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Biomedical subjects

Hitoshi Sakuraba

Publications and source records attributed to Hitoshi Sakuraba.

At least 19 recordsLinked to original sources
PubMed2006

Cytochemical analysis of storage materials in cultured skin fibroblasts from patients with I-cell disease.

BACKGROUND: In cultured fibroblasts from I-cell disease patients the transport of many lysosomal enzymes is defective, and affected cells contain inclusion bodies filled with undegraded substrates. However, the contents of these inclusion bodies have not been well characterized yet. We attempted to identify accumulated substances in cultured I-cell disease fibroblasts cytochemically. METHODS: Cultured fibroblasts from I-cell disease patients were double-stained with a monoclonal antibody to lysosome-associated membrane protein-1 (LAMP-1) and that to GM2 ganglioside, or a series of lectins that specifically bind to sugar moieties. RESULTS: The patients' cells were granularly stained with the antibody to GM2 ganglioside and the lectins including Maakia amurensis, Datura stramonium, and concanavalin A. Their localization was coincident with that of LAMP-1. CONCLUSIONS: GM2 ganglioside and various kinds of glycoconjugates having sialic acidalpha2-3galactose, galactosebeta1-4N-acetylglucosamine and mannose residues accumulate in enlarged lysosomes in I-cell disease fibroblasts.

Cells, Cultured↗
PubMed2006

Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 ganglioside antibodies.

BACKGROUND: GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 ganglioside activator protein associated with accumulation of GM2 ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. METHODS: We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. RESULTS: We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. CONCLUSIONS: This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.

Antibodies, Monoclonal↗
PubMed2006

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation.

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.

Abnormalities, Multiple↗
PubMed2006

Phospholipid storage in the myocardium of a unique Japanese case of idiopathic cardiomyopathy.

BACKGROUND: A unique adult male patient who developed cardiomyopathy was first suspected to have cardiac Fabry disease based on the pathological findings in heart tissues obtained on biopsy, but the alpha-galactosidase activity in his leukocytes was normal and no mutation was detected in the coding region of the alpha-galactosidase gene. We identified accumulated materials in the myocardium of this patient. METHODS: Pathological and biochemical analyses were performed using the autopsied heart tissues as samples. RESULTS: Although numerous lamellar and concentric inclusion bodies were ultrastructurally found in the autopsied myocardium, the alpha-galactosidase activity in the heart tissues was not decreased. Lipid analysis revealed the accumulation of phospholipids including phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, but not globotriaosylcereamide or gangliosides. CONCLUSIONS: We found that a large amount of phospholipids accumulated in the myocardium of a patient with idiopathic cardiomyopathy, and electron microscopic findings of lamellar and concentric inclusion bodies in cardiomyocytes. A cardiac phospholipid storage disorder should be considered as an important candidate disease on differential diagnosis of myocardiac disorders including cardiac Fabry disease.

Autopsy↗
PubMed2006

High incidence of later-onset fabry disease revealed by newborn screening.

The classic phenotype of Fabry disease, X-linked alpha -galactosidase A (alpha -Gal A) deficiency, has an estimated incidence of approximately 1 in 50,000 males. The recent recognition of later-onset variants suggested that this treatable lysosomal disease is more frequent. To determine the disease incidence, we undertook newborn screening by assaying the alpha-Gal A activity in blood spots from 37,104 consecutive Italian male neonates. Enzyme-deficient infants were retested, and "doubly screened-positive" infants and their relatives were diagnostically confirmed by enzyme and mutation analyses. Twelve (0.03%) neonates had deficient alpha-Gal A activities and specific mutations, including four novel missense mutations (M51I, E66G, A73V, and R118C), three missense mutations (F113L, A143T, and N215S) identified previously in later-onset patients, and one splicing defect (IVS5(+1G-->T)) reported in a patient with the classic phenotype. Molecular modeling and in vitro overexpression of the missense mutations demonstrated structures and residual activities, which were rescued/enhanced by an alpha-Gal A-specific pharmacologic chaperone, consistent with mutations that cause the later-onset phenotype. Family studies revealed undiagnosed Fabry disease in affected individuals. In this population, the incidence of alpha-Gal A deficiency was 1 in approximately 3,100, with an 11 : 1 ratio of patients with the later-onset : classic phenotypes. If only known disease-causing mutations were included, the incidence would be 1 in approximately 4,600, with a 7 : 1 ratio of patients with the later-onset : classic phenotypes. These results suggest that the later-onset phenotype of Fabry disease is underdiagnosed among males with cardiac, cerebrovascular, and/or renal disease. Recognition of these patients would permit family screening and earlier therapeutic intervention. However, the higher incidence of the later-onset phenotype in patients raises ethical issues related to when screening should be performed--in the neonatal period or at early maturity, perhaps in conjunction with screening for other treatable adult-onset disorders.

Adult↗
PubMed2006

Corrective effect on Fabry mice of yeast recombinant human alpha-galactosidase with N-linked sugar chains suitable for lysosomal delivery.

We have previously reported the production of a recombinant alpha-galactosidase with engineered N-linked sugar chains facilitating uptake and transport to lysosomes in a Saccharomyces cerevisiae mutant. In this study, we improved the purification procedure, allowing us to obtain a large amount of highly purified enzyme protein with mannose-6-phosphate residues at the non-reducing ends of sugar chains. The products were incorporated into cultured fibroblasts derived from a patient with Fabry disease via mannose-6-phosphate receptors. The ceramide trihexoside (CTH) accumulated in lysosomes was cleaved dose-dependently, and the disappearance of deposited CTH was maintained for at least 7 days after administration. We next examined the effect of the recombinant alpha-galactosidase on Fabry mice. Repeated intravascular administration of the enzyme led to successful degradation of CTH accumulated in the liver, kidneys, heart, and spleen. However, cleavage of the accumulated CTH in the dorsal root ganglia was insufficient. As the culture of yeast cells is easy and economical, and does not require fetal calf serum, the recombinant alpha-galactosidase produced in yeast cells is highly promising as an enzyme source for enzyme replacement therapy in Fabry disease.

Animals↗
PubMed2006

Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

Animals↗
PubMed2006

Elastogenesis in cultured dermal fibroblasts from patients with lysosomal beta-galactosidase, protective protein/cathepsin A and neuraminidase-1 deficiencies.

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.

Base Sequence↗
PubMed2006

Molecular pathologies of and enzyme replacement therapies for lysosomal diseases.

Lysosomal diseases comprise a group of inherited disorders resulting from defects of lysosomal enzymes and their cofactors, and in many of them the nervous system is affected. Recently, enzyme replacement therapy with recombinant lysosomal enzymes has been clinically available for several lysosomal diseases. Such enzyme replacement therapies can improve non-neurological disorders but is not effective for neurological ones. In this review, we discuss the molecular pathologies of lysosomal diseases from the protein structural aspect, current enzyme replacement therapies, and attempts to develop enzyme replacement therapies effective for lysosomal diseases associated with neurological disorders, i.e., production of enzymes, brain-specific delivery and incorporation of lysosomal enzymes into cells.

Animals↗
PubMed2005

Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

Animals↗
PubMed2005

Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement.

Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of beta-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.

Cathepsin A↗
PubMed2005

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human beta-hexosaminidase A on the accumulated GM2 ganglioside.

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl-beta-D-glucosamine (beta-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl-beta-D-glucosamine-6-sulfate (beta-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human beta-hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated beta-hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.

Animals↗
PubMed2005

Fabry disease: correlation between structural changes in alpha-galactosidase, and clinical and biochemical phenotypes.

Fabry disease comprises classic and variant phenotypes. The former needs early enzyme replacement therapy, and galactose infusion is effective for some variant cases. Attempts of early diagnosis before manifestations appear will begin in the near future. However, it is difficult to predict the phenotype, to determine the therapeutic approach, only from genetic information. Thus we attempted structural analysis from a novel viewpoint. We built structural models of mutant alpha-galactosidases resulting from 161 missense mutations (147 classic and 14 variant), and evaluated the influence of each replacement on the structure by calculating the numbers of atoms affected. Among them, 11 mutants, biochemically characterized, were further investigated by color imaging of the influenced atoms. In the variant group, the number of atoms influenced by amino-acid replacement was small, especially in the main chain. In 85% of the cases, less than three atoms in the main chain are influenced. In this group, small structural changes, located apart from the active site, result in destabilization of the mutant enzymes, but galactose can stabilize them. Structural changes caused by classic Fabry mutations are generally large or are located in functionally important regions. In 82% of the cases, three atoms or more in the main chain are affected. The classic group comprises dysfunctional and unstable types, and galactose is not expected to stabilize the mutant enzymes. This study demonstrated the correlation of structural changes, and clinical and biochemical phenotypes. Structural investigation is useful for elucidating the bases of Fabry disease and clinical treatment.

Amino Acid Substitution↗
PubMed2005

Distal myopathy with rimmed vacuoles: impaired O-glycan formation in muscular glycoproteins.

Distal myopathy with rimmed vacuoles (DMRV), is an autosomal recessive disorder with early adult onset, displays distal dominant muscular involvement and is characterized by the presence of numerous rimmed vacuoles in the affected muscle fibers. The pathophysiology of DMRV has not been clarified yet, although the responsible gene was identified as that encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase involved in the biosynthesis of sialic acids. To identify defective carbohydrate moieties of muscular glycoproteins from DMRV patients, frozen skeletal muscle sections from seven patients with DMRV, as well as normal and pathological controls, were treated with or without sialidase or N-glycosidase F followed by lectin staining and lectin blotting analysis. The sialic acid contents of the O-glycans in the skeletal muscle specimens from the DMRV patients were also measured. We found that Arachis hypogaea agglutinin (PNA) lectin reacted strongly with sarcolemmal glycoproteins in the DMRV patients but not with those in control subjects. alpha-Dystroglycan from the DMRV patients strongly associated with PNA lectin, although that from controls did not. The sialic acid level of the O-glycans in the DMRV muscular glycoproteins with molecular weights of 30 to 200 kd was reduced to 60 to 80% of the control level. The results show that impaired sialyl O-glycan formation in muscular glycoproteins, including alpha-dystroglycan, occurs in DMRV.

Adolescent↗
PubMed2005

An asymptomatic heterozygous female with fabry disease: implications for enzyme replacement therapy.

We report an asymptomatic female with Fabry disease immunohistochemically diagnosed by renal biopsy. She was initially diagnosed as having nephrotic syndrome, and renal biopsy was performed for pathological diagnosis. The renal specimen revealed non-specific findings (minor glomerular abnormalities) for nephrotic syndrome. Numerous laminated bodies in glomerular epithelial cells in electron microscopic findings and accumulations of ceramidetrihexoside immunohistochemically were observed and she was diagnosed with Fabry disease. However, no other laboratory data or clinical findings supported the diagnosis of Fabry disease. Since the efficacy of recombinant human alpha-galactosidase replacement therapy in this disease has been reported, whether enzyme replacement therapy for subclinical Fabry female patients is indicated or not is an important issue.

Adolescent↗
PubMed2004

Three dimensional structural studies of alpha-N-acetylgalactosaminidase (alpha-NAGA) in alpha-NAGA deficiency (Kanzaki disease): different gene mutations cause peculiar structural changes in alpha-NAGAs resulting in different substrate specificities and clinical phenotypes.

BACKGROUND: Kanzaki disease (OMIM#104170) is attributable to a deficiency in alpha-N-acetylgalactosaminidase (alpha-NAGA; E.C.3.2.1.49), which hydrolyzes GalNAcalpha1-O-Ser/Thr. Missense mutations, R329W or R329Q were identified in two Japanese Kanzaki patients. Although they are on the same codon, the clinical manifestation was more severe in R329W because an amino acid substitution led to protein instability resulting in structural change, which is greater in R329W than in R329Q. OBJECTIVE: To examine whether the different clinical phenotypes are attributable to the two mutations. METHODS: Plasma alpha-NAGA activity and urinary excreted glycopeptides were measured and three-dimensional models of human alpha-NAGA and its complexes with GalNAcalpha1-O-Ser and GalNAcalpha1-O-Thr were constructed by homology modeling. RESULTS: Residual enzyme activity was significantly higher in the R329Q- than the R329W mutant (0.022+/-0.005 versus 0.005+/-0.001 nmol/h/ml: p<0.05); the urinary ratios of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr were 2:10 and 8:10, respectively. GalNAcalpha1-O-Ser/Thr fit tightly in a narrow space of the active site pocket of alpha-NAGA. GalNAcalpha1-O-Thr requires a larger space to associate with alpha-NAGA because of the side chain (CH3) of the threonine residue. CONCLUSION: Our findings suggest that the association of alpha-NAGA with its substrates is strongly affected by the amino acid substitution at R329 and that the association with GalNAcalpha1-O-Thr is more highly susceptible to structural changes. The residual mutant enzyme in R329W could not associate with GalNAcalpha1-O-Thr and GalNAcalpha1-O-Ser. However, the residual mutant enzyme in R329Q catalyzed GalNAcalpha1-O-Ser to some extent. Therefore, the urinary ratio of GalNAcalpha1-O-Ser:GalNAcalpha1-O-Thr was lower and the clinical phenotype was milder in the R329Q mutation. Structural analysis revealed biochemical and phenotypic differences in these Kanzaki patients with the R329Q and R329W mutation.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2004

Clinical, biochemical, and cytochemical studies on a Japanese Salla disease case associated with a renal disorder.

We report the first Japanese case of Salla disease. A 5-year-old male patient developed unique proteinuria with other clinical manifestations, including coarse facies, dysostosis multiplex, mild mitral valve regurgitation, umbilical and inguinal herniation, and mild developmental delay. Pathological analysis of biopsied kidney tissues showed marked vacuolation of podocytes, mesangial cells, capillary endothelial cells, and tubular cells. Biochemical studies involving thin-layer chromatography and mass spectrometry revealed increased excretion of free sialic acid (N-acetylneuraminic acid) into the patient's urine. Immuno- and lectin staining of the patient's cells demonstrated the accumulation of sialyl and asialyl glycoconjugates in lysosomes and late endosomes. A defect in sialyl glycoconjugate metabolism is thought to have occurred in the patient's cells, besides impairment of the lysosomal transport of free sialic acid residues. A renal disorder should be considered as an important manifestation, not only in infantile free sialic acid storage disease but also in Salla disease.

Biological Transport↗