Serial MR imaging and 1H-MR spectroscopy in monozygotic twins with Tay-Sachs disease.

Four-year-old monozygotic female twins with early onset Tay-Sachs disease are described. The sisters showed similar slowly progressive clinical symptoms and deterioration, however the younger sister also demonstrated intractable myoclonus in the right leg. The serial MR images and (1)H-MR spectroscopy of the brain were obtained in both twins. MR images showed high intensity on T (2)-weighted image in the bilateral white matter, however there were no signal changes in the basal ganglia and thalamus during any of the phases. The ratio of N-acetylaspartate (NAA)/creatine (Cr) was decreased in the both white matter lesions and the corpus striatum, and that of myoinositol (mI)/Cr was increased in the damaged white matter on MR spectroscopy. The elevation of the lactate peak was clearly demonstrated in the left basal ganglia of the younger sister; however it was not shown in cerebral lesions of the elder sister. Changes in metabolites on MR spectroscopy were closely linked to the respective clinical features of each twin. Follow-up examination by (1)H-MR spectroscopy is useful for the evaluation of neuronal changes in children with Tay-Sachs disease.

Safety of ofloxacin (OFLX) and fosfomycin sodium (FOM) ear drops.

OBJECTIVE: The objective of this study is to evaluate the safety of two ear drops, Ofloxacin (OFLX: Taribid Otic Solution, Daiichi Seiyaku) and Fosfomycin sodium (FOM: Fosmicin S, Meiji Seiyaku). METHODS: Albino guinea pigs were used as experimental animals, and the ototoxicity was evaluated by means of threshold changes in the compound action potentials (CAP), when topically applied to the middle ear cavity of the guinea pig. The sound stimuli applied were; click sound, with tone bursts of 8 kHz, 4 kHz, and 2 kHz. In one group of animals, after one application of the ear drops in the right middle ear cavity, the change in CAP was compared with a contralateral saline control at 24h, one week, and four weeks. In other group of animals, the ear drops were applied into the middle ear cavity for seven consecutive days and the CAP was measured at 24h. RESULTS: At 24h the CAP threshold for click, 8 and 4 kHz elevated significantly for both the saline and ear drop treatment, but the threshold returned to normal when measured at 7 days and 28 days. Seven consecutive days of ear drops administration resulted in no reduction in the CAP for either ear drops. CONCLUSIONS: Based on the lack of changes in the CAP, these two ear drops studied did not show any significant ototoxicities.

Inflammatory gene signature in ulcerative colitis with cDNA macroarray analysis.

BACKGROUND: Most array analyses of ulcerative colitis have focused on identifying susceptibility genes for ulcerative colitis. AIM: To clarify the changes in gene expression during inflammation in ulcerative colitis colon mucosa using cDNA macroarray. METHODS: From 23 ulcerative colitis patients, 16 each of inflamed and non-inflamed specimens (total 32 samples for individual analysis) were obtained by colonoscopic biopsy. Eighteen of the 32 samples, used for pairwise analysis, consisted of nine sample pairs, each pair being from the same patient. We examined expression profiles of approximately 1300 genes with cDNA macroarray. Comparisons were made using two kinds of statistics, t-test and significance analysis of microarray in both analyses. The reproducibility of significant genes from the macroarray analysis was confirmed by real-time ploymerase chain reaction. RESULTS: We detected five upregulated genes, categorized into proinflammatory genes (MRP14, GRO gamma and SAA1) and anti-inflammatory genes (TIMP1 and Elafin) in inflamed mucosa, and one upregulated gene (L-FABP) in non-inflamed mucosa. CONCLUSIONS: As the cDNA macroarray analysis in this study exactly reflects the total profile of gene expression in the clinical setting of ulcerative colitis, the genes identified will be directly applicable to diagnostics or as novel therapeutic targets in active ulcerative colitis.

Genes encoding pseudo-response regulators: insight into His-to-Asp phosphorelay and circadian rhythm in Arabidopsis thaliana.

In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-to-Asp phosphorelay mechanisms are involved presumably in propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we identified and characterized a set of novel Arabidopsis genes whose products considerably resemble the authentic response regulators (ARR-series) of Arabidopsis in the sense that they have a phospho-accepting receiver-like domain. However, they should be discriminated from the classical ones in the strict sense that they lack the invariant phospho-accepting aspartate site. They were thus named APRRs (Arabidopsis pseudo-response regulators). Two such representatives, APRR1 and APRR2, were characterized extensively through cloning of the corresponding cDNAs, in terms of their structural designs, biochemical properties, subcellular localization in plant cells, and expression profiles at the transcriptional level. The result of in vitro phosphorylation experiment with the Arabidopsis AHP phosphotransmitter suggested that the pseudo-receivers have no ability to undergo phosphorylation. The result of transient expression assay with onion epidermal cells showed that the GFP-APRR1 fusion protein has an ability to enter into the nuclei. The C-terminal domain of APRR1, termed CONSTANS-motif, appears to be responsible for the nuclear-localization. The most intriguing result was that the accumulation of APRR1 transcript is subjected to a circadian rhythm. The APRR1 protein is identical to the one that was recently suggested to interact with the ABI3 (ABISCISIC ACID INSENSITIVE3) protein. These are discussed with special reference to the His-to-Asp phosphorelay signal transduction and circadian rhythm in Arabidopsis thaliana.

The 5'-flanking region of the mouse adenylyl cyclase type VIII gene imparts tissue-specific expression in transgenic mice.

The calcium-stimulated adenylyl cyclases (ACs) play a central role in stimulus-dependent modification of synaptic function. The type VIII AC (AC8) is one of three mammalian calcium-stimulated isoforms, each of which is expressed in a region-specific manner in the CNS. To delineate the DNA sequences responsible for appropriate targeting of AC8 expression, we report here the complete structure of the AC8 gene and define the pattern of expression of the full-length cDNA and its splice variants. In addition to expression within the brain, robust expression of AC8 was also found in the lung. By in situ hybridization, we have found the highest expression of AC8 mRNA within the olfactory bulb, thalamus, habenula, cerebral cortex, and hypothalamic supraoptic and paraventricular nuclei. By generating transgenic mice whose expression of beta-galactosidase is controlled by the AC8 5'-flanking DNA sequences, we demonstrate that the DNA sequences within the 10 kb preceding exon 1 are critical for establishment of this region-specific pattern. This spectrum of sites of production is unique to AC8 among the calcium-stimulated adenylyl cyclases and suggests nonredundant functions with other adenylyl cyclases in neuroendocrine regulation and/or behavior.

Peroxisome biogenesis disorders: identification of a new complementation group distinct from peroxisome-deficient CHO mutants and not complemented by human PEX 13.

Ten complementation groups of generalized peroxisome biogenesis disorders (PBD), (excluding rhizomelic chondrodysplasia punctata) have been identified using complementation analysis. Four of the genes involved have been identified using two different methods of (1) genetic functional complementation of peroxisome deficient CHO cell mutants and (2) homology searches for human dbEST, based on yeast genes involved in peroxisome biogenesis (PEX genes). We report here the first identification of a new complementation group which is genetically different from peroxisome deficient CHO mutants. There were no complementations by the human PEX 13 gene. The nature of the related gene is being investigated.

Trial of docosahexaenoic acid supplementation on a Japanese patient with a peroxisome biogenesis defect.

A female Japanese patient diagnosed with peroxisome biogenesis defect (PBD), who had hypotonia and craniofacial dysmorphism, was given supplementation of docosahexaenoic acid (DHA). Accumulation of very long chain fatty acids was revealed, and a diagnosis of PBD was made at 2 months of age because of the absence of peroxisomes, a defect in peroxisomal beta-oxidation enzymes and a decreased level of DHA in the erythrocytes. Supplementation of DHA was introduced at 3 months of age. For the first several months, psychomotor development was fairly good. The patient could laugh, brush off a blanket and play with toys at 6 months of age. However, neurological regression and convulsions occurred after 7 months of age. After recurrent respiratory infections and disturbance of the circadian rhythm, the patient died of liver failure and disseminated intravascular coagulopathy at 20 months of age. DHA may have a favorable effect on the early development of patients with PBD, but neurological deterioration cannot be prevented. Patients with a milder phenotype would be better candidates for DHA supplementation.

[Benign infantile mitochondrial myopathy caused by reversible cytochrome c oxidase deficiency].

A 2-month-old girl had generalized weakness, profound muscular hypotonia, hepatomegaly and severe lactic acidosis. She needed ventilatory support. Muscle specimen taken at 2 months showed ragged-red fibers, abnormal mitochondria, and reduced cytochrome c oxidase (CCO) staining Biochemical analysis showed CCO activity to be reduced to about 16% of the normal mean. She received carnitine and coenzyme Q10 supplementation from the age of 3 months and abnormal blood lactate values declined to near normal values during the first three weeks. Gradually her condition started to improved: she held her head at 9 months, and walked alone at 15 months. The second biopsy specimen at 3 years and 8 months showed almost normal CCO staining and she was free of clinical signs. This case is an example of a rare benign infantile mitochondrial myopathy caused by CCO deficiency. Early diagnosis is crucial to provide intensive treatment until spontaneous clinical improvement appears. We concluded that carnitine and coenzyme Q10 supplementation was a useful treatment for clinical improvement in patients with a benign infantile mitochondrial myopathy caused by CCO deficiency.

Identification of a nonsense mutation in ALD protein cDNA from a patient with adrenoleukodystrophy.

The molecular basis of X-linked adrenoleukodystrophy (ALD) was investigated. Six (A to 50) fragments of cDNA for ALD protein (Mosser et al. Nature 361: 726-730, 1993) from an adult patient with adrenomyeloneuropathy were amplified by PCR and mutations were screened by Mutation Detection Enhancement gel electrophoresis. A single base substitution (2154 C-->T, which resulted in the formation of a termination codon for glutamine (Q590STOP) and deletes Pst I site (CTGCAG-->CTGTAG), was detected. Eight other ALD patients did not have this mutation. A family study revealed the presence of both the mutant and normal alleles in the mother, a sister and a niece, indicating that these individuals were carriers. A nephew with childhood ALD who died 10 years earlier had the same mutant allele as detected by Pst I restriction assay. This report is the first description of a mutant allele for ALD, at the cDNA level, and presents confirmatory evidence of ALD protein as the primary etiology of ALD.

[Bone mineral content after successful renal transplantation].

In the present investigation, serial measurements of bone mineral content before and after successful renal transplantation were evaluated by single photon absorptiometry, using a Bone Mineral Analyser (Norland Corp., USA). Twenty-four renal transplant recipients, whose grafts were well functioning for more than 6 months, were the subjects of this study; they were divided into two groups: one was AZP group which includes 12 patients receiving azathioprine and high dose of steroid and another was CYA group which includes 12 patients receiving cyclosporin A and low dose steroid as immunosuppressants. In both groups patients were similar in regard to age, source of donor, duration on dialysis prior to transplant. Variation rate of bone mineral content was calculated as follows: b-a/a x 100 (a; value before transplantation, b; value after transplantation). Nonpaired t-test was used when comparing the values of both groups. The results were as follows: 1) AZP group showed no improvement of BMC after 24 months, but only one patient showed improvement of BMC after 36 months. 2) In CYA group, improvement of BMC was found in 5 out of 11 patients after 12 months, in 7 out of 10 patients after 24 months and in 4 out of 4 patients after 36 months. 3) The cumulative dose of methyl-prednisolone was significantly lower in CYA group than in AZP group both one and three months after transplantation (p less than 0.001). It is concluded that the cumulative dose of methyl-prednisolone may have an important factor in the recovery of bone.

Features of regenerated clones with or without fusion treatment between auxotrophic mutants of Streptomyces antibioticus and their antibiotic productivity.

During experiments on protoplast fusion of complementary auxotrophic mutants (194 and 11M-21) of Streptomyces antibioticus for strain improvement, the clones (typified by F-40) regenerated on minimal regeneration medium (MRM) were found to be prototrophs, and to produce an antibiotic different from those produced by the parent strain. The protoplast regeneration of each parent was examined as a negative control experiment. In the regenerated clones of 194, half of them produced actinomycins similar to those produced by the original mutant 194, but others (typified by R-20) seemed to produce antibiotics similar to those produced by F-40. In the taxonomic characterization of morphological, cultural, and physiological properties of each strain, F-40, R-20, and the parent mutant 194 had no significant differences with a few exceptions. The problem here is whether the antibiotic of R-20 is the same as that of F-40, which was first isolated and found to be a peptide antibiotic different from actinomycins, with activity against Gram-negative and Gram-positive bacteria.

The relation between Ames test data and electronic structure for a series of benzidine derivatives investigated by using cluster analysis.

The dose-response curves of benzidine derivatives calculated from Ames test data were analyzed by combining the least-squares method with cluster analysis. The calculations of the electronic structures of various benzidine derivatives have at the same time been performed by using the CNDO/2 method. The cluster analysis was carried out on various parameters concerning the electronic structure, such as total electron density, frontier electron density, the energy level of HOMO (highest occupied molecular orbital), and so on. It was found that the total electron density on the nitrogen atom gives a similar pattern of classification to that for mutagenic compounds in cluster analysis. In other words, it may be inferred that the nitrogen atoms of the benzidine derivatives play an important role in the metabolic activation of the compounds. Consequently, cluster analysis with a parameter of electronic structure should be a useful initial screening procedure for various analogous mutagenic compounds as well as carcinogenic compounds.

Carnitine depletion as a probable cause of hyperlipidemia in uremic patients on maintenance hemodialysis.

The mechanism of te development of hemodialysis hyperlipidemia was investigated in uremic patients on maintenance hemodialysis. Hemodialysis treatment lost large amounts of carnitine from blood into the dialysate fluid, resulting in the reduction in serum concentration of carnitine. After the treatments were repeated for more than 12 months, the serum concentration of carnitine reduced markedly and the serum triglyceride level increased significantly. In contrast, in patients who had been supplemented with commercial amino acids solution, the serum concentrations of carnitine and lipid were within normal ranges and remained unchanged even after repeated hemodialysis treatments. Carnitine administration also reduced the serum triglyceride level to or towards normal. The results suggest that carnitine depletion induced by hemodialysis treatments has a probable causal relationship to hyperlipidemia in uremic patients on long-term maintenance hemodialysis and that supplementation of carnitine or amino acids prevents carnitine depletion and improves hemodialysis hyperlipidemia.