Brain regional differences in the expansion of a CAG repeat in the spinocerebellar ataxias: dentatorubral-pallidoluysian atrophy, Machado-Joseph disease, and spinocerebellar ataxia type 1.

Three autosomal dominant spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy (DRPLA), Machado-Joseph disease (MJD), and spinocerebellar ataxia type 1 (SCA1), are associated with the expansion of a CAG repeat in the respective genes. To investigate the association between CAG repeat expansion and neuropathological findings, we analyzed several brain regions from 9 cases of DRPLA, 3 cases of MJD, and 1 case of SCA1. We found that the expanded alleles were smaller in the cerebellar cortex than in other brain regions, such as the frontal cortex, in these three diseases.

Expression of dentatorubral-pallidoluysian atrophy (DRPLA) proteins in patients.

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

A Japanese family carrying a novel mutation in the Emery-Dreifuss muscular dystrophy gene.

We report on a Japanese family affected by Emery-Dreifuss muscular dystrophy carrying a novel mutation of the emerin (STA) gene. The cardinal clinical feature of the family was cardiac conduction block and mild myopathy. A deletion of 11 bp with a frameshift was identified in exon 6, causing truncation of the predicted protein.

Regional and cellular expression of the Machado-Joseph disease gene in brains of normal and affected individuals.

Machado-Joseph disease (MJD) is an autosomal dominant disorder characterized pathologically by spinocerebellar degeneration. Recently, an expansion of CAG repeat in a gene located at the chromosome 14q32.1 was found to be responsible for the disease.

Preparation of specific antiserum to 17 alpha-estradiol 17-N-acetylglucosaminide.

A specific enzyme immunoassay (EIA) method has been developed for 17 alpha-estradiol 17-N-acetylglucosaminide as a model compound instead of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides. Two new haptens, 3-(omega-carboxyalkyl) ether derivatives of 17-alpha-estradiol 17-N-acetylglucosaminide, were synthesized and conjugated with bovine serum albumin (BSA). The EIA was newly established using specific antiserum elicited against 3-(1-carboxypropyl) ether of 17 alpha-estradiol 17-N-acetylglucosaminide (17NAG CPE)-BSA conjugate and beta-galactosidase-labeled 17NAG CPE as a labeled antigen.

Separation and detection of bile acid 3-glucuronides in human urine by liquid chromatography/electrospray ionization-mass spectrometry.

A method for the separation and detection of bile acid 3-glucuronides by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. On the ESI mode, glucuronides were characterized by an intense pseudo-molecular ion [M-h]- with a doubly charged ion [M-2H]2-, and the ratio of these negative ions were markedly influenced by an acidic component of salt added to a mobile phase, according to a pKa value of an acidic moiety at C-24. Bile acid 3-glucuronides in human urine were extracted with a SepPak C18 cartridge, followed by purification on lipophylic ion exchange gel, piperidinohydroxypropyl Sephadex LH-20.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations.

Cell death mechanisms in ALS.

Mutations in copper-zinc superoxide dismutase (CuZnSOD) that are associated with familial ALS (FALS) are dominant, gain-of-function mutations, but the nature of the function gained has not been identified. In addition to catalyzing the dismutation of superoxide, copper-zinc superoxide dismutase also displays peroxidase activity. Whereas mutants A4V and G93A retained superoxide dismutase activity, they demonstrated a markedly enhanced copper-dependent peroxidase activity in comparison with that of the wild type enzyme as detected by the spin trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) in electron paramagnetic resonance measurements.

[An autopsy case of late-onset chorea].

Senile chorea has been an ill-defined clinical entity because of the difficulty of differentiating it from Huntington's disease (HD) of late-onset type. The gene specific for HD has recently been found to contain an abnormal (CAG)n trinucleotide repeat which allows it to be differentiated from the other conditions. Our case of late-onset chorea was differentiated from HD by PCR.

A novel enzyme system for the reduction of 3-oxo bile acids in human red blood cells.

7 alpha,12 alpha-Dihydroxy-3-oxo- and 3,7,12-trioxo-5 beta-cholanoic acids labeled with 18O atoms were incubated with human red blood cells, and the biotransformation products were separated and characterized by gas chromatography-mass spectrometry as the pentafluorobenzyl ester-trimethylsilyl and -dimethylethylsilyl ether derivatives with the negative ion chemical ionization mode. The reduced products, 3 beta,7 alpha,12 alpha-trihydroxy-5 beta-cholanoic acid for the former, and 3 alpha-hydroxylated dioxo bile acid together with 3 beta-hydroxylated 7,12-dioxo-5 beta-cholanoic acid for the latter, were identified as metabolites. When 3-oxo bile acid was incubated with human blood denatured at 70 degrees C for 2 min, no metabolites were formed.

Synthesis of N-acetylcysteine conjugates of catechol estrogens.

The synthesis of N-acetylcysteine conjugates of 2-hydroxyestrone (2-OHE1) and 4-hydroxyestrone (4-OHE1) is described. The reaction of estrone 2,3-quinone with N-acetylcysteine provided 2-OHE1 and its C-4 and C-1 thioether conjugates in a ratio of 1:1, while estrone 3,4-quinone with N-acetylcysteine gave 4-OHE1 and its C-2 thioether conjugate as a sole product. Their structures were characterized by inspection of NMR spectra, chemical derivatization (methylation and acetylation), and comparison with the reactivity of 4-bromoestrone 2,3-quinone or 2-bromoestrone 3,4-quinone toward N-acetylcysteine.

Immunoaffinity extraction for liquid chromatographic determination of equilin and its metabolites in plasma.

Immunoaffinity extraction for the high-performance liquid chromatographic determination of equilin and its metabolites in plasma has been achieved. The antibody raised against an equilin 3-O-carboxymethyl ether-bovine serum albumin conjugate was characterized as having a high affinity for equilin and equilenin. One mL of the immunoaffinity adsorbent prepared by immobilization of an antibody was capable of retaining up to 1 microgram of equilin and equilenin, to 100 ng of other metabolites including 2-methoxylated and 17 beta-reduced compounds, and to 0.

Unusual trigonal-planar copper configuration revealed in the atomic structure of yeast copper-zinc superoxide dismutase.

The three-dimensional structure of yeast copper-zinc superoxide dismutase (CuZnSOD) has been determined in a new crystal form in space group R32 and refined against X-ray diffraction data using difference Fourier and restrained crystallographic refinement techniques. The unexpected result is that the copper ion has moved approximately 1 angstrom from its position in previously reported CuZnSOD models, the copper-imidazolate bridge is broken, and a roughly trigonal planar ligand geometry characteristic of Cu(I) rather than Cu(II) is revealed. Final R values for the two nearly identical room temperature structures are 18.

Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis.

A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5'-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD.

Epidemiological and genetic studies of Huntington's disease in the San-in area of Japan.

After the DNA diagnosis, we evaluated the prevalence of Huntington's disease (HD) in the San-in area of Japan, and confirmed the founder effect. There were 10 patients with HD in the San-in area, who were diagnosed clinically. The expansion of the CAG repeat was observed in 9 patients with HD members in their families, although those family members of the patients had already died.

[Molecular genetics of Huntington's disease].

Huntington's disease (HD) is a progressive neurodegenerative disorder which is clinically characterized by chorea, cognitive decline, and emotional disturbance; it is inherited in an autosomal dominant manner. The HD gene maps to chromosome 4p16.3.

Analysis of triplet repeats in the huntingtin gene in Japanese families affected with Huntington's disease.

Huntington's disease (HD) is associated with the expansion of a CAG repeat in the huntingtin gene. Molecular analysis of the repeat in Japanese HD patients and normal controls was performed. The size of the CAG repeat ranged from 37 to 95 repeats in affected subjects and from seven to 29 in normal controls.

Flow cytometric analysis of DNA synthetic phase fraction of the normal appearing colonic mucosa in patients with colorectal neoplasms.

DNA synthetic (S) phase fractions of normal appearing colonic mucosa in Japanese and British patients with colorectal neoplasms were compared with those in patients without colonic neoplasms. Normal crypts were isolated from fresh surgical specimens of the large intestine by the use of EDTA. After fixation with 70% ethanol, isolated crypts were digested with pepsin into single nuclei suspensions.

Stereospecific dehydrogenation of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids by acyl-CoA oxidase in rat liver light mitochondrial fraction.

From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) into (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R,25R)- or (24S,25S)-[24,25-2H2]THCA, synthesized from (24E)-delta 24-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5 beta-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and delta 24-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]- in the NICI mode.

Increased Cu/Zn superoxide dismutase-like immunoreactivity in the swollen axons of rats intoxicated chronically with beta,beta'-iminodipropionitrile.

Demonstration of a genetic linkage between the Cu/Zn superoxide dismutase (SOD1) gene and familial amyotrophic lateral sclerosis (ALS) has aroused interest in the role of SOD1 in spinal motoneuronal death. We used chronically beta,beta'-iminodipropionitrile (IDPN)-intoxicated rats as a model of ALS and investigated SOD1 changes in the spinal cord by immunocytochemical and in situ hybridization techniques. Compared with control rats, SOD1-like immunoreactivity (SOD1-IR) increased in swollen axons of the proximal spinal roots, but not in motoneuronal and dorsal root ganglion neuronal cell bodies where SOD1 gene transcription did not increase.

Stereoisomeric inversion of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acids in rat liver peroxisome.

The stereoisomeric inversion of (25R)- and (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) in rat liver peroxisome was studied. After incubation of an isomer of THCA-CoA thioester with a peroxisomal fraction, 5 beta-cholestanoic acids were extracted and optical antipodes were separated and determined by LC/APCI-MS. The transformation of (24E)-3alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid (delta 24-THCA) formed by acyl-CoA oxidase was also analyzed by GC/NICI-MS.

Abnormal gene product identified in hereditary dentatorubral-pallidoluysian atrophy (DRPLA) brain.

Dentatorubral-pallidoluysian atrophy (DRPLA) is associated with the expansion of an unstable CAG repeat. Using antibodies against a synthetic peptide corresponding to the sequence of the DRPLA gene product C terminus, we have identified the DRPLA gene product in normal human brains as a approximately 190 kD protein. We also find a larger approximately 205 kD protein specifically in DRPLA brains.

Abnormal gene product identified in Huntington's disease lymphocytes and brain.

Huntington's disease(HD) is associated with expansion of an unstable CAG repeat. Using antibodies against the synthetic peptide corresponding to the sequence of HD gene IT15, we have identified the HD gene product in normal lymphocytes as a approximately 350kDa protein by immunoblot analysis. Moreover, when a modified SDS-PAGE using a low concentration of methylenbisacrylamide was run longer, abnormal immunoreactive bands larger than normal ones were found exclusively in HD samples.

Synthesis of 15 alpha-hydroxyestrogen 15-N-acetylglucosaminides.

The synthesis of 15-N-acetylglucosaminides of 15 alpha-hydroxyesterone, 15 alpha-hydroxyestradiol, and 15 alpha-hydroxyestriol (estetrol) is described. The latter two were prepared by condensation of 2-acetamido-1 alpha-chloro-1,2-dideoxy-3,4,6-trio-O-acetyl-D-glucopyranose with appropriately protected 15 alpha-hydroxyestrogens by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Subsequent removal of protecting groups with methanolic potassium hydroxide provided the desired conjugates.

Potential bile acid metabolites. 23. Syntheses of 3-glucosides of nonamidated and glycine- and taurine-amidated bile acids.

The 3-glucosides of nonamidated lithocholic, chenodeoxycholic, ursodeoxycholic, deoxycholic, and cholic acids, and their double conjugate forms with glycine and taurine were synthesized. The key reactions used were 1) beta-D- glucosidation at C-3 by the Koenigs-Knorr condensation reaction of 3 alpha-hydroxylated bile acid methyl (or p-nitrophenyl) esters with 1 alpha-bromo-1-deoxy-2, 3, 4, 6-tetra-O-acetyl-D-glucopyranose in the presence of cadmium carbonate in refluxing benzene; 2) indirect and direct amidations at C-24 by the activated p-nitrophenyl ester and by the diethylphosphorylcyanide methods, respectively, using glycinate ester and taurine as coupling agents; and 3) simultaneous alkaline hydrolysis of the hydroxyl-protecting and ester groups in both the sugar and aglycone moieties.