Direct DNA sequencing from colony: analysis of multiple deletions of mitochondrial genome.

We have developed a rapid and efficient nucleotide sequencing technique, named the colony direct sequencing method, which combines both the conventional cloning method for picking up a single gene and the polymerase chain reaction (PCR) method for amplifying the gene directly from a colony. In the present study, the colony direct PCR product was used both for identification of the DNA insert and for nucleotide sequencing by an automated DNA analysis system. A nucleotide sequence of 300 to 400 bp could be determined within 13 h after picking the bacterial colonies on LB medium plates. We applied this method to sequencing of junctional regions of multiple deleted mtDNAs in two siblings with inherited recurrent myoglobinuria. Mitochondrial DNA fragments with deletions were amplified by PCR and then cloned into plasmids. Among 48 white colonies propagated on LB medium plates, nine different clones were identified by PCR directly from colonies. Determination of six different junctional sequences disclosed involvement of directly repeated sequences of 2 to 12 bp in length on each side of the deletions. We believe that the colony direct sequencing method will be a powerful tool in molecular genetics for identification of a single gene among polymorphic DNAs.

Ultrastructural localization of the terminal and lytic ninth complement component (C9) at the motor end-plate in myasthenia gravis.

The terminal and lytic complement component (C9) was localized at the motor end-plate in acquired autoimmune myasthenia gravis (MG) by the immunoperoxidase method, with adequate preservation of fine structure and negligible background staining. C9 was localized on short segments of the postsynaptic membrane on degenerated fragments of the junctional folds shed into the synaptic space, and on disintegrating junctional folds. An inverse relationship was noted between the structural integrity of the junctional folds and the abundance of C9 at a given end-plate region. Destruction of junctional folds by complement may induce relocation of the nerve terminal and increased spatial separation of end-plate regions on the muscle fiber. Destruction of junctional folds by the complement membrane attack complex is a cause of the acetylcholine receptor deficiency at the MG end-plate, but antibody-dependent modulation of the receptor may also contribute to deficiency of the receptor. In certain disorders other than autoimmune MG, pathological mechanisms other than complement-mediated lysis may affect the structural integrity of the postsynaptic region.

Ultrastructural localization of immune complexes (IgG and C3) at the end-plate in experimental autoimmune myasthenia gravis.

Rats immunized with purified torpedo acetylcholine receptor (AChR) plus adjuvants developed chronic experimental autoimmune myasthenia gravis (EAMG) after day 28. Forelimb muscles from EAMG rats 29 to 103 days after immunization and from control animals were used for the ultrastructural localization of IgG and C3. IgG was demonstrated with rabbit anti-rat IgG followed by treatment with peroxidase-labeled staphylococcal protein A; and C3 with peroxidase-labeled rabbit anti-rat C3, or with unlabeled rabbit anti-rat C3 followed by peroxidase-labeled protein A. In EAMG rats both IgG and C3 were localized on the terminal expansions of the junctional folds, where AChR is known to be located, and on detached, degenerated parts of the folds in the synaptic space. Background staining was negligible. The findings provide unambiguous evidence for a destructive autoimmune reaction involving the postsynaptic membrane in EAMG, implicate the complement system in this reaction and show that detachment of the tips of the junctional folds is one way by which immune complexes, and AChR, are eliminated from the postsynaptic membrane. The immuno-electron microscopic findings in chronic EAMG closely resemble those described in human myasthenia gravis.

Amyotrophic lateral sclerosis. Lack of central chromatolytic response of motor neurocytons corresponding to active axonal degeneration.

Ventral spinal roots and anterior horn cells in the lateral nuclear group of the fourth lumbar segment from 21 patients with amyotrophic lateral sclerosis (ALS) and 23 control patients were morphometrically analyzed. The number of large myelinated fibers was remarkably decreased, while small myelinated fibers were well preserved. The population of large myelinated fibers significantly correlated with the population of anterior horn cells. Numerous axonal degenerations were observed in the ventral spinal roots of patients with ALS, even in patients with severe loss of neurons and axons. In spite of this high frequency of active axonal degeneration, the incidence of central chromatolysis of anterior horn cells remained at the control level.

Passively transferred experimental autoimmune myasthenia gravis. Sequential and quantitative study of the motor end-plate fine structure and ultrastructural localization of immune complexes (IgG and C3), and of the acetylcholine receptor.

Experimental autoimmune myasthenia gravis (EAMG) was passively transferred with immunoglobulin from rats with chronic EAMG to normal recipients. IgG and C3 were localized on terminal expansions of junctional folds of end-plates by 6 hours. Segments of folds rich in acetylcholine receptor (AChR) and coated with IgG and C3 were shed into the synaptic space by 24 hours, resulting in AChR deficiency of the postsynaptic membrane. Many sensitized postsynaptic regions were destroyed by macrophages by day 2, but effective nerve-muscle contacts were reestablished by day 5. On day 10, end-plates were still structurally abnormal and showed AChR deficiency, but the animals were clinically recovered. On day 54, postsynaptic regions were still reduced in size, with slight reduction of postsynaptic AChR. Throughout the study, the miniature end-plate potential amplitude tended to vary directly with morphometric estimates of the abundance of the postsynaptic membrane reacting for AChR. Complement-mediated injury to the junctional folds and opsonization of the postsynaptic region can explain the morphologic changes. It is not yet known why phagocytic invasion of the end-plate occurs in acute EAMG and in passively transferred EAMG induced by chronic EAMG immuglobulin, but not in chronic EAMG and only rarely in the human disease.

Degenerating compartment and functioning compartment of motor neurons in ALS: possible process of motor neuron loss.

Using a morphometric method, we studied ventral spinal roots and anterior horn neurons of the fourth lumbar segment in 17 patients with ALS. Both populations of large myelinated fibers and anterior horn cells had significantly high correlations to muscle strength in the legs and duration of symptoms. However, active axonal degeneration was consistently present in terms of either large myelinated fibers or anterior horn cells.

Strong correlation between the number of CAG repeats in androgen receptor genes and the clinical onset of features of spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA), a motor neuron disease associated with androgen insensitivity, is caused by androgen receptor gene mutations with an increased number of tandem CAG repeats in exon 1. We investigated the increased number of CAG repeats in androgen receptor genes of 19 SBMA patients and found that this correlated strongly with the age at onset of muscle weakness. Thus, SBMA is the first genetic disease in which a strong correlation between the degree of genetic abnormality (number of CAG tandem repeats) and clinical phenotypic expression is demonstrable. The results further indicate that androgen gene mutation is directly involved in the degeneration of motor neurons.

DNA rearrangements in Japanese facioscapulohumeral muscular dystrophy patients: clinical correlations.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder in which the disease locus has been mapped to chromosome 4q35-qter. In most patients, the DNA rearrangements associated with FSHD have been found in the EcoRI fragment detected by the p13E-11 probe, and deletions of the 3.2 kb repeat units within the fragment are thought to cause the disease. To examine FSHD-associated DNA rearrangements in the Japanese population, we performed Southern blot analysis of the genomic DNA, using the p13E-11 and pFR-1 probes, in 158 Japanese individuals, including 38 FSHD patients from 19 families. We found that all but one (a possible affected recombinant) of the Japanese FSHD patients (97.4%) had specific smaller (< 28 kb) EcoRI fragments which cosegregated with the disease; this included four patients who had severe inflammatory changes in the muscle and eight patients with de novo DNA rearrangements. We found no FSHD patient who had a fragment larger than 28 kb. By contrast, only two of 35 Japanese controls (5.7%) had EcoRI fragments smaller than 28 kb. Our patients showed anticipation, i.e. decreased size of the EcoRI fragment in parallel with earlier onset of the disease (r = 0.531, P = 0.003, with younger age at onset in children (17.8 +/- 7.0) than their affected parents (31.5 +/- 14.8) (P = 0.019). However, since each family had a specific small EcoRI fragment associated with the disease, the differing clinical severity within a family cannot be explained by the size of the fragment alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of interleukin 6 in patients with sarcoidosis.

Interleukin 6 (IL-6) levels in various materials from patients with sarcoidosis were determined. The subjects of the study were 38 patients with sarcoidosis and 28 healthy controls. For detection of IL-6, an enzyme-linked immunosorbent assay method was used. Interleukin 6 activity in serum was detected in 4 of 30 patients, but not in 19 controls. In bronchoalveolar lavage (BAL) fluid, following 20-fold concentration, IL-6 activity was detected in four of ten patients (nonsmokers) and three of seven controls (two of two smokers and one of five nonsmokers). Interleukin 6 levels in the supernatants of cultured monocytes and alveolar macrophages (AMs) were significantly higher (p < 0.01 and p < 0.01, respectively) in patients with sarcoidosis than in controls. Interleukin 6 production from monocytes tended to correlate with that from AMs. A significant correlation (r = 0.70, p < 0.05) was found between IL-6 production from AMs and the ratio of CD4+/CD8+ in BAL fluid, although no correlation was observed between that from monocytes and CD4+/CD8+ ratio in BAL fluid. Taken together, IL-6 may be involved in the initiation and maintenance of alveolitis by activating and causing the proliferation of T cells.

The immunopathology of acquired myasthenia gravis.

Specific probes (alpha-bungarotoxin for acetylcholine receptor (AChR), staphylococcal protein A for IgG, monospecific antibodies against C3 and C9) labelled with peroxidase were applied to study of the ultrastructure of the MG end plate. In each case of MG there was postsynaptic AChR deficiency, usually greatest at end plates with marked degeneration of junctional folds. Morphometric estimates of postsynaptic AChR correlated linearly with the MEPP amplitude. In each case of MG, IgG was localized on the postsynaptic membrane where AChR is known to be located and on debris in the synaptic space. The abundance of antibody was proportionate to the amount of AChR remaining at the end plate. The localization of C3 was essentially identical with that of IgG. For most cases of MG it can be inferred that binding of IgG and C3 to AChR does not interfere with receptor function. C9, the terminal lytic complement component, was localized on debris in the synaptic space and on remnants of junctional folds. This proves that complement mediated destruction of junctional folds occurs in human MG. Studies in experimental auto-immune MG indicate that antibody-dependent internalization of AChR occurs in subclinical, mild and more severe diseases but increased AChR synthesis can compensate for this in subclinical and mild myasthenia. Complement-mediated injury of the postsynaptic membrane appears to be a requirement for induction of more severe MG.

Multiplicity of abnormal dystrophin in Becker muscular dystrophy. A Becker muscular dystrophy gene frequently produced two smaller sizes of dystrophin.

Dystrophin is a muscle cytoskeletal protein with a molecular mass (MM) of approximately 420 kDa and an isoelectric point (pI) of approximately 5.5, which is abnormal in size and/or abundance in Becker muscular dystrophy (BMD). We investigated the abnormality of dystrophin molecule in muscles biopsied from 23 BMD patients using the two-dimensional gel electrophoresis (TDGE). We found 7 protein spots which reacted specifically with the monoclonal anti-dystrophin antibody (mAb) A1C raised against N-terminal domain of the normal dystrophin. These spots were focused on the two-dimensional gel at the same position as the normal dystrophin (#1), at the position with MM approximately 480 kDa/pI approximately 5.35 (#2), the position with MM approximately 400-330 kDa/pI approximately 5.51-5.47 (#3), the position with MM approximately 300 kDa/pI approximately 5.4 (#4), the position with MM approximately 235-250 kDa/pI approximately 5.53-5.5 (#5), the position with MM approximately 165 kDa/pI approximately 6.0 (#6), and the position with MM approximately 160 kDa/pI approximately 5.75 (#7). These spots were classified into five patterns in individuals, that is, #1 alone in 3 patients, #3 alone in 1, the combination of #3 and 5 in 17, the combination of #1, 3 and 5 in 1 and the combination of #1, 2, 4, 6 and 7 in 1. The combination of #3 and 5 was observed in 17 of 23 patients (75%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic body and mitochondrial DNA deletion.

A patient with chronic progressive external ophthalmoplegia (CPEO) who had abundant cytoplasmic bodies in muscle fibers and a deletion of mitochondrial DNA is reported. The patient was a 26-year-old male suffering from ophthalmoplegia from age 21. He had a marfanoid skeletal abnormality and perceptive hearing loss, but had neither retinopathy, ataxia, nor dementia. In the mitochondria isolated from the biopsied skeletal muscle, NADH-ubiquinone oxidoreductase activity was slightly decreased, succinate-cytochrome c reductase activity was slightly increased, and cytochrome c oxidase activity remained normal. Southern blot analysis of the muscle DNA identified heteroplasmy composed of a normal-sized mitochondrial DNA and a mutant mitochondrial DNA with a 4.2-kilobase deletion. The PCR plus S1 analysis showed that the deletion extended from nucleotide position 7860 +/- 60 to 12,090 +/- 70. The histological studies of the biopsied muscle revealed ragged-red fibers and cytochrome c oxidase-negative fibers in 15.7% and 18.6% of the muscle fibers, respectively. Other conspicuous histological change was abundant cytoplasmic bodies surrounded by clusters of abnormal mitochondria. The cytoplasmic bodies were found preferentially in type 1 fibers, and exclusively in cytochrome c oxidase-negative fibers and in ragged-red fibers. Focal existence of cytoplasmic bodies in muscle fibers with abnormal mitochondria suggests that segregated distribution of the abnormal mitochondria with deleted mitochondrial DNA is involved in the pathogenesis of cytoplasmic bodies.

Thyrotropin releasing hormone levels in human cerebrospinal fluid in various neurologic diseases.

Thyrotropin releasing hormone (TRH) in the human cerebrospinal fluid (CSF) of 102 patients with non-neurologic and neurologic diseases was measured by a specific TRH radioimmunoassay. TRH levels in CSF were 17.6 +/- 3.4 pg/ml (mean +/- SE) in non-neurologic diseases. TRH levels in CSF were significantly elevated in cases of spinal cord tumor and multiple sclerosis (acute phase). Elevated TRH levels in CSF were decreased in these diseases by operation or prednisolone treatment, respectively. From these data, it is suggested that TRH is present in the human CSF and that measurement of TRH levels in CSF may be a useful indicator of the activity of certain neurologic diseases.

Immunostaining of dystrophin and utrophin in skeletal muscle of dystrophinopathies.

Immunostaining of biopsied skeletal muscle of 4 Duchenne (DMD), 12 Becker muscular dystrophy (BMD) and 3 DMD carriers' was performed using monoclonal antibodies against dystrophin and utrophin. In DMD, dystrophin-negative staining was observed except for revertant fibers which showed different stain patterns for each antibody. In 7 BMDs, there was faint/patchy stain in cases of deletion between exons 45-52, while in one case there was deletion between exons 12-17 and no stain was noted relevant to the deletion site. Moreover, in 2 cases of undetectable deletion, antibodies which recognize a terminal portion of the C-terminal domain revealed the absent stain. In DMD, the utrophin-positive fibers corresponded to dystrophin-negative fibers. In BMD, this relationship did not necessarily occur in each fiber. In DMD carriers, a cluster of dystrophin-negative fibers which was positive for utrophin were prominent. In dystrophinopathy, the immunostaining of dystrophin and utrophin is useful, in combination with dystrophin gene analysis to make a definite diagnosis.

Progressive external ophthalmoplegia and myositis.

We reported a senile male patient with progressive external ophthalmoplegia (PEO) and myositis. The ophthalmoplegia was severe, but other neuromuscular features were nearly normal. Muscle enzymes in serum were moderately elevated. Autoimmune, endocrinological or malignant diseases were not observed during the previous 4 years. Pathology of non-weak limb muscles biopsied twice was consistent with active inflammatory myopathy. The ragged-red or cytochrome c oxidase-negative fibers, which are a hallmark of mitochondrial myopathy with PEO, were not increased in comparison with age-matched control muscles. Analysis of mitochondrial DNA in muscle by the Southern blot method did not reveal any deletions. It was concluded that the inflammatory myopathy, myositis clinically localized at the ocular muscles, is an important and distinct disorder in PEO.

[Immunohistochemical localization of chymase; a mast cell marker and clinical significance in diseased human skeletal muscle].

UNLABELLED: In the advanced stage of dystrophinopathy, cardiac dysfunction is a serious complication for prognosis. Recently, an angiotensin converting enzyme (ACE), which converts angiotensin (A) 1 to A 2, has been reported to be effective for cardiac insufficiency. The A 2 is produced more dominantly in the path via the production of a neutral serine protease, chymase (MW 25,000), secreted from the mast cell. We have observed localization of chymase in diseased human skeletal muscle tissues, and evaluated its clinical significance. The frozen muscle biopsied specimens from 91 neuromuscular disorders (muscular dystrophies, inflammatory myopathies and neurogenic muscular disorders) were stained by using monoclonal antibody against the chymase, and the positive cells in a whole sectional field were counted. In the serial sections, we also performed routine histochemistry and immunostainings of immunological markers (CD4, CD8 and others) as well as the apoptotic proteins for comparison. RESULTS: The chymase-positive mast cells were scattered mainly in the endomysium, partly in the perimysium and around small vessels. Although the positivity was not disease specific, more numerous strongly positive cells were observed in dystrophinopathy and inflammatory myopathies, but less in myotonic dystrophy and neurogenic muscle disorders. In the normal control muscle, however, strongly positive cells appeared less frequently than in the above mentioned diseased muscles. The chymase-positive cells partly corresponded to the ubiquitin-positive ones, but perforin, granzyme A, Fas and Bcl-2 did not. In conclusion, the chymase-positive mast cell may play a primary or secondary role in the diseased muscle, and their more abundant appearance in dystrophinopathy and some other myopathies suggest the effectiveness of an ACE blocker, an anti-chymase drug.

[Immunostaining of anti-Fas IgG1 antibody in diseased human muscle].

Immunostaining of the Fas antigen using the anti-Fas IgG1 antibody was performed on biopsied human diseased muscles. The immunostaining showed negative results in the control muscles. In dystrophinopathy [DMD and BMD], positivity was seen mainly in type 2 fibers with no correlation to the opaque fibers and histochemical Ca2+ loading fibers in DMD. In DMD carriers, a relative correlation was seen between dystrophin-negative and Fas-positive fibers. In distal myopathy with rimmed vacuoles, fibers with positive staining in the vacuoles but negative in their membranes were seen at high frequency. In FSH, a very low frequency of positivity was seen. And in myotonic dystrophy, positivity was seen in the type 2 fibers containing the internal nuclei. In inflammatory myopathies, strong positivity was seen in the medium size fibers, and mild to moderate positivity in the fibers facing the perimysium. In neurogenic muscular disorders, fibers with concave borders or highly atrophic fibers showed Fas-positivity. In conclusion, there was no disease-specific Fas reaction in the human pathologic muscle samples, but the high positivity was apparent in some myopathies. This fact of Fas antigen would reflect a pathologic state in the skeletal muscle.

[Expression of MHC and cell adhesion molecules in muscular sarcoidosis].

Biopsied skeletal muscles from 5 patients with muscular sarcoidosis (nodular type; 1, and myopathic type; 4) were immunocytochemically examined. All biopsies presented granulomatous changes. Atrophic or regenerating muscle fibers adjacent to granuloma demonstrated compression or ischemic changes. In the center of the granuloma, CD68+ epitheloid cells and giant cells, and CD4+ T cells were localized. At the periphery of the granuloma, CD4+ T cells, CD8+ T cells, CD20+ B cells, and CD68+ macrophages were found. Expression of HLA-A,B,C was diffuse in the muscle fibers. Expression of HLA-DR and ICAM-1 was more prominent near the granuloma or perifascicular fibers, and that of LFA-3 was moderate in those lesions. VCAM-1 was expressed in endothelial cells and macrophages near the granuloma. Those findings indicate that interferon-gamma or TNF-alpha produced by infiltrating inflammatory cells may induce expression of these immunologic markers or adhesion molecules. Immunocytochemical differences between the nodular and myopathic forms of sarcoidosis are not evident, but either localization or abundance of granuloma in muscle bulks is relevant to weakness or atrophy of clinically affected muscle.

[Expression of the heat shock protein 70 in inflammatory myopathies].

Heat shock protein 70 (HSP 70) expression was immunohistochemically observed in diseased muscle fibers of 35 patients with dermatomyositis (DM) and 7 with polymyositis (PM). In DM, HSP 70 was localized in the sarcoplasm of type 1 fibers adjacent to the small vessels showing deposits of complement components in 13 patients and in the atrophic fibers at perifascicular regions in 7. HSP 70 was also expressed more preferentially in the small vessels rather than in the sarcoplasm in 13 DM patients. In PM, the expression of HSP 70 was blurred in all fibers including non-necrotic fibers invaded by T cells. In conclusion, HSP 70 is likely more frequently to be expressed in the sarcoplasm of DM than PM due to probable ischemic insults.

[Acquired unilateral blepharoptosis by wearing contact-lenses in young adult women].

Four young unmarried women developed unilateral non-fluctuating blepharoptosis by wearing contact-lenses. Past and family histories were unremarkable. Blepharoptosis insidiously occurred within a few years after wearing lenses. Contralateral lid was quite normal. No abnormalities was observed in pupils, extraocular muscles, ocular positioning and other systems. So far recognized causes of blepharoptosis were ruled out through extensive clinical and laboratory observations. Improvement was insufficient even after wearing lenses off. Pathogenesis is probably due to repeated minor trauma to the levator palpebral muscle and its tendon secondary to frequent wearing on/off contact-lenses.