Pupillary autonomic dysfunction in multiple system atrophy and Parkinson's disease: an assessment by eye-drop tests.

PURPOSE: To compare pupillary autonomic dysfunction in multiple system atrophy (MSA) and Parkinson's disease (PD). METHODS: We administered eye-drop tests to 40 MSA patients, 40 PD patients with similar disease duration, and 20 age-matched healthy controls. Pupillary supersensitivity to a parasympathomimetic agent (0.05% pilocarpine hydrochloride) and to a sympathomimetic agent (0.02% dipivefrine hydrochloride) was examined by assessing changes in pupil diameter. RESULTS: Pupillary supersensitivity to a parasympathomimetic agent (0.05% pilocarpine hydrochloride) and to a sympathomimetic agent (0.02% dipivefrine hydrochloride) was examined by assessing changes in pupil diameter. Pupillary supersensitivity to 0.05% pilocarpine was greatest among the PD patients (PD -23.1 +/- 14.4%, MSA -12.4 +/- 11.5%, control -9.5 +/- 8.2%, p < 0.05) but was not correlated with disease duration. Pupillary sensitivity to 0.02% dipivefrine was significantly greater in the PD and MSA patients versus controls (PD 10.5 +/- 12.0%, MSA 11.8 +/- 11.0%, control 3.1 +/- 5.8%, p < 0.05). MSA patients had pupillary sympathetic dysfunction from an early stage, whereas in PD patients it tended to gradually accelerate as the disease advanced. In MSA patients, pupillary sympathetic sensitivity to 0.02% dipivefrine was correlated with the severity of orthostatic hypotension during a head-up tilt test and with the elevation of systolic blood pressure during a noradrenaline infusion test. In PD patients, pupillary sympathetic sensitivity to 0.02% dipivefrine was correlated with a reduction of the heart-to-mediastinum (H/M) ratio using delayed-phase iodine-123 meta-iodobenzylguanidine ((123)I-MIBG) myocardial scintigraphy. CONCLUSION: These data indicate that eye-drop tests can reveal differences in the progression of pupillary autonomic dysfunction in patients with MSA and PD.

Autoantibodies against M1 muscarinic acetylcholine receptor in myasthenic disorders.

The Lambert-Eaton myasthenic syndrome (LEMS), often associated with small-cell lung carcinoma (SCLC), is a disorder of acetylcholine (ACh) release from motor nerve terminals. In most patients, it is caused by autoantibodies against the P/Q-type voltage-gated calcium channels (VGCC) that trigger ACh release. However, these antibodies are not detected in approximately 15% of clinically and electrophysiologically typical cases. The M1-type pre-synaptic muscarinic ACh receptor (M1 mAChR) modulates cholinergic neuromuscular transmission by linking to P/Q-type VGCC, and may partially compensate for the reduced calcium entry. Immunoblotting against solubilized human M1 mAChR, we detected autoantibodies in: (a) 14 of 20 (70%) anti-VGCC-positive LEMS patients; (b) all five anti-VGCC-negative LEMS patients, one of whose serum had previously passively transferred LEMS-type electrophysiological defects to mice; (c) all five LEMS patients with autonomic symptoms; (d) seven of 25 (28%) myasthenia gravis (MG) patients in whom increased ACh release partially compensates for post-synaptic defects; (e) none of 10 SCLC patients without LEMS. Although not proving primary pathogenicity of anti-M1 mAChR antibodies, the present results highlight their potential to affect synaptic compensatory mechanisms, more in LEMS than MG.

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas.

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and Km value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60 degrees C for 10 min.

Using breast radiographers' reports as a second opinion for radiologists' readings of microcalcifications in digital mammography.

OBJECTIVE: The aim of this study was to investigate a practical method for incorporating radiographers' reports with radiologists' readings of digital mammograms. METHODS: This simulation study was conducted using data from a free-response receiver operating characteristic observer study obtained with 75 cases (25 malignant, 25 benign and 25 normal cases) of digital mammograms. Each of the rating scores obtained by six breast radiographers was utilized as a second opinion for four radiologists' readings with the radiographers' reports. A logical "OR" operation with various criteria settings was simulated for deciding an appropriate method to select a radiographer's report in all combinations of radiologists and radiographers. The average figure of merit (FOM) of the radiologists' performances was statistically analysed using a jackknife procedure (JAFROC) to verify the clinical utility of using radiographers' reports. RESULTS: Potential improvement of the average FOM of the radiologists' performances for identifying malignant microcalcifications could be expected when using radiographers' reports as a second opinion. When the threshold value of 2.6 in Breast Imaging-Reporting and Data System (BI-RADS®) assessment was applied to adopt/reject a radiographer's report, FOMs of radiologists' performances were further improved. CONCLUSION: When using breast radiographers' reports as a second opinion, radiologists' performances potentially improved when reading digital mammograms. It could be anticipated that radiologists' performances were improved further by setting a threshold value on the BI-RADS assessment provided by the radiographers. ADVANCES IN KNOWLEDGE: For the effective use of a radiographer's report as a second opinion, radiographers' rating scores and its criteria setting for adoption/rejection would be necessary.

Effects of prostaglandin E1 in experimental autoimmune myasthenia gravis.

The effects of prostaglandin E1 (PGE1), a potent inhibitor of lymphocyte functions, were studied in rats immunized with acetylcholine receptor (AChR) to induce experimental autoimmune myasthenia gravis (EAMG). Daily injections of PGE1, 400 micrograms per day, prevented the development of acute EAMG, which is attributed to antibody-dependent, complement-mediated cytolysis. This was associated with suppression of delayed-type cutaneous hypersensitivity response to AChR. PGE1 did not prevent the subsequent onset of chronic EAMG, which reflects accelerated degradation of AChR by antibody and complement-mediated cell lysis in the postsynaptic membrane.

Experimental myasthenia due to alpha-bungarotoxin.

Neuromuscular transmission was studied electrophysiologically in rabbits intoxicated by alpha-bungarotoxin, a specific inhibitor of acetylcholine receptor. The findings consisted of a slight reduction in amplitude of single evoked muscle action potentials, a decrement in amplitude of successive evoked muscle action potentials post-tetanic potentiation and exhaustion, edrophonium reversal, and no change of muscle action potentials evoked by direct stimulation of the muscle. These were similar to characteristic electrophysiologic phenomena seen in 40 patients with myasthenia gravis. The receptor abnormality may be responsible for the underlying defect of myasthenia.

Hyperthyroidism and myasthenia gravis with features of Eaton-Lambert syndrome.

In a 50-year-old man with hyperthyroidism and myasthenic weakness electrophysiologic phenomena similar to Eaton-Lambert syndrome were seen in classical myasthenia gravis. The orbicularis oculi showed an abnormally small muscle action potential in response to nerve stimulus and unusual facilitation of the response with activity. These signs are compatible with Eaton-Lambert syndrome, but the findings obtained from the adductor pollicis were typical of classical myasthenia. A possible common basis for hyperthyroidism and such an electrophysiologic complexity of transmission failure was considered as a pathogenic hypothesis.

Presynaptic modification of neuromuscular transmission by antiacetylcholine receptor antibody: myasthenic serum and monoclonal antibody produced by transformed lymphocytes.

When myasthenic serum or a monoclonal antiacetylcholine receptor (anti-AChR) antibody produced by human transformed lymphocytes and transferable to an animal was applied to rat diaphragms in vitro, presynaptic facilitation was demonstrated by changes in ACh quantal content of endplate potentials. The results correlated with ability of the antibody to block binding of alpha-bungarotoxin to AChR, but not with titers of anti-AChR antibody by immunoprecipitation assay and AChR degradation rate. Antibody to the receptor site near the ACh-binding site may act presynaptically to compensate for the postsynaptic failure in myasthenia gravis.

Neuromuscular defect after suppression of ion conductance.

In rat skeletal muscle, trimetazidine (TMZ) caused a transmission defect without directly blocking binding of acetylcholine--ionophore impairment. In vivo, TMZ produced low-amplitude and cumulative depression of successive muscle responses, and immediate posttetanic exhaustion. These features differed from the effects of alpha-bungarotoxin (alpha-BuTx) or immunization with acetylcholine receptor (experimental autoimmune myasthenia gravis [EAMG]). In vitro, TMZ-induced block was similar to both alpha-BuTx-induced block and EAMG in many respects, but there were differences in endplate potentials evoked during and after rapid repetitive activations. These differences suggest that antibodies to the acetylcholine receptor do not affect the ionophore.

Experimental autoimmune myasthenia gravis and cholinergic ionophore: an electrophysiologic study.

Rabbits were immunized with purified acetylcholine receptor from Narke electroplax japonica. A defect of neuromuscular transmission, physiologically identical to human myasthenia gravis, developed when antibodies against the receptor were found in serum. To clarify the mode of action of these antibodies, changes in the endplate current of frog muscle fibers were recorded after exposure to immune rabbit sera. The rabbit sera depressed the amplitude of the endplate current, but caused no change in the time course or the dependence of amplitude and half-decay time on membrane potential. Antibodies may affect acetylcholine binding without impairing ionophore function.

Active state properties of denervated and immobilized muscle: comparison with dystrophic muscle.

Possible roles of neurotrophic mechanisms and muscle activity in the contractile abnormalities of muscular dystrophy were studied by comparing human dystrophic muscle to denervated and immobilized muscle. As evident in denervated muscle from the decreased acceleration of twitch development (decreased active state intensity of shortening), and isoproterenol-induced decrease of twitch with shortened decay of the active state, part of the abnormality in the subcellular calcium transport system in dystrophic muscle seems to be influenced by disordered neural regulation. Other active state abnormalities relating to activation processes and contractile proteins in dystrophic muscle were also demonstrated in both denervated and immobilized muscle, with some being more marked in immobilized muscle. The findings indicate that a neurogenic hypothesis cannot entirely explain the pathogenesis of progressive muscular dystrophy.

Antibodies to synthetic peptides of the alpha1A subunit of the voltage-gated calcium channel in Lambert-Eaton myasthenic syndrome.

To search for antigenic sites in the molecular structure of alpha1A subunit of the voltage-gated calcium channel (VGCC) (P/Q-type) in the Lambert-Eaton myasthenic syndrome (LEMS), we studied by immunoprecipitation assay serum samples from 30 LEMS patients (16 with small cell lung carcinoma (SCLC), 20 disease controls (10 with SCLC without LEMS and 10 with myasthenia gravis), and 15 healthy controls. Synthetic peptide antigens corresponded to the extracellular region (S5-S6 linker region) of each of the four domains forming the alpha1 subunit of P/Q-type VGCC. In addition, we studied serum samples for anti-P/Q-type VGCC antibodies by using omega-conotoxin MVIIC-labeled extract of human cerebellum as an antigen. Among sera of 30 LEMS patients, nine samples (30%) (six with SCLC) were positive for antibodies to the domain IV S5-S6 linker peptide, and six samples (20%) (five with SCLC) were positive for antibodies to the domain II S5-S6 linker peptide. Only two of 15 antipeptide-positive sera were positive for both antibodies. Titers for antibodies to domain IV, as well as those for antibodies to domain II, correlated with those of anti-P/Q-type VGCC (human cerebellum extract) antibodies. The antipeptide antibody was present in only one of 20 disease controls, a patient with SCLC without LEMS. Our observations suggest two potential epitopes of LEMS antibodies.

Recombinant calcium channel is recognized by Lambert-Eaton myasthenic syndrome antibodies.

The authors studied sera from 36 patients with Lambert-Eaton myasthenic syndrome (LEMS) by immunoblots using the recombinant protein derived from the DNA sequence encoding for the domain III S5-S6 linker of the P/Q-type voltage-gated calcium channel al subunit. The results of 18 patients were positive for antibodies to this recombinant protein. The results of 2 of 10 patients with lung cancer without LEMS were also positive.

Effects of epinephrine, ouabain, and insulin in experimental autoimmune myasthenia gravis.

To gain a clue to the target of anti-AChR antibody, rats with acute and chronic experimental autoimmune myasthenia gravis (EAMG) that was induced by immunization with Narke anti-acetylcholine receptor (AChR) were studied using agents acting on active Na-k transport. Postsynaptic response to epinephrine was defective in chronic EAMG with high titers of antibody, suggesting that active Na-K transport system modulated by cyclic adenosine monophosphate (AMP) may be affected primarily by antibody. Sensitivity to ouabain was less than normal in acute EAMG and became close to normal when treated with anticomplementary factor. Findings suggest that acute EAMG is a case of functional denervation. Normal response to insulin occurred in all phases of EAMG.

Role of prostaglandin E2 in contractile abnormality induced by calcium ionophore, A23187.

We evaluated the possible role of prostaglandin E2 (PGE2) in the calcium(Ca++)-mediated damage of skeletal muscle by a calcium ionophore (A23187) that induces excessive Ca++ influx. Twitch and tetanus of rat diaphragms were depressed with either PGE2 or A23187. A23187-induced depression was reduced by PG synthesis inhibitors, aspirin, or indomethacin, though less than that by a protease inhibitor, leupeptin. PGE2-induced depression was also inhibited by leupeptin. Damage of the muscle cell by excessive intracellular free Ca++ may thus be mediated via a PGE2 pathway besides other mechanisms including non-lysosomal, Ca++-activated proteases.

Ultrastructural localization of acetylcholine receptor at the motor endplate: myasthenia gravis and other neuromuscular diseases.

Peroxidase-conjugated alpha-bungarotoxin (P-BGT) was used for the ultrastructural localization of the acetylcholine receptor in motor endplates. Brachial biceps muscle specimens were obtained from six patients with myasthenia gravis (MG) (two ocular and four generalized), five other patients with neuromuscular diseases (limb-girdle dystrophy, polymyositis, and amyotrophic lateral sclerosis) and two controls. In all patients with generalized MG, most of the endplates showed a marked decrease in P-BGT binding. In one of two patients with ocular MG, the amount and distribution of P-BGT binding appeared normal, whereas the other patient showed a slight decrease in P-BGT binding. There was a loose correlation between clinical severity of MG and acetylcholine receptor index or antiacetylcholine receptor antibodies. On the other hand, the amount and distribution of acetylcholine receptor in other neuromuscular diseases was well preserved, even at the endplates denuded of their nerve terminals in amyotrophic lateral sclerosis (ALS) cases.

Limb muscle endplates in ocular myasthenia gravis: quantitative ultrastructural study.

Motor endplate ultrastructure in the biceps brachii was quantitatively analyzed in five patients with ocular myasthenia gravis (56 endplates), six patients with generalized myasthenia gravis (83 endplates), and five controls (64 endplates). Ultrastructural changes of the motor endplates were observed in nonweak limb muscles of patients with ocular myasthenia gravis as well as in generalized myasthenia; these changes were mostly restricted to the postsynaptic region. Decrease of the mean presynaptic and postsynaptic membrane length, and postsynaptic membrane density, were more remarkable in generalized myasthenia than in ocular myasthenia. Widening of the primary and secondary synaptic clefts was also observed more frequently in generalized myasthenia. There was no correlation between ultrastructural alterations and the duration of symptoms or treatment, severity of disease, or titers of antiacetylcholine receptor antibody.

Lambert-Eaton myasthenic syndrome as an autoimmune calcium-channelopathy.

Lambert-Eaton myasthenic syndrome (LEMS), often associated with small cell lung carcinoma (SCLC), is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. We focused attention on the P/Q-type VGCC, against which a majority of LEMS patients carry the specific antibody. Since the P/Q-type VGCC expresses in SCLC, the motor nerve terminal and SCLC may share a common VGCC antigen. In search for antigenic sites at the molecular level, We employed peptides or recombinant protein corresponding to the S5-S6 linker of each of four domains forming the alpha 1A subunit and tested their antigenicity. As the result, we specified the domain II, III and IV as immunodominant sites by the induction of an immune-mediated animal model of LEMS and the assay for antibodies in LEMS patients. Also, by use of peptides or recombinant protein corresponding to the synaptotagmin I, we found that in this VGCC-associated protein, the segment which exposes extracellularly during exocytosis can be antigenic for LEMS.

Effect of calcitonin gene-related peptide on skeletal muscle via specific binding site and G protein.

In curarized rat skeletal muscle, rat calcitonin gene-related peptide (CGRP), a peptide coexisted with acetylcholine in the motor nerve terminal, increased the isometric twitch force, accompanied by an increase in the active state intensity of shortening, prolonged duration of the active state and additive effect of a phosphodiesterase inhibitor; the results reflect a potentiation in the sarcoplasmic calcium transport system. This CGRP effect was enhanced by cholera toxin, suggesting the activation of guanine nucleotide binding regulatory protein (G protein) that stimulates adenylate cyclase (Gs). The pertussis toxin (IAP), a factor to prevent the cyclic AMP decrease by inactivating the G protein that inhibits adenylate cyclase (Gi), provided no effect on the action of CGRP. The existence of CGRP binding site in the sarcolemmal membrane was confirmed by Scatchard analysis of binding data; affinity of the binding site for CGRP was decreased in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP gamma S). The Gs protein is thus implicated in the CGRP binding site and intracellular processes of signal transduction. CGRP did not modify the neuromuscular transmission and cable properties of the muscle membrane.

Myasthenogenicity in the main immunogenic region of acetylcholine receptor as modified by conformational design: an approach to antigenic synthetic peptides.

Myasthenogenic regions in the acetylcholine receptor (AChR) alpha-subunit were studied in view of the conformation-dependent B-cell epitope expected at beta-turn and the MHC class II-restricted T-cell epitope expected at alpha-helix. Torpedo AChR alpha 67-76 and alpha 107-116 were synthesized as the main immunogenic region and the site specific for T-cell epitope in Lewis rat, respectively. Model peptides, synthesized by combining these natural sequence segments or by intervening the segment aligned as Asn-Pro-Gly-Gly (NPGG) in natural sequence segments, were tested in terms of antigenic conformation. The model peptide, alpha 107-116.alpha 67-76.alpha 107-116, was immunogenic in the induction of the animal model of myasthenia, accompanied by the anti-peptide antibody cross-reactive with the native AChR. High antigenicity in antibody assays for various peptide- and native AChR-immunized rats was found when the model peptides, alpha 107-116.alpha 67-76 and/or alpha 107-116.NPGG.alpha 67-76 were used for measurement as antigens. Antigenic conformation for the induction of the disease may thus be different from that for the reactivity to antibody.