Myasthenic crisis with delayed recovery after plasmapheresis.

We report on 2 elderly patients with myasthenia gravis in whom recovery from crisis was prolonged despite intensive plasmapheresis (PP). In both patients, the anti-acetylcholine (anti-AChR) titer failed to fall sufficiently after completing PP. These patients might have had antibodies that produced a more pronounced effect on the degradation of AChR, or the synthesis of AChR might have been reduced by aging. The anti-AChR titer did not correlate with a reduction of IgG after PP in 1 patient. Successful treatment was achieved by keeping the anti-AChR titer at a low level via the concomitant use of prednisolone with PP.

Acquired idiopathic generalized anhidrosis with isolated sudomotor neuropathy.

The pathogenesis and underlying lesion of acquired idiopathic generalized anhidrosis (AIGA) are apparently heterogeneous. We report a patient with AIGA in whom the eccrine glands were histologically normal. However, electron microscopic examination showed markedly low numbers of nerve terminals and unmyelinated axons associated with the eccrine glands. Our laboratory investigations suggest that degeneration of postganglionic sympathetic cholinergic nerve may be the underlying pathogenetic mechanism of anhidrosis in this patient.

Treatment of experimental allergic encephalomyelitis by selective removal of CD4+ T cells.

Experimental allergic encephalomyelitis (EAE) is a major animal model of human multiple sclerosis (MS). The pathogenesis of both MS and EAE directly involves CD4+ helper T cells. To remove CD4+ T cells selectively from the circulation, we designed a new column in which anti-CD4 monoclonal antibody was immobilized on the activated substance. Most of the CD4+ T cells, including pathogenic T cells, were selectively adsorbed from whole blood with a direct perfusion through the column in vitro, resulting in depletion of the antigen-specific T cell responses. A preliminary trial of ex vivo treatment with the column in EAE rats lowered the percentages of CD4+ populations but failed to alter the course of the disease, suggesting repeated treatment would be necessary to suppress the development of the disease. We conclude that this new column is potentially useful, and repeated treatment would be beneficial in MS and other CD4+ T cell dependent autoimmune diseases.

[Microsatellite analysis of Duchenne muscular dystrophy].

Duchenne muscular dystrophy is X-linked recessive neuromuscular disorders caused by mutations in the dystrophin gene. Prenatal diagnosis and carrier detection are usually performed using diallelic RFLP-markers which are not always informative. Now 30 of microsatellite marker have reported, these microsatellite polymorphism can easily be amplified using PCR technique. If mutations are known to localize in this region of the dystrophin gene or if routine RFLP-analysis is uninformative, the analysis of microsatellite markers is the preferable technique in prenatal diagnosis and carrier detection.

Methodologies for specific intron and exon RNA localization in cultured cells by haptenized and fluorochromized probes.

We have determined optimal conditions for the detection of mRNA sequences in cultured cells by nonradioactive in situ hybridization. For this purpose a number of different cell lines have been used: rat 9G cells for the detection of human cytomegalovirus immediate early mRNA, and HeLa as well as 5637 carcinoma cells for the detection of housekeeping gene mRNAs. Extensive optimization of fixation and pretreatment conditions revealed that most intense hybridization signals are obtained when cells are grown on glass microscope slides, fixed with a mixture of formaldehyde and acetic acid, pretreated with pepsin and denatured prior to hybridization. In addition, we also studied the potential of fluorochromized probes for the direct detection of multiple RNA sequences. The optimized in situ hybridization procedure revealed that immediate early mRNA transcripts are, in addition to a cytoplasmic localization, localized within nuclei of rat 9G cells. Double hybridization experiments showed that intron and exon sequences colocalize within the main nuclear signal. In addition, the presence of small, intron-specific, fluorescent spots scattered around the main nuclear signals indicates that intron sequences which are spliced out can be visualized. Additional information about the functioning of cells could be obtained by the detection of mRNA simultaneously with bromodeoxyuridine, incorporated during S-phase, or its cognate protein. The sensitivity of these methods is such that mRNAs of abundantly expressed housekeeping genes can be detected in a variety of cell lines with high signal to noise ratios.

Gene-deletion and carrier detections, and prenatal diagnosis of Duchenne muscular dystrophy by analysis of the dystrophin gene amplified by polymerase chain reaction.

Polymerase chain reaction (PCR)-based diagnosis was carried out in 62 patients (57 probands) with Duchenne or Becker muscular dystrophy (DMD or BMD) and 226 members in 57 families. The PCR studies were also performed for carrier detection in 57 mothers and 58 sisters, and prenatal diagnosis of 4 fetuses at risk of DMD. The PCR with 7 sets of primers, which amplify 7 different exon-sequences of the dystrophin gene, detected gene deletion of at least one exon in 49% of the probands. The PCR with the other 4 primer sets, which amplify 3 intragenic loci, and subsequent endonuclease digestion detected in 84% of the mothers a heterozygous pattern in at least one such locus/segment. Using the same primer sets, carrier detection was successful in 5 sisters of familial DMD cases, while recombination between the ERT87 and the 3' end intragenic loci was observed in 11% of family members studied. Prenatal diagnosis was made in all the 4 fetuses; two males were affected, one male fetus non-affected, and the remaining one female fetus a carrier. Thus, the PCR study and the primers used in the present study are useful and convincing for rapid diagnosis of DMD and/or BMD.

Correlation between clinical features and deletions of the gene for dystrophin in Duchenne muscular dystrophy.

The clinical course and prognosis of Duchenne muscular dystrophy (DMD) was compared in patients with deletions of the gene for dystrophin (cDMD) and those without such deletions. A total of 24 patients was followed for at least 2 yrs. At age 12 the rating of the activities of daily life (ADL) and disease stage were less favorable in those patients with deletions of the gene for cDMD. At age 14, no difference in ADL and disease stage was observed between the two groups. The percent vital capacity was lower in those patients with the cDMD deficit. When the prognosis was evaluated by multivariate analysis of the data obtained at age 12, the percent of patients predicted as dying before the age of 20 was 40% for those without the cDMD deficit but 76% for those who were cDMD defective. None of the cDMD defective patients lived longer than 20 yrs, whereas 5 of 14 patients without the cDND deficit survived longer than 20 yrs. Disorders such as cardiac and respiratory failure were also seen more frequently in the cDND defective patients. These results suggest that patients with Duchenne muscular dystrophy with defective cDMD have more severe disease than those without cDMD deficit.

Changes of egg retinoids during the development of Xenopus laevis.

The changes of egg retinoids during the development of Xenopus laevis were investigated by high-performance liquid chromatography (HPLC). All-trans retinal and 3-dehydroretinal are endogenous in the egg and are distributed to both the eyes and the ventral portion of the larval body. These retinals are converted to all-trans retinyl palmitate and 3-dehydroretinyl palmitate during the development up to stage 46. 11-cis retinal and 3-dehydroretinal can be detected after stage 40 in the eyes but not in the larval ventral portion. It is suggested that retinoids are transported from the larval ventral portion to the eyes after stage 41/42.

Molecular-genetic study of Duchenne and Becker muscular dystrophies: deletion analyses of 45 Japanese patients and segregation analyses in their families with RFLPs based on the data from normal Japanese females.

This study consisted of 1) molecular deletion analyses in patients with Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) using the entire cDNA for the DMD gene as hybridization probes, 2) RFLP analyses in a large number of Japanese normal women using 11 DMD-linked cloned DNAs as probes, and 3) segregation analyses with these RFLP data in 17 DMD families in which prenatal or carrier diagnosis was required. The deletion study showed that 18 (43%) of 42 male DMD patients had a deletion within the DMD gene, while no detectable deletion was found in 3 BMD patients. These deletions were preferentially observed at the 5' end of the DMD gene, while no deletion was found in the 3' portion of the gene. Of a total of 15 RFLPs detected with the 11 probes, one was a new RFLP (probe/enzyme: P20/MspI). In 6 RFLPs, the allele frequencies in the Japanese were statistically different from those in the Caucasian. Based on the RFLP data combined with the result of the deletion study, an estimated diagnostic rate for prenatal diagnosis and/or carrier detection in the Japanese DMD families was 63%. The real diagnostic rate obtained from the prenatal and carrier diagnoses, which were practically performed in 17 families, corresponded to the estimation. A protocol useful for the diagnosis in Japanese DMD families is presented.

[Strategy for molecular diagnosis of Duchenne muscular dystrophy: alleles frequency in RFLPs detected with X-linked probes in normal Japanese].

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder. Recently it has been suggested that the protein named "dystrophin" is not produced in the DMD muscle because of their mutant of defective DMD gene. Recent molecular studies using subclones from the DMD-cDNA as probes revealed a DNA deletion in about a half of the DMD patients. Since not all patients showed deletion, segregation analysis with restriction fragment length polymorphisms (RFLPs) is still necessary and important for diagnosis not only patients but female carriers as well. Therefore, we tried to detect RFLPs with the use of many probes localized within and/or flanked with the DMD gene and made a protocol for the diagnosis with our RFLP data. Among 50 normal Japanese females, the frequencies of rare alleles which identified with enzyme/probe combinations in each were as follows (RFLPs practically available for diagnosis are underlined): 0.38 for p782/EcoRI; 0.14 for p99-6/Pst I: 0.45 for pJ66-HI/Pst I: 0.16 for pERT87-30/Bgl II; 0.44 for 87-15/Xmn I: 0.07 for 87-15/Taq I; 0.33 for 87-8/Taq I; 0.44 for 87-1/Xmn I; 0.45 for 87-1/Mva I; 0.43 for Xj-1.1/Taq I; 0.34 for 754-11/EcoR I; 0.41 for 11.28/Taq I. Some of our results were different from the Caucasian both on the allele frequencies and the restriction fragment length. We proposed a diagnostic protocol of DMD and it's carriers in Japan. Firstly, segregation analysis should be performed with five different combinations of double probe/enzyme, such as Yj-1,.1/L1.28/Taq I, ERT87-1/87-15/Xmn I, 87-1/Mva I, J66-HI/99-6/Pst I, and 782/754-11/EcoR I.(ABSTRACT TRUNCATED AT 250 WORDS)

Composition and distribution of retinal and 3-hydroxyretinal in the compound eye of the dragonfly.

Retinoids in the compound eyes of nymphs and adult dragonflies in 11 families of the 3 suborders were extracted by the oxime method, and analysed by high performance liquid chromatography. Almost all of the species examined contained both retinal and 3-hydroxyretinal in the compound eye. The ratio of 11-cis 3-hydroxyretinal to 11-cis retinal (3-OH ratio) was calculated as an index of the retinoid composition. The 3-OH ratios of the whole eye of nymphs in all the suborders and of adults of the suborder Zygoptera were very high, 2.2 at the minimum, but in Anisozygoptera and Anisoptera most of the ratios were distributed between 1 and 2.7. In the family Gomphidae, exceptionally low 3-OH ratios, less than 1, were observed in several species. The regional distributions of the retinals in the adult compound eyes were also examined. In the Zygopteran compound eye, both retinals were distributed evenly all over the eye, while in the compound eye of the other two suborders, the 3-OH ratios in the dorsal area of the eye were extremely low. In several species of Gomphidae and Libellulidae the ratios in the dorsal areas were zero. From the correspondence of these results and the compartment of the compound eye, it appeared that the large ommatidia in the dorsal area contained only retinal. This was confirmed when the large facet region in the dorsal part of the compound eye of an Anax was excised and examined, and only retinal was detected. However, the ventral area of the true dragonflies' compound eye which did not include the large ommatidia contained both retinals, and the 3-OH ratio was more than ten. The biological significance of using both retinals as chromophores of visual pigments in the dragonfly eye is discussed in relation to the structure of the ommatidia and to the vision of dragonflies.

Formation of visual pigment chromophores during the development of Xenopus laevis.

Retinoids in the eyes of Xenopus laevis at several developmental stages, were analyzed by high-performance liquid chromatography (HPLC). At stage 37/38, larval eyes contained mainly all-trans isomers of retinal, 3-dehydroretinal, retinyl ester and 3-dehydroretinyl ester. Ratios of all-trans 3-dehydroretinal to retinal and of all-trans 3-dehydroretinyl ester to retinyl ester were almost 1 at the stage. With the advance of development, the amounts of all-trans retinal and 3-dehydroretinal decreased; however, those of all-trans retinyl ester and 3-dehydroretinyl ester increased. The chromophores of visual pigments, 11-cis retinal and 3-dehydroretinal, were detected at stage 40 (total; 0.2 pmol/eye) and their amounts increased after that stage. The ratio of 11-cis 3-dehydroretinal to retinal was almost 1 at stages 40-42. The ratio became larger after stage 43 and was almost 19 at stage 46. The ratio of all-trans 3-dehydroretinyl ester to retinyl ester, also, increased after stage 42 and reached 11 at stage 46. The mechanism of 11-cis formation during development is discussed in relation to retinoid conversions in the eyes.

Retinoid composition in the compound eyes of insects.

Retinoids in the compound eyes of insects in ten orders were extracted by the oxime method and analysed by HPLC. Four geometrical isomers (13-cis, 11-cis, 9-cis and all-trans) of syn and anti retinal oximes, and syn and anti 3-hydroxyretinal oximes were separated in a single analysis by a stepwise eluent condition. The amounts of the two isomers, syn 11-cis and syn all-trans, were quantified. 11-Cis 3-hydroxyretinal was detected in six orders: Lepidoptera, Diptera, Coleoptera, Neuroptera, Hemiptera and Odonata, and retinal and 3-hydroxyretinal were found together in the compound eyes of some species of Coleoptera and Odonata. We conclude that early in their phylogeny, insects had the ability to use 3-hydroxyretinal as the chromophore of visual pigment. Peaks corresponding to syn 9-cis and 13-cis 3-hydroxyretinal oximes were observed on the chromatogram of extracts from fly heads and compound eyes of cicadas. Retinol and 3-hydroxyretinol were also analysed and quantified relative to retinal and 3-hydroxyretinal. Larger amounts of the alcohols than the aldehydes were found in the compound eyes of butterflies, hornets, cicadas and grasshoppers, which are diurnal insects. 3-Dehydroretinal has not been detected in insects.

A fly, Drosophila melanogaster, forms 11-cis 3-hydroxyretinal in the dark.

All-trans and 11-cis 3-hydroxyretinals were synthesized and the presence of these substances in the head of Drosophila melanogaster was shown by using high performance liquid chromatography. Even when the head extract was prepared in the dark from the flies reared successively in the dark, both of the 3-hydroxyretinal isomers were detected. In the culture medium, they were not present. D. melanogaster must have an 11-cis 3-hydroxyretinal forming-system that does not need light.