain cultured MEs long-term. This new system allows study of motor neurons and the influence on them of putative growth, survival and maturation factors in a tissue culture milieu that more closely resembles in vivo conditions than other culture systems. Our established parameters can now serve as a basis for such studies.

Acetylcholinesterase↗
PubMed1993

beta-Amyloid precursor protein mRNA is increased in inclusion-body myositis muscle.

Vacuolated muscle fibers in muscle biopsies of 8 out of 8 inclusion body myositis (IBM) patients, including 2 hereditary patients, manifested increased mRNA for the beta-amyloid precursor protein (beta APP) that contains Kunitz-type protease inhibitor motif. In affected fibers, increased beta APP-mRNA correspond to abnormally accumulated beta APP immunoreactivity (including beta-amyloid protein epitope). In normal human muscle fibers increased beta APP-mRNA was present only at the neuromuscular junctions. Our study (a) suggests that abnormally accumulated beta APP in IBM vacuolated fibers results, at least partly, from increased beta APP generation, and (b) provides the first demonstration of up-regulated beta APP-mRNA in pathologic human tissue other than brain of Alzheimer's disease and Down's syndrome.

Adolescent↗
PubMed1993

New advances in inclusion-body myositis.

The major new advances in seeking the pathogenic mechanisms of sporadic inclusion-body myositis and hereditary inclusion-body myopathy are discussed. Hypotheses are presented regarding the possible causes and significance of amyloid deposits in sporadic inclusion-body myositis and the roles of abnormally accumulated ubiquitin, beta-amyloid protein, beta-amyloid precursor protein, alpha 1-antichymotrypsin, hyperphosphorylated tau, and prion protein in the vacuolated muscle fibers in both sporadic inclusion-body myositis and hereditary inclusion-body myopathy. Because hereditary inclusion-body myopathy is virtually free of Congophilic amyloid deposits but, like sporadic inclusion-body myositis, contains large accumulations of beta-amyloid protein, it is possible that the lesions in hereditary inclusion-body myopathy may represent "early" changes. There are striking similarities between the pathology of inclusion-body myositis muscle and brains affected by Alzheimer's disease in regard to accumulation of ubiquitin, beta-amyloid protein and its precursor protein, alpha 1-antichymotrypsin, and hyperphosphorylated tau.

Amyloid↗
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↗
PubMed1993

The effect of wrist rest, caffeine, and oral timolol on the hand steadiness of ophthalmologists.

Because hand steadiness is an important aspect of microsurgical technique, hand movement, amplified by a hand-held laser pointer, was measured in 14 ophthalmology trainees on four separate mornings when each had ingested (1) 200mg of caffeine, (2) 12.5mg of timolol (2.5mL of timolol 0.5% ophthalmic solution), (3) a combination of caffeine and the beta-adrenergic blocker, or (4) a placebo. Caffeine increased the resting pulse and hand movement. Although the beta-adrenergic blocker and the combination of caffeine and this drug decreased the resting heart rate from that of placebo, they did not significantly change hand steadiness. However, compared with caffeine alone, the beta-adrenergic blocker-caffeine combination significantly decreased hand movement and the pulse. Resting the wrist significantly steadied hand movements. Hand movement was not correlated with the level of ophthalmology training, the trainee's weight, or their daily caffeine consumption.

Administration, Oral↗
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 multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.

Adolescent↗
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↗