While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the... more While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the high bone turnover with increased bone resorption by osteoclasts, little is known about the mechanism of intramembranous bone loss contributed by AR in osteoblasts. Here, we discovered a dramatic decrease in the area of calcification, new bone, and the number of osteocytes in calvaria from AR-deficient mice related to a reduction in mineralization caused, in part, by the diminished activity of AR-deficient osteoblasts. Enforced AR expression in differentiated osteoblasts boosts mineralization while knockdown of AR expression prevents androgen-induced mineralization. We identified the tissue-nonspecific alkaline phosphatase (TNSALP) and several members of small integrin binding ligand N-linked glycoprotein (SIBLING) gene family as androgen target genes required for AR-mediated bone formation. We show that inorganic phosphate (Pi) levels and TNSALP activity increased in response to androg...
The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with ch... more The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.
Transferrin (Tf) was found immunologically in pancreatic juice of normal rats at a concentration ... more Transferrin (Tf) was found immunologically in pancreatic juice of normal rats at a concentration of 0.28 +/- 0.10 mg/ml but was found to be approximately 4-times higher in iron-deficient rats. Iron saturation of pancreatic Tf of normal rats was 40% and similar to that of serum Tf. Approximately 27% of a dose of iron from 59Fe-diferric Tf was absorbed through the ligated segments of proximal intestine in normal rats. The iron absorption ratio of 59Fe-diferric Tf was higher from the duodenal and jejunal segments than the ileum and significantly inhibited by monodansylcadaverine (MDC) or 20-times excess of unlabeled diferric Tf. The presence of Tf receptors was demonstrated by specific binding of 125I-diferric Tf to the brush border membrane vesicles of the small intestine in normal rats. The binding was not inhibited by the presence of albumin or IgG. The association constant (Ka) was 1.03 +/- 0.50 x 10(8) M-1 and number of binding sites was 5.10 +/- 0.96 x 10(12) sites/mg protein in ...
Journal of Nutritional Science and Vitaminology, 1995
Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine w... more Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine were studied. Specific Fe(II) and Fe(III) binding sites were detected by iron binding assay using immobilized BBM from the upper intestine on a nitrocellulose sheet and the binding to Fe(II) was considerably higher than that to Fe(III). The dissociation constants for Fe(II) were 0.26+/-0.02 nm (M+/-SE) for high-affinity binding sites and 2.70+/-0.26 nm forlow-affinity binding sites. For Fe(II),the number of high-affinity binding sites was 0.35+/-0.04 x 10(16)/mg of protein (M+/-SE) and that of low-affinity binding sites was 1.07+/-0.11 x 10(16)/mg of protein. Bound Fe(II) could not be replaced with Fe (II), Fe(III) or transferrin-bound iron and approximately 50% of bound Fe(II) was removed by chelators. The apparent molecular weights of Fe(II) binding peaks were 250 and 120 kDa by gel filtration. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detected three bands with iron-binding activity. The distribution of the Fe(II) binding sites showed that the number of low-affinity sites was significantly higher in the proximal third of the intestine compared with the rest of the intestine. Iron deficiency increased the number of Fe(II) binding sites. These findings suggest that the Fe(II) binding sites might play a physiological role in iron absorption in the rat.
The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profile analysis... more The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profile analysis could be applied to the prenatal diagnosis of steroid 21-hydroxylase deficiency. We designed PCR primers to amplify most of the 21-hydroxylase gene, including all the mutations previously reported. PCR-SSCP analysis in eight patients showed at least one polymorphic site in each case. We confirmed that the mobility shifts in SSCP in an affected kindred were transmitted as a Mendelian trait. As these results indicated that PCR-SSCP profiles could be used for DNA-based diagnosis, we attempted to use this technique for prenatal diagnosis. DNA was obtained by chorionic villus sampling of a fetus and PCR-SSCP profiles were analysed in the PCR-amplified fragments in which the mobility shifts had been observed in the SSCP of the proband. We concluded that the fetus was a carrier. Direct nucleotide sequencing and allele-specific oligonucleotide hybridization confirmed that the fetus was heterozygous. At birth, the infant showed no signs of virilization or of abnormal endocrine findings on laboratory study. The results suggest that this new application of PCR-SSCP has advantages over conventional RFLP analysis and is useful in making a prenatal diagnosis of steroid 21-hydroxylase deficiency both rapidly and accurately.
Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineraliza... more Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineralization, deficiency of tissue-non-specific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In this study we analysed the genomic TNSALP gene from a patient with HOPS, her family, and unrelated normal controls. The proband was a 52-year-old Japanese woman with adult onset HOPS. The patient showed deficiency in alkaline phosphatase (ALP) activity, increased urinary excretion of phosphoethanolamine and severe periodontal disease. Genomic DNA was extracted from the peripheral leukocytes of the subjects. Based on published sequence data in the TNSALP gene, 11 pairs of polymerase chain reaction (PCR) primers were used to amplify the coding exons. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. By PCR-SSCP analysis of the patient's genomic DNA, fragments containing exon 5 revealed abnormal mobility. This abnormal mobility (exon 5) was also found in the genomic DNA in her mother's sister, but were not detected in her father, brothers or sisters, and unrelated normal controls. Sequencing analysis of the abnormal band extracted from the SSCP gel revealed a C to T transition at nucleotide position 571 (C571T) in exon 5. This mutation resulted in a substitution of Ala-115 with a Val in the mature TNSALP polypeptide. PCR-ASO analysis also confirmed this missense point mutation. The result of this study showed that the pro-band has inherited the C571T mutation in exon 5 from her mother alone and the disease in this family was inherited as an autosomal dominant trait from the pedigree. The C571T mutation is a new missense point mutation and appears to cause significant changes in the structure and function of TNSALP because Ala-115 is highly conserved in rat TNSALP and human tissue-non-specific, intestinal and placental ALPs.
Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related ... more Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.
Biochemical and Biophysical Research Communications, 1995
Human MSH3 (hMSH3), previously named human mismatch repair protein 1 (MRP1), is one of the human ... more Human MSH3 (hMSH3), previously named human mismatch repair protein 1 (MRP1), is one of the human homologs of the bacterial DNA mismatch repair protein MutS. The hMSH3 gene is expressed at low level in most types of cells. Using the RT-PCR technique, we examined the expression of the hMSH3 gene in bone marrow cells from 40 patients with various hematological malignancies. The hMSH3 mRNA was not detectable in 7 cases including 3 of chronic myelogenous leukemia, 2 of acute myelogenous leukemia, and 1 of acute lymphocytic leukemia, and 1 of myelodysplastic syndrome. In addition, 17 cases showed significantly reduced expression of the hMSH3 gene. Southern blot analysis of genomic DNA demonstrated no remarkable changes in the structure and the copy number of the hMSH3 gene in all cases. These results suggest that inactivation of the hMSH3 gene may be involved in the development of hematological malignancies.
While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the... more While androgen receptor (AR)-deficient mice developed osteopenia in endochondral bones due to the high bone turnover with increased bone resorption by osteoclasts, little is known about the mechanism of intramembranous bone loss contributed by AR in osteoblasts. Here, we discovered a dramatic decrease in the area of calcification, new bone, and the number of osteocytes in calvaria from AR-deficient mice related to a reduction in mineralization caused, in part, by the diminished activity of AR-deficient osteoblasts. Enforced AR expression in differentiated osteoblasts boosts mineralization while knockdown of AR expression prevents androgen-induced mineralization. We identified the tissue-nonspecific alkaline phosphatase (TNSALP) and several members of small integrin binding ligand N-linked glycoprotein (SIBLING) gene family as androgen target genes required for AR-mediated bone formation. We show that inorganic phosphate (Pi) levels and TNSALP activity increased in response to androg...
The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with ch... more The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.
Transferrin (Tf) was found immunologically in pancreatic juice of normal rats at a concentration ... more Transferrin (Tf) was found immunologically in pancreatic juice of normal rats at a concentration of 0.28 +/- 0.10 mg/ml but was found to be approximately 4-times higher in iron-deficient rats. Iron saturation of pancreatic Tf of normal rats was 40% and similar to that of serum Tf. Approximately 27% of a dose of iron from 59Fe-diferric Tf was absorbed through the ligated segments of proximal intestine in normal rats. The iron absorption ratio of 59Fe-diferric Tf was higher from the duodenal and jejunal segments than the ileum and significantly inhibited by monodansylcadaverine (MDC) or 20-times excess of unlabeled diferric Tf. The presence of Tf receptors was demonstrated by specific binding of 125I-diferric Tf to the brush border membrane vesicles of the small intestine in normal rats. The binding was not inhibited by the presence of albumin or IgG. The association constant (Ka) was 1.03 +/- 0.50 x 10(8) M-1 and number of binding sites was 5.10 +/- 0.96 x 10(12) sites/mg protein in ...
Journal of Nutritional Science and Vitaminology, 1995
Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine w... more Characteristics of iron binding to the solubilized brush border membrane (BBM) of rat intestine were studied. Specific Fe(II) and Fe(III) binding sites were detected by iron binding assay using immobilized BBM from the upper intestine on a nitrocellulose sheet and the binding to Fe(II) was considerably higher than that to Fe(III). The dissociation constants for Fe(II) were 0.26+/-0.02 nm (M+/-SE) for high-affinity binding sites and 2.70+/-0.26 nm forlow-affinity binding sites. For Fe(II),the number of high-affinity binding sites was 0.35+/-0.04 x 10(16)/mg of protein (M+/-SE) and that of low-affinity binding sites was 1.07+/-0.11 x 10(16)/mg of protein. Bound Fe(II) could not be replaced with Fe (II), Fe(III) or transferrin-bound iron and approximately 50% of bound Fe(II) was removed by chelators. The apparent molecular weights of Fe(II) binding peaks were 250 and 120 kDa by gel filtration. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) detected three bands with iron-binding activity. The distribution of the Fe(II) binding sites showed that the number of low-affinity sites was significantly higher in the proximal third of the intestine compared with the rest of the intestine. Iron deficiency increased the number of Fe(II) binding sites. These findings suggest that the Fe(II) binding sites might play a physiological role in iron absorption in the rat.
The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profile analysis... more The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profile analysis could be applied to the prenatal diagnosis of steroid 21-hydroxylase deficiency. We designed PCR primers to amplify most of the 21-hydroxylase gene, including all the mutations previously reported. PCR-SSCP analysis in eight patients showed at least one polymorphic site in each case. We confirmed that the mobility shifts in SSCP in an affected kindred were transmitted as a Mendelian trait. As these results indicated that PCR-SSCP profiles could be used for DNA-based diagnosis, we attempted to use this technique for prenatal diagnosis. DNA was obtained by chorionic villus sampling of a fetus and PCR-SSCP profiles were analysed in the PCR-amplified fragments in which the mobility shifts had been observed in the SSCP of the proband. We concluded that the fetus was a carrier. Direct nucleotide sequencing and allele-specific oligonucleotide hybridization confirmed that the fetus was heterozygous. At birth, the infant showed no signs of virilization or of abnormal endocrine findings on laboratory study. The results suggest that this new application of PCR-SSCP has advantages over conventional RFLP analysis and is useful in making a prenatal diagnosis of steroid 21-hydroxylase deficiency both rapidly and accurately.
Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineraliza... more Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineralization, deficiency of tissue-non-specific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In this study we analysed the genomic TNSALP gene from a patient with HOPS, her family, and unrelated normal controls. The proband was a 52-year-old Japanese woman with adult onset HOPS. The patient showed deficiency in alkaline phosphatase (ALP) activity, increased urinary excretion of phosphoethanolamine and severe periodontal disease. Genomic DNA was extracted from the peripheral leukocytes of the subjects. Based on published sequence data in the TNSALP gene, 11 pairs of polymerase chain reaction (PCR) primers were used to amplify the coding exons. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. By PCR-SSCP analysis of the patient's genomic DNA, fragments containing exon 5 revealed abnormal mobility. This abnormal mobility (exon 5) was also found in the genomic DNA in her mother's sister, but were not detected in her father, brothers or sisters, and unrelated normal controls. Sequencing analysis of the abnormal band extracted from the SSCP gel revealed a C to T transition at nucleotide position 571 (C571T) in exon 5. This mutation resulted in a substitution of Ala-115 with a Val in the mature TNSALP polypeptide. PCR-ASO analysis also confirmed this missense point mutation. The result of this study showed that the pro-band has inherited the C571T mutation in exon 5 from her mother alone and the disease in this family was inherited as an autosomal dominant trait from the pedigree. The C571T mutation is a new missense point mutation and appears to cause significant changes in the structure and function of TNSALP because Ala-115 is highly conserved in rat TNSALP and human tissue-non-specific, intestinal and placental ALPs.
Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related ... more Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.
Biochemical and Biophysical Research Communications, 1995
Human MSH3 (hMSH3), previously named human mismatch repair protein 1 (MRP1), is one of the human ... more Human MSH3 (hMSH3), previously named human mismatch repair protein 1 (MRP1), is one of the human homologs of the bacterial DNA mismatch repair protein MutS. The hMSH3 gene is expressed at low level in most types of cells. Using the RT-PCR technique, we examined the expression of the hMSH3 gene in bone marrow cells from 40 patients with various hematological malignancies. The hMSH3 mRNA was not detectable in 7 cases including 3 of chronic myelogenous leukemia, 2 of acute myelogenous leukemia, and 1 of acute lymphocytic leukemia, and 1 of myelodysplastic syndrome. In addition, 17 cases showed significantly reduced expression of the hMSH3 gene. Southern blot analysis of genomic DNA demonstrated no remarkable changes in the structure and the copy number of the hMSH3 gene in all cases. These results suggest that inactivation of the hMSH3 gene may be involved in the development of hematological malignancies.
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Papers by H. Orimo