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Biomedical subjects

O Sakamoto

Publications and source records attributed to O Sakamoto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency.

Serum phenylalanine concentrations decreased in 4 patients with hyperphenylalaninemia after loading with tetrahydrobiopterin. There were no abnormalities in urinary pteridine excretion or in dihydropteridine reductase activity. However, mutations were detected in the phenylalanine hydroxylase gene, suggesting a novel subtype of phenylalanine hydroxylase deficiency that may respond to treatment with cofactor supplementation.

Administration, Oral↗
PubMed1999

Relationship between kinetic properties of mutant enzyme and biochemical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HCS) deficiency is a metabolic disorder that causes a biotin-responsive multiple carboxylase deficiency. We analyzed the kinetic properties of seven mutant HCS proteins. Two of these enzymes harbored mutations within the putative biotin-binding region of HCS and showed elevated Km values for biotin compared with that of the wild-type form (Km mutant; Gly581Ser: 45 times, delThr610: 3 times). The remaining five mutations (Arg183Pro, Leu216Arg, Leu237Pro, Val333Glu, and Val363Asp) were located outside the biotin-binding region. The enzymes containing these mutations showed normal or low Km values for biotin (non-Km mutant). Symptoms of patients who have the non-Km, mutants, as well as those of patients who have the Km, mutants, responded to biotin therapy. This is probably because the Km value for biotin of normal HCS is higher than the physiologic concentration of biotin in human cells. The Vmax values of all mutant HCS proteins were considerably decreased, but to a variable degree. The responsiveness to biotin supplementation of propionyl-CoA carboxylase activity in cultured cells bearing the mutations correlated well with the degree of reduction in the Vmax of HCS. Patients who have mutant HCS proteins with lower Vmax showed poorer clinical and biochemical responses to biotin therapy. These observations suggest that the reduction of Vmax is an essential factor for pathophysiology and prognosis of HCS deficiency under treatment with large amounts of biotin. The determination of HCS genotype can be valuable for characterizing the clinical phenotype in HCS deficient patients.

Biotin↗
PubMed1999

Satisfaction of patients treated surgically for intractable aspiration.

STUDY OBJECTIVE: Impaired laryngeal protective function can result in intractable aspiration requiring surgical treatment. There are, however, few reports evaluating the satisfaction of patients and the efficacy of surgical therapy. The purpose of this study is to determine whether surgery for intractable aspiration is beneficial for alleviating depression and improving the mood of patients who have undergone surgical treatment and whether patients and their families are satisfied with the therapy. PATIENTS AND STUDY DESIGN: Seven patients with recurrent aspiration pneumonia that could not be controlled by appropriate medical therapies participated in the study. These patients had no hope of recovering laryngeal function. Six underwent laryngectomy and one underwent laryngotracheal separation. After surgery, we evaluated the efficacy of the therapy and the patients' satisfaction with the therapy. METHODS: The following clinical variables concerning surgical procedure were examined: operation time, time until oral intake, videofluorographic study, and surgical complications. The treatment methods including feeding status were also examined before and after surgery. In addition, the following markers were examined to evaluate the efficacy of the surgery: score of aspiration pneumonia, body mass index, total protein, albumin, hematocrit, WBC count, C-reactive protein, erythrocyte sedimentation rate, and the Barthel Index, an indicator of daily activity. Furthermore, the grade of depression and mood, and satisfaction of patients and their caretakers among family members were scored by the Zung self-rating depression scale, a 20-picture face scale, and the visual analog scale. RESULTS: After surgical therapy, we confirmed by videofluorography that aspiration was completely prevented. No surgical complications occurred. By 18 +/- 6 days, all seven patients were able to ingest a meal orally. The need for extensive medical care and repeated hospitalizations became unnecessary after surgery. The control of pneumonia and albumin improved significantly. The grade of depression and mood of patients and their families also improved significantly. Satisfaction scores of patients receiving therapy were very high. CONCLUSIONS: Our study shows that surgical therapy to prevent aspiration improves the depression and mood of patients and their families as well as feeding status and clinical outlook. Surgical therapy for patients with intractable aspiration is effective and beneficial.

Aged↗
PubMed1999

Presence of RON receptor tyrosine kinase and its splicing variant in malignant and non-malignant human colonic mucosa.

The presence of RON and its variant isoform in malignant and non-malignant human colonic tissues was examined by immunohistochemistry using paraffin-embedded sections and RT-PCR analysis followed by direct sequencing of PCR product using RNAs isolated from frozen tissues. In normal colonic mucosa, RON was uniformly expressed in crypt cells, especially in the bottom of crypta. On the other hand, the expression was distributed heterogeneously in adenomas and in colon cancer. The expression of RON was significantly related to the degree of differentiation of colon cancer and the deletion of the expression was observed in colon cancer specimens with high incidence. The RT-PCR analysis of RNA isolated from non-malignant and malignant colonic tissue revealed the presence of two RON mRNA isoforms with 432-bp and 286-bp. Direct sequencing of major product of 432-bp was revealed to be identical to that of human wild-type RON. On the other hand, major product of 286-bp was revealed to be almost identical to that of a splicing variant of RON transcript which has been found in human gastric cancer cell line, KATO-III. The results obtained in this study may indicate that both wild-type RON and its variant isoform play an important role in regulating the normal function of colonic mucosa such as differentiation and motile activity and the expression of both wild-type RON and its variant isoform could be considered to be reduced during malignancy of human colonic mucosa.

Adenocarcinoma↗
PubMed1998

Molecular analysis of glycogen storage disease type Ib: identification of a prevalent mutation among Japanese patients and assignment of a putative glucose-6-phosphate translocase gene to chromosome 11.

Glycogen storage disease type Ib (GSD-Ib) is an inborn error of metabolism with autosomal recessive inheritance, caused by defects in microsomal transport of glucose-6-phosphate. Recently, Gerin et al isolated a human cDNA encoding a putative transporter homologous to bacterial transporters of hexose-6-phosphate, and identified two mutations in its gene in two patients with GSD-Ib (9). Independently, a linkage analysis mapped the GSD-Ib gene on chromosome 11q23 (10). It remains to be elucidated whether the two genes are identical or GSD-Ib is genetically heterogeneous. We first mapped the transporter gene on chromosome 11 by using a DNA panel of human/hamster hybridoma cells. The result suggested that the GSD-Ib genes identified by the two distinct approaches may be identical and GSD-Ib was allelic. We then studied four unrelated Japanese families with GSD-Ib, and found three novel mutations: a four-base deletion/two-base insertion, a point mutation within a consensus splicing donor site, and a missense mutation (W118R). The W118R mutation was found in 4 out of 8 mutant alleles, suggesting that it is prevalent among Japanese patients.

Amino Acid Sequence↗
PubMed1998

Identification of holocarboxylase synthetase (HCS) proteins in human placenta.

Holocarboxylase synthetase (HCS) is a key enzyme in biotin utilization in eukaryotic cells. In a previous work from our laboratory, we described the cloning and sequencing of a full-length human HCS cDNA. Due to the presence of three candidate sites for initiation of translation, the identification of full-length HCS proteins remains uncertain. Using antibodies directed against human HCS sequences, we have identified, in human placenta, three cytosolic HCS proteins, of 86, 82 and 76 kDa. Similar results were observed in lysates of cells transfected with an HCS expression vector, as well as with human HCS cDNA transcribed and translated in a cell-free system. When anti-HCS antibodies were tested for their ability to inhibit HCS enzymatic activity, only the antibody directed against a region of HCS from Ile128 to Pro398, and not the antibodies against more proximal N-terminal regions inhibited HCS activity, suggesting that the sequence from Ile128 to Pro398 is essential for the catalytic activity of this enzyme. HCS synthesized in a cell-free system was not translocated into rat liver mitochondria. These results suggest that our human HCS cDNA encodes the cytosolic forms of the enzyme. These results also suggest that mRNA encoding cytosolic HCS can be translated from all three translation initiation codons, Met1, Met7 and Met58.

Animals↗
PubMed1997

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Animals↗
PubMed1997

Characterization of mutant holocarboxylase synthetase (HCS): a Km for biotin was not elevated in a patient with HCS deficiency.

Holocarboxylase synthetase (HCS) is an essential enzyme for the biotinylation of several mammalian carboxylases. A deficiency of HCS is accountable for early onset biotin-responsive multiple carboxylase deficiency. To address the mechanism of biotin responsiveness, we analyzed the kinetic properties of the previously identified mutant, L237P, and another mutant, V550M, described in this report. The V550M mutant contains a G to A transition at position 1935, which is within the putative biotin binding site, whereas the mutation in L237P occurs outside the biotin binding site. Km and Vmax values for the mutant proteins were determined by overexpressing cDNAs encoding the mutants in transformed fibroblasts from an HCS-deficient patient. Enzyme activity assays were performed using apo-carboxyl carrier protein as a substrate. A Km for biotin that was larger than the value found for the wild-type cDNA was observed in fibroblasts transfected with the V550M cDNA, but not the L237P cDNA. The Vmax for the expressed L237P cDNA was 4.3% of that observed for the wild-type cDNA. Biotin-responsiveness in the patient with the L237P mutation was neither due to an increased affinity for biotin nor a restoration of stability of the mutant by biotin treatment. A new mechanism of biotin responsiveness in HCS deficiency is presented.

Biotin↗
PubMed1996

Molecular cloning of rat macrophage-stimulating protein and its involvement in the male reproductive system.

Macrophage-stimulating protein (MSP), a member of the hepatocyte growth factor family, is a ligand for receptor tyrosine kinase STK/RON. Here we isolated a full-length cDNA of rat MSP and a partial cDNA of rat STK/RON, then characterized their expression in the male reproductive system. In situ hybridization revealed that MSP mRNA was localized to spermatogonia and spermatocytes in the testis and the epithelium lining the lumen of the epididymis. On the other hand, RT-PCR analysis showed that STK/RON mRNA was expressed in sperm collected from both testis and epididymis. These findings suggest that locally produced MSP may play a vital role in germ cell-germ cell interaction during spermatogenesis as well as in the acquisition of sperm motility and/or fertilizing capacity in the epididymis. Our findings reveal new possible roles of the MSP-STK/RON signaling pathway.

Amino Acid Sequence↗
PubMed1996

Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrier protein as a substrate.

We developed a simple and sensitive method for assessing holocarboxylase synthetase (HCS) activity that is based on measuring incorporation of [3H]biotin into apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E. coli. Kinetic analysis of HCS from normal fibroblasts showed that the Km for biotin was 260 +/- 94 nmol/l (mean +/- S.D.; n = 5). In contrast, the Km values of HCS from two cell lines derived from patients with HCS deficiency were 7200 and 3700, clearly distinguishable from the control value. The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected. Our method is useful for enzymatic diagnosis of HCS deficiency and characterization of HCS.

Acetyl-CoA Carboxylase↗
PubMed1995

Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients.

Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.

Amino Acid Sequence↗
PubMed1995

Interleukin-13 selectively suppresses the growth of human macrophage progenitors at the late stage.

Interleukin-13 (IL-13) is a pleiotropic cytokine that inhibits the production of inflammatory cytokines of monocytes. We investigated the effects of IL-13 on the clonal growth of human hematopoietic progenitors. IL-13 alone did not support any colony formation. IL-13 markedly suppressed macrophage colonies that were formed in the presence of IL-3 and erythropoietin, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor. Macrophage colony cells showed dendritic cell-line morphology and cellular aggregates. IL-13 did not affect granulocyte colony and erythroid burst formation. Delayed addition of IL-13 and replating onto the culture dishes with IL-13 showed that macrophage colony formation was suppressed during days 8 and 14 of culture. These results indicate that IL-13 affects the growth of the late stage of committed macrophage progenitors. Single-cell culture of isolated CD34+CD33+ cells with IL-13 confirmed that macrophage colony formation was significantly suppressed. These results show that IL-13 directly suppresses the proliferation of differentiating macrophages. In addition, these suppressive effects of IL-13 were synergistic with IL-4. Furthermore, in the liquid culture of bone marrow cells in the presence of IL-13, the number of CD14 (monocyte-macrophage antigen)-positive cells decreased and CD18 (LFA-1 beta)-positive cells increased. It is concluded that IL-13 affects the growth of the late stage of macrophage precursors as well as mature monocytes. Induction of differentiation of human monocytes may be correlated with the suppression of their progenitors.

Bone Marrow Cells↗
PubMed1994

Monocyte chemoattractant protein-1 in idiopathic pulmonary fibrosis and other interstitial lung diseases.

Macrophages play a crucial role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). To examine the mechanisms for increased monocyte/macrophage recruitment in IPF and nonIPF interstitial lung diseases (nonIPF) the localization of monocyte chemoattractant protein-1 (MCP-1) was investigated in 14 cases of IPF, seven cases of nonIPF, and seven normal control lungs (CTRL) by immunohistochemistry using a specific anti-MCP-1 monoclonal antibody, F9. By double immunohistochemical staining using F9 and one of the cell type specific antibodies significant differences in the staining pattern of MCP-1 were observed between IPF and nonIPF. In IPF MCP-1 was observed in cuboidal and flattened metaplastic epithelial cells, alveolar macrophages, and vascular endothelial cells. In contrast, no epithelial cells were stained for MCP-1 in nonIPF cases, although alveolar macrophages and vascular endothelial cells were labeled. Northern hybridization analysis of selected cases showed marked expression of MCP-1 messenger RNA (mRNA) in IPF and nonIPF compared with CTRL. These findings suggest that the MCP-1 production in IPF and nonIPF plays an important role in the recruitment of monocyte/macrophages. Monocyte chemoattractant protein-1 production by epithelial cells in IPF may be caused by the metaplastic nature of the epithelial cells and may be one of the key factors inducing the irreversible progression of IPF.

Adult↗
PubMed1994

Pulmonary granulomatosis caused by aspirated green tea.

We describe the first reported case (to our knowledge) of pulmonary granulomatosis caused by aspirated green tea. In this case, we found granulomatous alveolitis with lymph follicles, T lymphocytosis with predominantly CD8+ cells in the bronchoalveolar lavage fluids, positive serum precipitin and proliferative response of peripheral blood lymphocytes to the tea infusion, and efficacy of steroid therapy. These results indicate that the pathogenesis of the disease was due to both humoral and cellular immunities to the aspirated green tea.

Aged↗
PubMed1993

Novel low immunosuppressive derivatives of the antitumor drug fluoropyrimidine, UK-21 and UK-25: effect on delayed type hypersensitivity and tumor immunity.

Previously, we reported that two novel 5-fluoropyrimidine derivatives, 2',3',5'-tris-O-[N-(2-n-propyl-n-pentanoyl)glycyl]-5-fluorouridine (UK-21) and 1-(6-[N-(2-n-propyl-n-pentanoyl)glycyl]amino-n-hexylcarbamoyl)- 5-fluorouracil (UK-25), show potent antitumor activity with low immunotoxicological effects. The purpose of this paper was to evaluate the effect of these drugs on delayed type hypersensitivity (DTH). Not only UK-21 and UK-25 but also tegafur (FT-207) and 5-fluorouracil (5-FU) produced no suppression of picryl chloride (PC)-induced DTH in mice but rather enhanced it. It is known that variation of the sensitizing antigen dose alters the effect of drugs on the immune response. Because it was difficult to control the antigen dose in PC-DTH, the sheep erythrocyte (SRBC)-induced response was used to examine the effect of drugs on delayed type hypersensitivity in the succeeding experiments. Either a therapeutic dose or an over-dose of the respective drug was given to mice sensitized with 5 x 10(5) or 5 x 10(7) SRBC. The suppressive effects of UK-21 and UK-25 on the DTH were lower than those of FT-207 and 5-FU. UK-21 and UK-25 enhanced Meth A tumor-specific DTH in BALB/c mice, but FT-207 and 5-FU did not. UK-21, UK-25 and FT-207 showed a tendency to enhance or restore the Meth A tumor neutralizing activity of spleen cells in mice bearing the tumor, but carmofur (HCFU) did not. These results indicated that the suppressive effects of UK-21 and UK-25 on the tumor immune response were also low.

Animals↗