Haruo Shintaku

Trophic effect of multiple growth factors in amniotic fluid or human milk on cultured human fetal small intestinal cells

Objectives To evaluate the role of growth factors in amniotic fluid and in human milk on gastrointestinal adaptation of the fetus and very low-birth-weight infants, the effects of these fluids and multiple growth factors were investigated in a human fetal small intestinal cell line (FHs 74 Int). Methods After FHs 74 Int cells were incubated with amniotic fluid, human milk, or recombinant growth factors, growth-promoting activity was measured by [3H]-thymidine incorporation into cells. Results Incubating cells with amniotic fluid or human milk promoted growth dose dependently. Genistein almost completely inhibited growth-promoting activity in amniotic fluid P = 0.002), and growth was partially inhibited by antibodies against epidermal growth factor (EGF) (P = 0.047), insulin-like growth factor-1 (IGF-1, P = 0.047), or fibroblast growth factor (FGF, P = 0.014). This activity in human milk was inhibited almost completely by genistein (P h 0.0001) and partially inhibited by antibodies against EGF (P = 0.036), IGF-1 (P = 0.009), FGF (P = 0.004), hepatocyte growth factor (HGF, P = 0.001), or transforming growth factor-&agr; (TGF-&agr;, P = 0.001). Although recombinant EGF, IGF-1, FGF, HGF, and TGF-&agr; elicited a synergistic trophic response on cultured cells, the response was much less than with amniotic fluid or with human milk. Conclusion In aminiotic fluid and in human milk, EGF, IGF-1, FGF, HGF, and TGF-&agr; have a strong trophic effect on immature intestinal cells and may be involved in perinatal gastrointestinal adaptation.

Long-Term Treatment and Diagnosis of Tetrahydrobiopterin-Responsive Hyperphenylalaninemia with a Mutant Phenylalanine Hydroxylase Gene

A novel therapeutic strategy for phenylketonuria (PKU) has been initiated in Japan. A total of 12 patients who met the criteria for tetrahydrobiopterin (BH4)-responsive hyperphenylalaninemia (HPA) with a mutant phenylalanine hydroxylase (PAH) (EC 1.14.16.1) gene were recruited at 12 medical centers in Japan between June 1995 and July 2001. Therapeutic efficacy of BH4 was evaluated in single-dose, four-dose, and 1-wk BH4 loading tests followed by long-term BH4 treatment, and also examined in relation to the PAH gene mutations. The endpoints were determined as the percentage decline in serum phenylalanine from initial values after single-dose (>20%), four-dose (>30%), and 1-wk BH4 (>50%) loading tests. Patients with mild PKU exhibiting decreases in blood phenylalanine concentrations of >20% in the single-dose test also demonstrated decreases of >30% in the four-dose test. The 1-wk test elicited BH4 responsiveness even in patients with poor responses in the shorter tests. Patients with mild HPA, many of whom carry the R241C allele, responded to BH4 administration. No clear correlation was noted between the degree of decrease in serum phenylalanine concentrations in the single- or four-dose tests and specific PAH mutations. The 1-wk test (20 mg/kg of BH4 per day) is the most sensitive test for the diagnosis of BH4-responsive PAH deficiency. Responsiveness apparently depends on mutations in the PAH gene causing mild PKU, such as R241C. BH4 proved to be an effective therapy that may be able to replace or liberalize the phenylalanine-restricted diets for a considerable number of patients with mild PKU.

Brain magnetic resonance imaging in 23 patients with mucopolysaccharidoses and the effect of bone marrow transplantation.

A longitudinal study of cranial magnetic resonance imaging (MRI) was carried out in 23 patients with mucopolysaccharidoses (MPS); 1 each of types IH, VI, and VII; 2 of type IS; 10 of type II; and 4 each of types IIIB and IVA. Six types of distinct abnormalities were 1) cribriform changes or spotty changes in the corpus callosum, basal ganglia, and white matter; 2) high‐intensity signal in the white matter on T2‐weighted image; 3) ventriculomegaly; 4) diffuse cerebral cortical atrophy; 5) spinal cord compression; and 6) megacisterna magna. The cribriform changes that corresponded to dilated perivascular spaces were found in the patients with MPS IS, II, and VI. The patchy and diffuse intensity changes were found in the patient with MPS II and IIIB, respectively. MPS IH and the severe type of MPS II showed marked ventriculomegaly. Marked cerebral atrophy was observed in all MPS IIIB patients and in the severe type of MPS II patients. Spinal cord compression was a feature usually observed in MPS IH, IVA, VI, and VII. Megacisterna magna was frequent in the patients with MPS II (6/10). In 2 of 5 patients, the therapeutic effect of bone marrow transplantation (BMT) was remarkable. Both the cribriform changes and the intensity change of the white matter in a MPS VI patient disappeared 8 years after the BMT. Slight improvement of cribriform change was noted in 1 patient with MPS II 3 years after the BMT. MRS was not sufficient to estimate the accumulation of glycosaminoglycans but was useful for evaluating neuronal damages.

Novel Mechanism and Role of Angiotensin II–Induced Vascular Endothelial Injury in Hypertensive Diastolic Heart Failure

Objective—The mechanism and role of angiotensin II–induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure. Methods and Results—We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1−/− mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1−/− mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure. Conclusion—AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.

Disorders of Tetrahydrobiopterin Metabolism and their Treatment

Tetrahydrobiopterin (BH4) deficiencies are disorders affecting phenylalanine metabolism in liver and neurotransmitters biosynthesis in brain. BH4 is the essential cofactor in the enzymatic hydroxylation of 3 aromatic amino acids (phenylalanine, tyrosine, and tryptophan). BH4 is synthesized from guanosine triphosphate (GTP) catalyzed by GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase (SPR), and in aromatic amino acids hydoxylating system is regenerated by pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). To date, 4 enzyme deficiencies (GTPCH, PTPS, DHPR, PCD) have been reported and they all follow an autosomal recessive mode of inheritance. The incidence of BH4 deficiency is at 1 in 1,000,000, except that in Taiwanese (much higher than in Japanese and Caucasians). BH4 deficiency has been diagnosed in patients with hyperphenylalaninemia (HPA) by neonatal mass-screening based on BH4 oral loading tests, analysis of urinary or serum pteridines, and measurement of dihydropterindine reductase (DHPR) activity in blood from a Guthrie card. BH4 deficiency without treatment causes combined symptoms of HPA and neurotransmitter (dopamine, norepinephrine, epinephrine, and serotonin) deficiency, such as red hair, psychomotor retardation, and progressive neurological deterioration. Treatment of BH4 deficiencies consists of BH4 supplementation (2-20 mg/kg per day) or diet to control blood phenylalanine concentration and replacement therapy with neurotransmitters precausers (L-dopa/CarbiDOPA and 5-hydroxytryptophan), and supplements of folinic acid in DHPR deficiency.

A new Japanese case of succinyl-CoA: 3-ketoacid CoA-transferase deficiency

Succinyl-CoA:3-ketoacid CoA-transferase (SCOT; EC 2.8.3.5) deficiency (McKusick 245050) is a rare inherited metabolic disease. This enzyme is a key enzyme for utilization in peripheral tissues of ketone bodies that are produced by the liver. The absence of SCOT activity blocks peripheral ketone body utilization and causes recurrent attacks of severe ketoacidosis beginning in the neonatal or infantile period. Five cases of SCOT deficiency have been reported (Tildon and Cornblath 1972; Spence et al 1973; Middleton et al 1987; Saudubray et al 1987; Perez-Cerda et al 1992). The prognosis of these patients seems to parallel the severity of the SCOT deficiency. Here we report a new case of SCOT deficiency in Japan. We also report SCOT activity in the lymphocytes of the patient and his family.

Phospho‐Ser727 of STAT3 regulates STAT3 activity by enhancing dephosphorylation of phospho‐Tyr705 largely through TC45

Signal transducer and activator of transcription 3 (STAT3) is a latent cytoplasmic transcription factor. It is activated by cytokines, including interleukin‐6 (IL‐6) through phosphorylation at Tyr705 (pY705), which is required for its dimerization and nuclear translocation. However, the role of Ser727 phosphorylation, occurring during activation, remains poorly understood. Using a combination of HepG2‐stat3‐knockdown cells reconstituted with various STAT3 mutants and protein kinase inhibitors, we showed that phospho‐S727 has an intrinsic mechanism for shortening the duration of STAT3 activity, in turn shortening the duration of socs3 mRNA expression. Both STAT3WT and STAT3Ser727Asp (S727D) but not STAT3Ser727Ala (S727A) showed rapid dephosphorylation of pY705 after the inhibition of tyrosine kinases. We found that the nuclear TC45 phosphatase is most likely responsible for the phospho‐S727‐dependent pY705 dephosphorylation because TC45 knockdown caused prolonged pY705 with sustained socs3 mRNA expression in STAT3WT but not in STAT3S727A, and overexpressed TC45 caused rapid dephosphorylation of pY705 in STAT3WT but not in STAT3S727A. We further showed that phospho‐S727 did not affect the interaction of TC45 with STAT3, and that a reported methylation at K140 of STAT3 occurring after phospho‐S727 was not involved in the pY705 regulation. These findings indicate that phospho‐Ser727 determines the duration of STAT3 activity largely through TC45.

Patients Homozygous for the T435N Mutation of Succinyl-CoA:3-Ketoacid CoA Transferase (SCOT) Do Not Show Permanent Ketosis

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; locus symbol OXCT; E.C. 2.8.3.5) is the main determinant of the ketolytic capacity of tissues. Hereditary SCOT deficiency causes episodic ketoacidosis. Permanent ketosis has been regarded as a pathognomonic feature of SCOT deficiency. There are three SCOT-deficient patients from a small region in Japan and they have not manifested permanent ketosis, even though their ketoacidotic crises were as severe as those of other SCOT-deficient patients. All three were homozygous for the T435N mutation. Transient expression analysis of wild-type and mutant cDNA showed that the T435N mutant retained significant residual SCOT activities (20% for that of the wild-type at 39.5°C, 25% at 37°C, and 50% at 30°C). The difference of residual SCOT activities at these temperatures in expression analyses was due to differences in the level of the mutant protein. SCOT activity of the T435N protein was more vulnerable than the wild-type to heat treatment at 42°C and 55°C. These temperature-sensitive characteristics of the mutant protein may explain, in part, why the patients developed ketoacidotic crises during febrile illness. In SCOT-deficient patients retaining some residual activity, permanent ketosis may be absent.

Molecular and clinical analyses of Japanese patients with carbamoylphosphate synthetase 1 (CPS1) deficiency.

AbstractCarbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

Succinyl‐CoA:3‐ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT‐deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT‐deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G→T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C→T) and V133E (c398T→A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20–35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl‐CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates. Hum Mutat 12:83–88, 1998.