Direct observation of the critical relaxation of polarization clusters in BaTiO3 using a pulsed x-ray laser technique.

We have developed a new method to investigate the relaxation time of the dipole moment in polarization clusters in BaTiO3. Time correlation of speckle intensities was measured by the use of a double pulsed soft x-ray laser. The evolution of the relaxation time of the dipole moment near the Curie temperature (T(C)) was investigated. The maximum relaxation time (approximately 90 ps) is shown to appear at a temperature of 4.5 K above the T(C), being coincident with the one where the maximum polarization takes place. This method is widely applicable to any other critical decay processes at phase transitions.

Mucopolysaccharidosis type IVA. N-acetylgalactosamine-6-sulfate sulfatase exonic point mutations in classical Morquio and mild cases.

Mucopolysaccharidosis type IVA (MPS IVA) results from a genetic deficiency of N-acetylgalactosamine-6-sulfate (Gal-NAc6S) sulfatase. We have identified two different exonic mutations causing GalNAc6S sulfatase deficiency in two unrelated Japanese families, in one patient with classical Morquio disease, and in two brothers with a mild form of MPS IVA. The nucleotide sequence of the full-length cDNA derived from a patient with classical Morquio disease revealed a two-base deletion at nucleotide position 1343-1344 (1344-1345 or 1345-1346) that altered the reading frame (designated 1342delCA). This mutation, inherited from the proband's consanguineous parents, was revealed by TaqI restriction analysis of a cDNA fragment amplified by the polymerase chain reaction. In the proband with the mild form of the disease, a C to G transversion at nucleotide 667 predicted the substitution of Lys for Asn204 (N204K). Since a new AluI site was created by the N204K mutation, restriction analysis indicated that the affected brothers were homozygous for this mutation, as confirmed by the finding that both their parents had this lesion. Transient expression in GalNAc6S sulfatase deficient fibroblasts of these two mutant alleles showed completely deficient or markedly decreased enzyme activities, thereby indicating that these two mutations were responsible for the enzyme deficiency.

Enhancement of double auger decay probability in xenon clusters irradiated with a soft-x-ray laser pulse.

The interaction of large Xe clusters with a soft x-ray laser pulse having a wavelength of 13.9 nm and an intensity of up to 2x10(10) W/cm2 was investigated using a time-of-flight ion mass spectrometer. The corresponding laser photon energy was sufficiently high to photoionize Xe 4d innershell electrons. It was found that Xe3+ ions (which result from double Auger decay of 4d vacancies) became the dominant final ionic product with increasing cluster size and x-ray intensity. This is in contrast to the results of synchrotron radiation experiments involving free Xe atoms, in which Xe2+ is the dominant resultant ion species. Possible mechanisms responsible for the enhancement of the double Auger transition probability in x-ray laser and cluster interaction are discussed.

Effect of 'attenuated' mutations in mucopolysaccharidosis IVA on molecular phenotypes of N-acetylgalactosamine-6-sulfate sulfatase.

Mucopolysaccharidosis IVA is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS gene was performed for seven MPS IVA patients with attenuated phenotypes from three unrelated families. Four of 5 missense mutations identified in this study (p.F167V, p.R253W, p.R380S, p.P484S) and two reported (p.F97V, p.N204K), associated with attenuated phenotypes, were characterized using in vitro stable expression experiments, enzyme kinetic study, protein processing and structural analysis. The stably expressed mutant enzymes defining the attenuated phenotype exhibited a considerable residual activity (1.2-36.7% of the wild-type GALNS activity) except for p.R380S. Enzyme kinetic studies showed that p.F97V, p.F167V and p.N204K have lower affinity to the substrate compared with other mutants. The p.F97V enzyme was the most thermolabile at 55 degrees C. Immunoblot analyses indicated a rapid degradation and/or an insufficiency in processing in the mutant proteins. Tertiary structure analysis revealed that although there was a tendency for 'attenuated' mutant residues to be located on the surface of GALNS, they have a different effect on the protein including modification of the hydrophobic core and salt-bridge formation and different potential energy. This study demonstrates that 'attenuated' mutant enzymes are heterogeneous in molecular phenotypes, including biochemical properties and tertiary structure.

The mouse N-acetylgalactosamine-6-sulfate sulfatase (Galns) gene: cDNA isolation, genomic characterization, chromosomal assignment and analysis of the 5'-flanking region.

Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.

Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro.

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.

Molecular basis of mucopolysaccharidosis type VII: replacement of Ala619 in beta-glucuronidase with Val.

We have identified a mutation causing beta-glucuronidase (beta Gl) deficiency in a 6-year-old girl with mucopolysaccharidosis type VII. Enzyme assay of lysates of a girl's lymphocytes or cultured fibroblasts showed little residual activity and a normal beta Gl-specific mRNA level, as revealed by Northern-blot analysis. Sequencing of the full-length mutated cDNA revealed a C----T transition, an event causing a single Ala619----Val change (we designated this variant beta GGifu). This change is detected by loss of the cleavage site for the enzyme Fnu4HI in the mutated cDNA. On the basis of the loss of Fnu4HI restriction site, the patient was shown to be a homozygote with the beta GGifu mutation and her parents and brother were heterozygotes. This mutation disrupts a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gls, and it reduces the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA.

Heteroallelic missense mutations of the galactosamine-6-sulfate sulfatase (GALNS) gene in a mild form of Morquio disease (MPS IVA).

Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 +/- 5% of controls (as compared to 5 +/- 3% of controls in severe MPS IVA, P < .003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K(m) and increased Vmax in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mild GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines.

Treatment of MPS VII (Sly disease) by allogeneic BMT in a female with homozygous A619V mutation.

A 12-year-old girl with Sly disease (mucopolysaccharidosis VII; beta-glucuronidase deficiency), who is homozygous for the A619V mutation, had a successful allogeneic BMT, donored by an HLA-identical unrelated female to replace the deficient enzyme. Within 5 months after BMT, the enzyme activity of the recipient's lymphocytes increased to normal range. No signs of acute or chronic GVHD were observed. For the successive 31 months post-BMT, beta-glucuronidase activity in her lymphocytes was maintained at almost normal levels and excretion of glycosaminoglycans in the urine was greatly diminished. Ultrastructural findings demonstrated no abnormal vacuoles and inclusion bodies in the cytoplasm of her rectal mucosal cells. Coincident with the restoration of the enzyme activity, clinical improvement was dramatic. Especially notable were improvements in motor function. The patient was able to walk alone for a long time without aid, and she even became able to ride a bicycle and take a bath. In addition, recurrent infections of the upper respiratory tract and the middle ears decreased in frequency and severity, and dyspnea on exertion, severe snoring and vertigo have substantially improved. Thus, allogeneic BMT in this patient produced a better quality of life and provided a more promising outlook.

N-acetylgalactosamine-6-sulfate sulfatase in human placenta: purification and characteristics.

N-Acetylgalactosamine-6-sulfate sulfatase from human placenta was purified 33,600-fold using beta-N-acetyl-D-galactosamine 6-sulfate-(1----4)-beta-D-glucuronic acid-(1----3)-N-acetyl-D-[3H]galactosaminitol 6-sulfate as the substrate. This enzyme is an oligomer with a molecular mass of 120 kDa and consists of polypeptides of 40 and 15 kDa. The 15 kDa polypeptide is a glycoprotein. This purified protein has activities of N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. Rabbit antiserum was raised against the purified protein. The antibody titrated N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase. The size of the precursor of the enzyme is 60 kDa, as determined by cell-free translation. The optimal pH values of the N-acetylgalactosamine-6-sulfate sulfatase and galactose-6-sulfate sulfatase activities are pH 3.8-4.0, and the Kms are 8 and 13 microM, respectively. Sulfate and phosphate ions are potent competitive inhibitors for the enzyme and their inhibition constants are 35 and 200 microM, respectively. Cross-reactive materials of 40 and 15 kDa were detected by immunoblot analysis, in the placenta, liver, and normal fibroblasts, but not in fibroblasts from a patient with Morquio disease.

Purification and partial characterization of alpha-N-acetylglucosaminidase from human liver.

A deficiency in alpha-N-acetylglucosaminidase is known as mucopolysaccharidosis IIIB or Sanfilippo B syndrome. We purified this enzyme almost 39,000-fold from liver to homogeneity with 3% recovery. Use of concanavalin A (Con A)-Sepharose and heparin-Sepharose resulted in 13.4-fold and 11.6-fold purifications of the enzymatic activity, respectively. The molecular mass was estimated to be 300 kDa by gel filtration and 80 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The isoelectric point was 5.1, optimal pH was 4.5, and the Km for p-nitrophenyl alpha-N-acetylglucosamine was 0.13-0.20 mM. The purified enzyme was stable at 50 degrees C for 1 h and within the pH range of 6.5-8.5. Anti-serum against the purified enzyme raised in BALB/c mice inhibited the activities of alpha-N-acetylglucosaminidase.

Mucolipidosis III (pseudo-Hurler polydystrophy); clinical studies in aged patients in one family.

Three adult patients (38-year-old male, 86-year-old female, and 61-year-old male) in a family with mucolipidosis III (ML-III) were described. They had characteristic features of ML-III and they survived a long time. N-acetylglucosaminyl 1-phosphotransferase activity was low in fibroblasts of a patient, but its residual activity remained at a relatively high level (24.5-35.3% of controls), which may explain the benign clinical course. Odontoid dysplasia and atlanto-axial dislocation was found in one patient, and surgical treatment improved his physical disability. Bilateral carpal tunnel syndrome as well as claw hand deformities were common in all of the patients. The clinical manifestations were important for the diagnosis and the management of the patients.

Adult Sandhoff's disease: R505Q and I207V substitutions in the HEXB gene of the first Japanese case.

We describe a 31-year-old Japanese man with adult Sandhoff s disease presenting as spinocerebellar degeneration. There was a marked cerebellar atrophy on MRI, and proliferation of abundant PAS-positive foamy macrophages in the rectal mucosa. The activities of total beta-Hex, beta-Hex A, and beta-Hex B in leucocytes of the patient were 14%, 15%, and 6% of control values, respectively. However, oligosacchariduria or ultrastructural storage materials in liver tissue were nil. Direct sequencing of cDNA and genomic DNA, and restriction digestion revealed two different homozygous base substitutions in the HEXB gene: the G1514-->A substitution (R505Q) and the A619-->G substitution (1207V). The parents were consanguineous. His healthy mother, an enzymatic heterozygous carrier, was homozygous for 1207V, but heterozygous for R505Q mutation. Thus, the patient is probably homozygous for both base substitutions and a R505Q mutation may be linked to the phenotype of adult Sandhoff's disease.

Glycosaminoglycan excretion in random samples of urine.

The measurement of glycosaminoglycans (GAGs) on a single random urine specimen in 405 healthy individuals and in 79 patients with mucopolysaccharidoses (MPSs) is described. The uronic acid (UA) content of the precipitated GAGs was estimated by the method of Bitter and Muir. The results were expressed as uronic acid to creatinine (UA/C) ratio. We found that the UA/C ratio decreased with age but had a rather constant low value between the ages of 20 and 33 in healthy individuals. Except for most patients with Morquio syndrome, the UA/C ratio in random urine samples was higher in patients with MPSs than in healthy individuals.

Screening test for urinary glycosaminoglycans and differentiation of various mucopolysaccharidoses.

The Ames MPS paper spot test and the cetylpyridinium chloride (CPC)-citrate turbidity test were used to detect urinary glycosaminoglycans (GAGs) as screening tests for the diagnosis of mucopolysaccharidoses (MPSs). The simplicity and reliability of these two methods were evaluated. The MPS paper spot test correlated more closely with the carbazole test than did the CPC-citrate turbidity test. The monodimensional electrophoresis of Cappelletti et al was also used to screen the subtypes of MPSs using differences in electrophoretic patterns. MPS VII cannot be distinguished from normal by its electrophoretic pattern, but can be detected by the MPS paper spot test. Therefore, the MPS paper spot test combined with monodimensional electrophoresis can detect these subtypes of MPSs (MPS I-II, III, IV, VI, VII). Since only 20 ml of random urine is needed and the result can be obtained with complete reliability in only 7 hours, this screening test appears to be useful for the early diagnosis of MPSs.

Activities of sulfatases for the degradation of acidic glycosaminoglycans in cultured skin fibroblasts from two siblings with multiple sulfatase deficiency.

Cultured skin fibroblasts from two siblings with multiple sulfatase deficiency (MSD) were assayed for the activities of sulfatases known to degrade acidic glycosaminoglycans (AGAG). There were iduronate sulfatase, arylsulfatase B, heparan sulfate (HS) sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, HS-derived N-acetylglucosamine-6-sulfate sulfatase, and two keratan sulfate (KS)-derived N-acetylglucosamine-6-sulfate sulfatases. The activities of sulfatases required for the degradation of HS were reduced to a greater extent than those for the degradation of dermatan sulfate (DS), and those of sulfatases associated with basic defect of Morquio disease type A were moderately decreased or normal. On the other hand, urinary excretion of AGAG in both patients was increased about 10-fold compared to controls, and especially, the excretion of HS and DS was increased about 150-fold and 50-fold, respectively. Keratan sulfate was not detected. The results suggest that in patients with MSD the degradation of HS might be affected to a greater extent than that of DS.

[Molecular basis of mucopolysaccaridosis type VII (beta-glucuronidase deficiency)].

We have identified several mutations causing beta-glucuronidase (beta Gl) deficiency in three cases with mucopolysaccaridosis type VII (MPS VII). Enzyme assay of lysates of these lymphocytes or cultured fibroblasts showed little residual activity and that the beta Gl-specific mRNA levels were normal, as revealed by Northern blot analysis. Mutated cDNA clones including the entire coding sequencing were isolated from a library in case 1 and PCR (polymerase chain reaction) products in case 2 and 3 derived from cultured fibroblasts. Sequencing of the full-length mutated cDNA revealed C----T transitions, an event causing a single Ala619----Val change (cases 1 and 2) and Arg382----Cys and Pro649----Leu changes (case 3). The former change is detected by loss of the cleavage site for the enzyme Fnu 4 HI in the mutated cDNA. On the basis of the loss of Fnu 4 HI restriction site, the patients (cases 1 and 2) were shown to be a homozygote with this mutation and the parents and brother in case 1 were heterozygotes. The Ala619----Val and Arg382----Cys mutations disrupt a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gl's, and they lower the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA. However the Pro649----Leu mutation does not lower the enzyme activity.

[Clinical and neuroradiological evaluation of the long-term surviving siblings of Sanfilippo syndrome A type].

The siblings of Sanfilippo syndrome type A (MPS III A) have been reported. The relationship of their parents was the first cousins. Case 1: A 30-year-old Japanese man was hospitalized because of gait disturbance and mental impairment. His early somatic and mental development was normal until 9 years of age when mental deterioration had developed. Speech and gait disturbances and double incontinence occurred at 18 years of age. He could not walk at 21 years of age. Those symptoms were slowly progressive. Case 2: A 32-year-old Japanese man, the elder brother of case 1, had a similar clinical history to that of case 1. Their neurological findings revealed mental impairment, coarse face, positive forced grasp and sucking reflexes, and pyramidal signs. Lumbar X-ray showed platyspondylitis, compression fracture of L 1 and osteoporotic changes. Brain MRI of both cases showed brain atrophy, ventricular dilatation and abnormal high intensity signals near the posterior horn of the lateral ventricles on T2 weighted image. Low perfusion images of fronto-parietal regions were seen in the early phase of SPECT using 123I-IMP. This siblings were diagnosed as Sanfilippo syndrome type A because of heparan sulfaturia and deficiency of heparan sulfate sulfamidase of the lymphocytes. Average life span of Sanfilippo syndrome type A is not so long but the age of our cases is over 30 years of age.

[Mucopolysaccharidoses].

Mucopolysaccharidoses (MPS) are a lysosomal storage disorders caused by deficiency of several enzymes needed for degradation of mucopolysaccharides (glycosaminoglycans). Undegraded glycosaminoglycans accumulate in the cell, part of which are excreted into the urine. There are 10 known enzyme deficiencies that give rise to six distinct MPS. Despite the different enzyme defect, there is clinical similarity between different deficiencies and conversely, wide phenotypic variety among even one enzyme deficiency. Most of the cDNAs corresponding the defect enzyme as well as genomic DNA have been cloned. Mutational analyses of the patient gene have revealed the molecular basis of the disease, correlation of the genotype and phenotype and made the accurate heterozygote diagnosis feasible. Supportive management can greatly improve the quality of life and moreover, development of therapies, such as BMT, administration of recombinant enzyme and gene transfer are in progress for the patients suffering from MPS.

[Mucopolysaccharidosis type VII: characterization of exonic point mutations and molecular heterogeneity].

We identified two different exonic point mutations causing beta-glucuronidase (beta G1) deficiency in three Japanese patients with mucopolysaccharidosis type VII. The beta G1-specific mRNA levels were normal. Sequence analysis of the full-length mutated cDNAs showed C-->T transitions, which resulted in a single Ala619-->Val change (case 1, a 8-year old female and case 2, a 24-year old male) and a Arg382-->Cys change (case 3, a 7-year-old female). Each of these two amino acid changes reduced the beta G1 activity of the corresponding mutant beta G1 expressed following transfection of COS cells with expression vectors harboring the mutated cDNAs.