Biomedical subjects
M Fujieda
Publications and source records attributed to M Fujieda.
Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action.
We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha (RXRalpha) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha.
Lysosomal glycogen storage disease with normal acid maltase with early fatal outcome.
In a male infant who had cardiomyopathy, generalized muscle weakness and increased serum creatine kinase levels, his muscle biopsy revealed myopathic changes with tiny intracytoplasmic vacuoles containing PAS-positive material and high acid phosphatase activity, but had normal acid maltase activity biochemically. These findings were consistent with those seen in lysosomal glycogen storage disease with normal acid maltase (Danon disease). Sarcolemmal indentations commonly seen in this disease were missing, but a complement membrane attack complex, C5b-9 was positive along the surface membrane of the muscle fibers as seen in X-linked vacuolar myopathy. The patient was on a respirator and died at 27 months of age from pneumonia and hypertrophic cardiomyopathy. Lysosomal glycogen storage disease with normal acid maltase may be manifested at birth with marked skeletal and cardiac involvement leading to death in early infancy.
Requirement of corepressor binding of thyroid hormone receptor mutants for dominant negative inhibition.
Resistance to thyroid hormone (RTH) is mainly caused by dominant negative inhibition of wild type thyroid hormone receptor (TR) function due to mutations in the ligand binding domain of the TR beta. Because no RTH mutant was identified in the hinge region of the TR, the contribution of this region for dominant negative inhibition was explored. In transient transfection assays, the dominant negative activity of a RTH-type mutant (P453X) was abolished by an introduction of a mutation into the hinge region (P214R). Although this hinge mutation did not alter homo- or heterodimerization with retinoid X receptor (RXR), its association with nuclear receptor corepressor (N-CoR) was impaired. These results indicate that association of corepressor with the RTH mutants through the hinge region is crucial for their dominant negative activity.
Phorbol ester-induced production of prostaglandin E2 from phosphatidylcholine through the activation of phospholipase D in UMR-106 cells.
To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2 (PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2 production. The TPA-induced PGE2 production was not affected by the pretreatment of cells with a phospholipase A2 inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2 production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2 production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2 by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2 nor phospholipase C is involved in the TPA-induced PGE2 production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2 via a PLD/PAP/DAG lipase/COX pathway.
Antineutrophil cytoplasmic antibody-positive crescentic glomerulonephritis associated with propylthiouracil therapy.
A teenage girl with crescentic glomerulonephritis had antineutrophil cytoplasmic antibody (ANCA) detected after she had received propylthiouracil (PTU) for hyperthyroidism without cutaneous vasculitis. ANCA was detected on admission; renal biopsy showed crescentic glomerulonephritis with focal necrotizing glomerulonephritis but no immune deposits. Administration of steroid and decreasing the dose of PTU produced a good clinical response and the ANCA disappeared. It was concluded that ANCA is closely related to the pathogenesis of crescentic glomerulonephritis and that treatment with PTU appeared to induce ANCA.
Periodic discharge of adrenocorticotropin and vasopressin associated with focal glomerulosclerosis.
We report the first case of the syndrome of periodic adrenocorticotropin (ACTH) and vasopressin (ADH) discharge associated with focal glomerulosclerosis. Approximately 30 cases of this syndrome have so far been reported in Japan, but no cases associated with renal dysfunction have yet been reported. The patient, a 10-year-old Japanese boy, was referred to our hospital because of recurrent attacks of vomiting. He was diagnosed as having this syndrome from clinical and laboratory findings. While various drugs were tried to manage his vomiting attacks, only valproic acid appeared to be effective in reducing the frequency of the attacks. Chronic nephritis was manifested when the patient was 12 years old, which required treatment with continuous ambulatory peritoneal dialysis. Valproic acid was proved to be effective in reducing the number of attacks over 4 months.
Soluble thrombomodulin and antibodies to bovine glomerular endothelial cells in patients with Henoch-Schönlein purpura.
AIM: To evaluate the clinical significance of soluble thrombomodulin and antiendothelial cell antibodies (AECA) in children with Henoch-Schönlein purpura. METHODS: Binding of serum AECA to bovine glomerular endothelial cells was evaluated by enzyme linked immunosorbent assay, cytotoxicity against glomerular endothelial cells by spectrophotometric assay, and soluble thrombomodulin concentrations by sandwich enzyme immunoassay. RESULTS: IgA AECA were detected in seven of 15 patients with Henoch-Schönlein purpura and nephritis, but were not detected in patients without nephritis or in controls. Patients with Henoch-Schönlein nephritis had raised titres of IgA AECA and serum thrombomodulin; severe proteinuria and renal histological changes were associated with raised titres of IgA AECA and raised serum thrombomodulin. No subjects had complement dependent cytotoxicity against glomerular endothelial cells. CONCLUSIONS: High titres of IgA AECA and raised serum thrombomodulin may be clinically useful markers of renal involvement in patients with Henoch-Schönlein purpura.
Alport syndrome with a peculiar pattern of distribution of the alpha3-alpha6 chains of type IV collagen in renal basement membrane.
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Antibodies to endothelial cells in Kawasaki disease lyse endothelial cells without cytokine pretreatment.
Kawasaki disease (KD) is characterized by diffuse vasculitis and marked immune activation. To confirm the presence of antiendothelial cell antibodies (AECA) and cytotoxicity of AECA, we investigated the presence of AECA using ELISA and cytotoxicity of AECA in KD. Sera from patients with acute KD were assessed for binding to human umbilical vein endothelial cells (HUVEC) using a cell-based ELISA, and for cytotoxicity against HUVEC as indicated by the conversion of a tetrazolium salt (MTT) into a coloured product. IgM AECA were detected in 8/19 KD sera, IgG AECA were detected in 5/19 KD sera. Significant differences in both AECA titres existed between patients and febrile and afebrile controls. Six out of 19 patients showed complement-dependent cytotoxicity against HUVEC. Cytotoxicity was significantly enhanced by pretreating HUVEC with tumour necrosis factor (TNF), and reduced by incubation with gammaglobulin. Serum titres of IgM AECA in the KD patients were positively correlated with cytotoxicity. Findings suggest that IgM AECA mediates complement-dependent cytotoxicity against endothelial cells in patients with KD, and gammaglobulin may reduce complement-dependent cytotoxicity of AECA against endothelial cells.
Heterodimerization preferences of thyroid hormone receptor alpha isoforms.
Thyroid hormone receptors (TRs) are members of the steroid hormone receptor superfamily and are encoded by two different genes, alpha and beta. Three isoforms (alpha 1, alpha 2, and alpha 3) are created by alternative splicing of the TR alpha gene. In TR alpha 2 and alpha 3, the distal half of the putative dimerization domain is disrupted and the carboxy terminus of the protein is substituted with different amino acids. To evaluate the properties of these alterations in the dimerization region, DNA binding and dimerization of TR alpha isoforms were studied by electrophoretic mobility shift assays. TR alpha 1 formed a monomer or a homodimer on certain thyroid hormone responsive elements (TREs), whereas TR alpha 2 and alpha 3 did not bind effectively to any of the TREs studied. TR alpha 1 formed a heterodimer with 9-cis retinoic acid receptor alpha (RXR alpha) on all TREs studied. Although TR alpha 2 did not bind as a homodimer, it did bind as a heterodimer with RXR alpha to DR4 and MHC-TRE. TR alpha 3 bound as a heterodimer to a broader repertoire of TREs, including DR4, MHC, ME, and F2-TRE. These results indicate that the alterations in the dimerization region in TR alpha 2 and alpha 3 abrogated homodimer binding, but differentially affected heterodimerization with RXR alpha on various TREs.
[Pharmacokinetic and clinical evaluations of azithromycin in the pediatric field].
Azithromycin (AZM), an oral macrolide antibiotic drug, was studied for its efficacy and pharmacokinetics in the treatment of pediatric patients with respiratory tract infections. The results of the study are presented. Fifteen pediatric patients in an age range between 1 and 10 years were given 8.9 to 14.7 mg/ kg body weight of AZM once daily for 3 to 4 days. Pharmacokinetics of AZM in three patients were examined. AZM concentration in plasma was 0.037 microgram/ml at 72 hours after final dosing, while urine concentration was 10.9 micrograms/ml measured during a period between 72 and 96 hours post-dosing. Fourteen patient cases were included in analysis of drug efficacy for AZM. AZM was found "markedly effective" in all seven patients with pneumonia, and "effective" in all three patients with acute bronchitis, two patients with mycoplasmal pneumonia, and one patient with tonsillitis. In one patient with mycoplasmal bronchitis, AZM was found slightly effective. The efficacy rate was 92.9% (13/14). The study found one case of side effect, moderate diarrhea. No laboratory abnormality was documented. In conclusion, an antibiotic AZM was found useful in a treatment of pediatric respiratory tract infections.
Acute renal failure after exercise in a child with renal hypouricemia.
We describe a 15 year old boy with renal hypouricemia who developed acute renal failure after a school athletics meeting, accompanied by appendicitis. During acute renal failure, the highest level of uric acid was 5.0 mg/dL, creatinine 7.9 mg/dL and urea nitrogen 58.6 mg/dL. After recovery, the serum uric acid fell to 0.9 mg/dL and the fractional excretion of uric acid (FEuA) exceeded the normal range. The probenecid and pyrazinamide tests showed that the patient had a total defect of uric acid reabsorption. This case suggested that strenuous exercise could be responsible for acute renal failure in patients with renal hypouricemia.
Defective natural killer cell activity and deficient production of interferon-gamma in children with acute lymphoblastic leukemia.
Natural killer (NK) cell activity, OK-432-augmented-NK cell activity, concentrations of interferon-gamma (IFN-gamma) in the culture supernatants of lymphocytes stimulated with OK-432, and subsets of NK cells and memory T cells were analyzed in 42 children with acute lymphoblastic leukemia (ALL) receiving maintenance chemotherapy. Natural killer and augmented-NK cell activities, and concentrations of IFN-gamma in the supernatants of cultured lymphocytes, were significantly lower in the patients with ALL than in age-matched control children. Among the NK cell subsets, proportions of CD57+ cells in the patients with ALL were significantly higher than in the controls, and proportions of a memory T cell subset (CD4+ CD29+ T cells) in the patients were also significantly higher than in the controls. These results suggest that the function of NK cells and memory T cells that are considered as IFN-gamma producing cells, may be defective in ALL, and that CD57+ cells and CD4+ CD29+ cells may be resistant to or recover rapidly from suppression by cytotoxic chemotherapy.
[Clinical studies on cefozopran in pediatrics].
We conducted clinical studies on cefozopran (CZOP), a newly developed parenteral cephalosporin, for its clinical application in the field of pediatrics. 1. A clinical study was performed on 16 children with infections, including 9 with pneumonia, 1 each with acute bronchitis, enterocolitis, purulent lymphadenitis, 4 with skin and soft tissue infections. CZOP was administered by 30 minutes intravenous drip infusion. Doses varying from 20 to 35 mg/kg body weight were given t.i.d. Lengths of treatment ranged from 4 to 14 days. 2. Clinical efficacies were excellent in 10 and good in 6 cases, with an efficacy rate of 100%. The overall bacterial eradication rate for the pathogenic bacteria was also 100%. 3. Side effect was noted in 1 case with skin rash. Abnormal laboratory test data were found in 5 cases including slight elevations of GPT in 4 cases and GOT in 2 cases.
Defective activity of Epstein-Barr virus (EBV) specific cytotoxic T lymphocytes in children with chronic active EBV infection and in their parents.
Antibodies of Epstein-Barr virus (EBV), EBV-specific cytotoxic T lymphocyte (EBVCTL) activity and the lymphocyte subset of CTL were examined in 13 Japanese children with chronic active EBV infection (CAEBV) and their parents (eight fathers and 10 mothers). Anti-virus-capsid antigen (VCA)-IgG antibody titers ranged from 1:640 to 1:5120 in the patients with CAEBV and from 1:40 to 1:640 in the parents. While anti-VCA-IgM antibody was detected in three patients, anti-VCA-IgA antibody in five and anti-early-antigen (EA)-IgG antibody in 11, no antibody was detected in the parents except anti-EA antibody, which was positive in the mothers of cases 5 and 13 (1:10 and 1:40). Anti-EBV-associated nuclear antigen (EBNA) antibody was < or = 1:10 in six out of 13 patients with CAEBV and in 10 out of 18 parents tested. Epstein-Barr virus activity was significantly lower (P < 0.005) both in the children with CAEBV and in their parents than in seropositive age-matched controls. Proportions of a CTL subset (CD8+ CD11- lymphocytes) in the patients with CAEBV were significantly higher (P < 0.005) than in controls, while those in the parents were at the same level as in controls. Defective EBVCTL activity and anti-EBNA-antibody responses were frequently observed both in children with CAEBV and in their parents, which may suggest that the abnormal immune response to EBV may be based on a familial disorder, though no familial involvement has been reported in Japanese children with CAEBV.
Effect of thyroid hormone on epidermal growth factor gene expression in mouse submandibular gland.
Epidermal growth factor (EGF) in mouse submandibular gland (SMG) is synthesized in the granular convoluted tubular (GCT) cells. The synthesis of EGF in SMG has been shown to be increased by thyroid hormone. This increase was attributed to the increase in EGF mRNA. Not known is how thyroid hormone increases the mRNA level. In the present study the effect of thyroid hormone administration on EGF gene expression in SMG was studied in hypothyroid mice. Hypothyroidism was induced by treating the mice with propylthiouracil. The amount of SMG EGF mRNA was markedly decreased in hypothyroid mice. Administration of T3 increased the mRNA in a dose-dependent manner. The increase in EGF mRNA by T3 was evident as early as 6 h after T3 administration. A nuclear run-off assay indicated that the induction of EGF gene expression by T3 is at a transcriptional level. Bromodeoxyuridine incorporation into GCT cells was not affected by T3 administration, suggesting that T3 does not cause the proliferation of these cells. In situ hybridization revealed that T3 increases EGF mRNA in GCT cells at a single cell level. These results suggest that thyroid hormone increases EGF gene transcription without affecting cellular proliferation.