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Immunohistochemical localization of Copper-transporting P-type ATPase (ATP7B), a gene product responsible for Wilson disease, was visualized in the brain tissues of the Long-Evans agouti rat in detail using tissue-blotting technique and confocal laser microscopy. The ATP7B was intensely detected in neuronal cells of the hippocampal formation, olfactory bulbs, cerebellum, cerebral cortex and nuclei in the brainstem in which high amounts of copper and cuproenzymes, dopamine beta hydroxylase and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were detected. The present results suggest that ATP7B plays key roles in neurotransmissions of catecholamine pathway and preventing brain tissues from injury by superoxide radicals to regulate the cellular Cu concentration and/or activities of cuproenzymes related to neurotransmissions and a free radical metabolism. Furthermore, it is reasonable to assume that neurotoxicity due to abnormal copper accumulation or irregular regulation of cuproenzymes in the critical brain regions by mutation of the ATP7B gene leads to neurological failures of Wilson disease.

巣状糸球体硬化症 (focal glomerular sclerosis; FGS) による慢性腎不全のため自動腹膜透析を施行中にAlcaligenes xylosoxidans単独感染による汎発性腹膜炎を合併した症例を経験した。各種抗生剤の腹腔内，経静脈，経口投与によってもCAPD排液中の細胞数が減少せず，原因菌が排液，チューブ出口部から検出され続けた。CAPDチューブ入れ換え術により腹膜炎は軽快した。CAPDチューブ先端およびチタニウムアダプタとの接続部にバイオフィルムを確認した。腹膜透析中の腹膜炎に対する内科的治療が奏効せず，バイオフィルムの存在が予測された場合には，早期にCAPDチューブ入れ換え術を考慮すべきだと考えられる。

本研究の目的は，地域在住女性高齢者のバランス能力と下肢筋力，歩行速度の関係を検討することである。歩行補助具を使用せずに屋外歩行が可能な，60歳以上の女性高齢者を対象に，バランス能力，下肢筋力，歩行速度を測定した。バランス能力では静的立位時重心動揺(開眼・閉眼)と機能的バランス評価としてFunctional Balance Scale(FBS)，Functional Reach(FR)を測定した。下肢筋力はCybex770にて膝関節伸展ピークトルク値(体重比;%BW)を測定し，歩行速度は10m最大歩行速度とその際の歩行率，歩幅を測定した。統計学的分析の結果，機能的バランス評価，下肢筋力，歩行速度，歩行率，歩幅は年齢と有意な負の相関関係を認めたが，重心動揺は相関を認めなかった。年齢を制御変数とした場合，機能的バランス評価と歩行速度，歩行率，歩幅はそれぞれ有意な正の相関を認めた。以上より，健常女性高齢者の歩行能力には機能的バランス能力が重要な要因となることが示唆された。

Background Nedd4 is a ubiquitin-protein ligase containing a calcium/lipid-binding domain, multiple WW domains and a C-terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin-conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport. Results Three mRNA species for human Nedd4 were found to be 6.4-, 7.8- and 9.5-kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue- and cell-specificities of Nedd4 expression. The Nedd4 protein, ≈120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down-regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N-terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full-length Nedd4 and E2 enzyme revealed that both proteins mostly colocalized in the cytoplasmic periphery, while the N-terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme. Conclusion Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.

Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder. It is X-linked and hemizygous new-born males usually suffer fatal hyperammonemia. In contrast, carrier females manifest variable phenotypes, ranging from asymptomatic carriers to those with severe hyperammonemia. In order to understand the correlation between X-inactivation status and the clinical phenotype of carrier females with this disorder, we analyzed the X-inactivation pattern of peripheral blood leukocytes in a family consisting of a clinically normal mother and two daughters with severe manifestation. In addition, we obtained tissue samples from various parts of the liver of one of these daughters and analyzed X-inactivation patterns and the residual OTC activities. The X-inactivation of peripheral blood leukocytes was nearly random in these carrier females and showed no correlation with the disease phenotype. However, the X-inactivation of the liver was much more skewed and correlated well with the OTC activity of all samples. Interestingly, the degree of X-inactivation varied considerably, even within the same liver.

In Japan, urea cycle disorders (UCDs) are one of the most frequent inborn errors of metabolism, estimated to have a prevalence of 1 per 50,000 live births. In an attempt to develop more effective treatment and enhance the quality of life, we investigated the clinical manifestations and prognosis of 216 patients with UCDs diagnosed and treated between 1978 and 1995. These included 92 cases of neonatal-onset UCD and 116 of late-onset UCD. Two cases of ornithine transcarbamylase (OTC) deficiency in males and 2 cases of argininosuccinase (AL) deficiency were diagnosed prospectively. By far the most common disorder was OTC deficiency, accounting for 2/3 of all cases. At the end of 1995, the 5-year survival rate was 22% for the neonatal-onset type and 41% for the late-onset type. Among the 20 long-term survivors with neonatal-onset UCD, 18 (90%) had moderate to severe neurodevelopmental deficits; this contrasts with 13 of 47 (28%) survivors with the late-onset type. In analysing 108 UCD cases, peak blood ammonia level during the first hyperammonaemic attack was correlated with neurodevelopmental outcome. When the concentration of blood ammonia was less than 180 mumol/L (5 times normal), there was no severe neurological damage. When the concentration of blood ammonia exceeded 350 mumol/L (10 times normal) at the first hyperammonaemic attack, the patients died or had severe neurological deficits. Our data point to the importance of early diagnosis and aggressive treatment.

Background It has been suggested that quantitative analysis of urinary bile acids may help to distinguish primary 3-oxo-delta 4-steroid 5 beta-reductase deficiency from the excretion of 3-oxo-delta 4 bile acids that occurs as a result of liver damage. Methods Urinary bile acids were quantitatively analyzed by gas chromatography-mass spectrometry in four Japanese patients with severe neonatal cholestasis associated with hypertyrosinemia without urinary succinylacetone (i.e. tyrosinemia type I-like disease). These four patients represented sporadic cases. Results Large amounts of 3-oxo-delta 4 bile acids were detected, which comprised greater than 80% of the total urinary bile acids. Small amounts of allo-bile acids and primary bile acids were also detected, comprising less than 1% and 15% of the total urinary bile acids, respectively. Conclusions It was suspected that these four patients had a primary 3-oxo-delta 4-steroid 5 beta-reductase deficiency. However, it is possible that some patients in this study may have had a secondary 3-oxo-delta 4-steroid 5 beta-reductase deficiency, caused by idiopathic neonatal cholestatic liver failure.

The purpose of this study was to assess the construct validity of the Berg Balance Scale (BBS) in stroke inpatients. We measured the BBS score of 46 stroke patients, and simultaneously assessed the following neurological conditions: attention, mental status, emotion, agnosia, visual perception, sensation and motor status of the affected lower extremity. We also assessed functional independence in the basic activities of daily living using the Barthel Index. Patients’ functional walking abilities were categorized into 4 levels (from with assistance to independent within hospital). The mean BBS scores were associated with attentional deficits, emotional distress, proprioceptive sensation problems and motor status. These neurological conditions explained 43% of the BBS scores. The BBS scores correlated with the Barthel Index and were associated with functional walking ability. The BBS scores and walking ability explained 67% of the Barthel Index. 83% of patients scoring above a score of 40 were independent within hospital in functional walking ability. These results provide information to support construct validity of the BBS in stroke inpatients.

健常女性における，膝屈伸筋群の等速性筋出力特性と年齢や歩行能力との関係を検討することを本研究の目的とし，若年群18名(22.0±1.6歳)と高齢群47名(70.6±5.1歳)を対象とした。筋出力特性はCybex770を用いて60・180deg/sにおけるpeak torqueの体重比(%BW)，angle of peak torque，acceleration time(AT)を全対象者について測定し，最大歩行速度と6分間歩行距離(6MD)は高齢群のみ測定した。2群の比較において，有意な%BWの低下とATの延長が認められた。また，高齢群では%BWとATは有意な負の相関関係にあり，ATは年齢，最大歩行速度，6MDと有意な相関関係にあった。最大歩行速度，6MDを従属変数とし，年齢，%BW，ATを独立変数とした重回帰分析では，最大歩行速度はATと年齢の影響を強く受け，6MDでは年齢のみの影響を強く受けていた。従って，高齢者における膝屈伸筋群の等速性筋出力にはATの考慮が重要であることが明らかとなった。

家族性特発性低分子蛋白尿症 (familial idiopathic low-molecular-weight proteinuria: FILMWP) は，低分子蛋白尿を呈する腎尿細管障害で本邦にて報告された。一方，腎結石症を伴うDent病などの3つの遺伝性腎尿細管障害が欧米で報告され，クロライドチャンネルCLCN5遺伝子の異常であることが報告された。これらの疾患には臨床上の類似性がみられるが，FILMWPでは腎不全やくる病がないことで区別される。 私達はFILMWP患者5例についてCLCN5遺伝子を解析し，一塩基挿入2例，一塩基欠失2例，ナンセンス変異1例を認めた。FILMWPの多くは本遺伝子の異常でおこると考えられ，これら4つの腎尿細管疾患は1つの疾患の亜型と考えられる。またCLC5蛋白のドメイン11に変異が多く存在する (自験例5例中2例)。遺伝子型と表現型の関係はまだ明らかではない。本遺伝子の変異だけでなく，修飾遺伝子，環境因子が表現型に影響を与えるものと考えられる。

We studied the effects of Shikaron, which is composed of 8 Chinese herb extracts, on carcinogenesis and the cytotoxic activity and cytokine production of lymphocytes in mice treated with a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). We found a significantly lower incidence of urinary bladder carcinoma in mice treated with BBN plus Shikaron 200 mg/kg/day (5 of 20 mice, 25%), than in mice treated with BBN alone (16 of 20 mice, 80%). Shikaron protected the cytotoxic activity of lymphocytes against YAC-1 cells from suppression by BBN. Cytotoxic activity against P-815 cells significantly increased in mice treated with BBN plus Shikaron, as compared with normal mice and BBN-treated mice. Shikaron also protected production of interleukin-2 and interferon-gamma by lymphocytes from suppression by BBN. These findings strongly suggest that Shikaron exerted an anticarcinogenic effect in carcinogen-treated mice through activation of NK and LAK cells and cytokine production by T lymphocytes.

Mutations in the CLCN5 gene in Japanese patients with familial idiopathic low-molecular-weight proteinuria. Familial idiopathic low-molecular-weight proteinuria (FILMWP) is a renal proximal tubulopathy that occurs predominantly in males. FILMWP is characterized by mild proteinuria consisting of low-molecular-weight proteinuria, aminoaciduria and relatively conserved renal function, but without rickets. To determine whether FILMWP is related to the CLCN5 gene, which is responsible for Dent's disease and two related disorders, we analyzed the CLCN5 gene from four Japanese families with FILMWP. We identified two novel mutations: one was a single base insertion at codon 520 serine in exon 10 and the other was a single base deletion at codon 403 tyrosine in exon 8. These mutations caused a shift in the reading frame, resulting in synthesis of truncated CLC5 proteins that lacked 220 (29%) and 314 (42%) amino acids, respectively. These mutations were demonstrated to cosegregate with the disease in two families, respectively. We conclude that the CLCN5 gene is responsible for this proximal renal tubulopathy in some Japanese families and that FILMWP is possibly a variant of Dent's disease.

Hereditary tyrosinemia 1 (HT1) is characterized by progressive liver damage, from infancy, and by a high risk for hepatocellular carcinoma. HT1 is due to mutations in the fumarylacetoacetate hydrolase gene Fah, encoding the last enzyme in the tyrosine catabolic pathway. Lethal albino deletionc 14CoS mice and mice with target-disruptedFah are models for HT1, but they die in the perinatal period, albeit with a different phenotype from that seen in HT1 in humans. We first asked whether homozygous null mutation of the 4-hydroxyphenylpyruvate dioxygenase gene Hpd could rescue the homozygous c 14CoS mice (c 14CoS/c 14CoS orFah −/−). The double mutantFah −/− Hpd −/− mice appeared normal, at least until age 18 months, and there was no evidence of liver disease, findings that facilitated examination of the effect of Fah −/− on mature and unmodified hepatocytes in vivo. The hepatocytes ofFah −/− undergo rapid apoptosis, and acute death follows. Essentially the same phenomena were observed whenFah −/− Hpd −/− mice were administered homogentisate intraperitoneally. These changes in liver pathology in Fah −/− Hpd −/− mice after the administration of homogentisate were associated with massive urinary excretion of succinylacetone. These results suggest that accumulation of fumarylacetoacetate, maleylacetoacetate, or succinylacetone seems to trigger the endogenous process of apoptosis in hepatocytes that lack fumarylacetoacetate hydrolase activity. This apoptosis may be related to the development of hepatocellular carcinomas seen in HT1 patients and pharmaceutically treated fumarylacetoacetate hydrolase-deficient mice.

Prolidase deficiency is an autosomal recessive inherited disease characterized clinically by frequent infections, mental retardation, and various skin lesions. Fundamental treatments for these manifestations have not been established. We performed adenovirus-mediated gene transfer of human prolidase cDNA into fibroblasts from patients with prolidase deficiency. Infection with the adenovirus vector carrying human prolidase cDNA increased prolidase activity in fibroblasts up to approximately 7.5 times of that of normal control fibroblasts. This indicates the feasibility of adenovirus-mediated gene therapy to treat patients with prolidase deficiency in the future.

In our on-going studies on gene therapy, we constructed an expression vector pCAGGS-WDeu bearing full-length human ATP7B cDNA. We also developed a series of monoclonal antibodies directed to human ATPase7B by immunizing mice with recombinant ATPase7B developed in Escherichia coli. Immunochemical analysis with these antibodies revealed that the expression vector produced human ATPase7B protein with 155 Kd in cultured COS-1 cells. Immunohistochemical analysis revealed that the protein is localized on membranes of cellular organelle and the recombinant ATPase7B in the cells could be solubilized with TritonX-100. These studies provided pertinent evidence that human ATPase7B, defective in patients with Wilson's disease, and Long-Evans cinnamon (LEC) rats, is a membranous protein with 155 Kd. The plasmind vector and specific antibodies developed in this study will facilitate in vivo studies on gene therapy for Wilson's disease, using LEC rats. J. Trace Elem. Exp. Med. 10:111–117, 1997. © 1997 Wiley-Liss, Inc.

ICR mice were treated with a carcinogen, N-butyl-N'-butanolnitrosoamine BBN), every day for 8 consecutive weeks and the effects of oral administration of edible mushrooms on the induction of urinary bladder carcinoma and on the activities of macrophages and lymphocytes were studied. Bladder carcinoma were found in all 10 mice (100%) treated with BBN alone, while we observed carcinoma only in 9 of 17 mice (52.9%), in 7 of 15 mice (46.7%) and 13 of 20 mice (65.0%) treated with Lentinus edodes, Grifola frondosa and Pleurotus ostreatus, respectively. Chemotactic activity of macrophages was suppressed in mice treated with BBN alone but maintained almost the normal level in mice treated with BBN plus Lentinus, Grifola or Pleurotus. Lymphocytes collected from mice treated with BBN plus each mushroom showed almost normal blastogenic response against concanavalin A, although those from mice treated with BBN alone completely retarded their response. Cytotoxic activity of lymphocytes against Yac-1 cells was also maintained at a normal level in mice treated with BBN plus each mushroom. Whereas in mice treated with BBN alone significant depression of NK cell activity occurred. Significantly higher cytotoxic activity against P-815 cells was observed in lymphocytes from mice treated with BBN plus each mushroom than that in lymphocytes from normal mice or mice treated with BBN alone.

Imatinib mesylate, a selective tyrosine kinase inhibitor, is the frontline therapeutic agent used for the treatment of chronic myeloid leukemia (CML), and its therapeutic efficacy is associated with trough concentrations. Therefore, monitoring imatinib trough concentrations is strongly recommended for successful treatment of CML patients. It has been recently shown that some drugs altered imatinib plasma levels in adult patients. However, drug interactions with imatinib in children are still unknown. Here, we report a case of a 12-year-old child with epilepsy who was also diagnosed with CML and given imatinib in addition to an enzyme-inducing antiepileptic drug, carbamazepine. Compared to population kinetics data, the data obtained for the patient showed a significant decrease of imatinib plasma concentrations. Our findings suggest that monitoring imatinib plasma concentrations in children receiving enzyme-inducing antiepileptic drugs is needed to optimize the therapeutic efficacy of imatinib.

SummaryMouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known regarding amino acid metabolism in human ESCs/iPSCs. We show that human ESCs/iPSCs require high amounts of methionine (Met) and express high levels of enzymes involved in Met metabolism. Met deprivation results in a rapid decrease in intracellular S-adenosylmethionine (SAM), triggering the activation of p53-p38 signaling, reducing NANOG expression, and poising human iPSC/ESCs for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells undergo apoptosis. We also show that human ESCs/iPSCs have regulatory systems to maintain constant intracellular Met and SAM levels. Our findings show that SAM is a key regulator for maintaining undifferentiated pluripotent stem cells and regulating their differentiation.

We report the case of a family with late-onset ornithine transcarbamylase deficiency (OTCD). Several family members had died from OTCD, and the c.221GA, p.Lys221Lys mutation was detected at the 3' end of exon 6 of OTC in the X-chromosome of some members. We provided genetic counseling on pregnancy, delivery, and neonate management to a 4th-generation female carrier and decided on metabolic management of her child from birth. Two male patients were diagnosed with late-onset OTCD on the basis of blood amino acid and genetic analysis, and they received arginine supplementation from the asymptomatic, early neonatal period. These children grew and developed normally, without decompensation. Patients with late-onset OTCD can and should be diagnosed and treated in the early neonatal period, especially those from families already diagnosed with late-onset OTCD, and family members must be provided with genetic counseling.

Amino acids are a group of metabolites that are important substrates for protein synthesis, are important as signaling molecules and play central roles as highly connected metabolic hubs, and therefore, there are many reports that describe disease-specific abnormalities in plasma amino acids profile. However, the causes of progression from a healthy control to a manifestation of the plasma amino acid changes remain obscure. Here, we extended the plasma amino acids profile to relationships that have interactive properties, and found remarkable differences in the longitudinal transition of hyperglycemia as a diabetes emergency. What is especially important is to understand pathogenesis for better treatment and early diagnosis of diabetes. In this study, we performed interactive analysis using time course data of the plasma samples of AKITA mice, which develop hyperglycemia. Primarily, we decided to analyze the interactive property of amino acids which had highly significant association with hyperglycemia, namely alanine, glycine, leucine, isoleucine and valine. Next, we inferred the interactive network structure, which reproduces the actual time course within an error allowance of 10% using an S-system model (a conceptual mathematical model for analyzing and simulating networks). The emphasis of this study was altered interactions of plasma amino acids that show stabilizing and destabilizing features in a variety of clinical settings. By performing sensitivity analysis, the most dominant relations in this network were selected; the control paths from glycine to isoleucine in healthy control and from alanine to glycine in hyperglycemia. This result is in good agreement with the biological knowledge regarding branched-chain amino acids, and suggests the biological importance of the effect from alanine to glycine.

Organic acidemias (OAs) are rare inborn errors of metabolism. The clinical presentations of methylmalonic acidemia (MMA) and propionic acidemia (PA) in Japan have not yet been examined in detail. We aimed to investigate the clinical presentations of OAs in Japan and evaluate current therapies for improving long-term outcomes, especially in MMA and PA cases. Questionnaires were sent to 928 institutions in 2009 inquiring about OAs, and secondary questionnaires were sent to those who confirmed that they had diagnosed and/or treated such cases; 119 cases were eventually included for analysis. In Japan, the majority of OAs was MMA, which was associated with a high mortality rate. The survival rates at 20 years of age in vitamin B12-unresponsive MMA, vitamin B12-responsive MMA and PA patients were 69.8%, 94.4% and 95.8%, respectively. Factors associated with mortality in MMA were failure to thrive, hypoglycemia and pancreatitis. Factors associated with mental retardation in vitamin B12-unresponsive MMA, vitamin B12-responsive MMA, and PA were seizure and liver dysfunction, seizure and failure to thrive, and failure to thrive, respectively. We advocated that avoiding failure to thrive due to too restricted protein diet, hypoglycemia and pancreatitis associated with mortality lead to improve outcome, especially in vitamin B12-unresponsive MMA patients.

Many reports have been published on the long-term outcome and treatment of hepatic glycogen storage diseases (GSDs) overseas; however, none have been published from Japan. We investigated the clinical manifestations, treatment, and prognosis of 127 hepatic GSD patients who were evaluated and treated between January 1999 and December 2009. A characteristic genetic pattern was noted in the Japanese GSD patients: most GSD Ia patients had the g727t mutation, and many GSD Ib patients had the W118R mutation. Forty-one percent (14/34) of GSD Ia patients and 18% (2/11) of GSD Ib patients of ages 13 years 4 months had liver adenoma. Among subjects aged 10 years, 19% (7/36) of the GSD Ia patients and none of the GSD Ib patients had renal dysfunction. The mean height of male GSD Ia patients aged 18 years was 160.8±10.6 cm (n=14), and that of their female counterparts was 147.8±3.80 cm (n=9). Patients with hepatic GSDs develop a variety of symptoms but can survive in the long term by diet therapy, corn starch treatment and supportive care. Liver transplantation for hepatic GSDs is an important treatment strategy and can help improve the patients'quality of life.

Three Japanese brothers with progressive deterioration of hepatic function and fatty degeneration of the liver eventually leading to hepatic encephalopathy were recently encountered. Each of them had a congenital intrahepatic portosystemic venous shunt due to a patent ductus venosus. Liver dysfunction and hepatic steatosis reverted to normal with surgical correction of the ductus venosus. Our observations suggest that impairment of liver function occurs first in the presence of malnutrition related to a reduction of blood in the portal vein. Hepatic steatosis is a consequence of a depleted metabolism of hepatocytes. Congenital portosystemic venous shunt due to a patent ductus venosus in this family has a genetic background.

In this study, forty-one hemodialysis outpatients performed a daily walking exercise. For this exercise, patients were instructed to walk beyond a daily target number of steps for three consecutive weeks. The target number of steps was defined as 120% of the average number of steps per day prior to the exercise. The effects of this exercise were examined by comparing the six-minute walking distance (6MD), and physiological cost index (PCI) before and after performing the exercise. The results were as follows: the target numberof steps during the exercise period was achieved by 19 of 41 patients; 6MD of target achieving patients was significantly increased by this exercise; the difference in PCI before and after this exercise was not statistically significant. This study suggests that the present exercise triggered an increase in the number of steps per day and improved the actual walking performance as measured by 6MD.

Branching morphogenesis (BrM), an essential step for salivary gland development, requires epithelial-mesenchymal interactions. BrM is impaired when the surrounding mesenchyme is detached from the salivary epithelium during the pseudoglandular stage. It is believed that the salivary mesenchyme is indispensable for BrM, however, an extracellular matrix gel with exogenous EGF can be used as a substitute for the mesenchyme during BrM in the developing salivary epithelium. Stem/progenitor cells isolated from salivary glands in humans and rodents can be classified as mesenchymal stem cell-like, bone-marrow-derived, duct cell-like, and embryonic epithelium-like cells. Salivary-gland-derived progenitor (SGP) cells isolated from duct-ligated rats, mice, and swine submandibular glands share similar characteristics, including intracellular laminin andα6β1-integrin expression, similar to the embryonic salivary epithelia during the pseudoglandular stage. Progenitor cells also isolated from human salivary glands (human SGP cells) having the same characteristics differentiate into hepatocyte-like cells when transplanted into the liver. Similar to the dissociated embryonic salivary epithelium, human SGP cells aggregate to self-organize into branching organ-like structures on Matrigel plus exogenous EGF. These results suggest the possibility that tissue stem cells organize rudiment-like structures, and the embryonic cells that organize into whole tissues during development are preserved even in adult tissues.

Background and purpose In adult brain, it has been reported newly generated neural stem / progenitor cells survived and differentiated to mature neurons in the severely damaged area after stroke. In neonatal brain, however, it was demonstrated that neurogenesis was stimulated after hypoxia ischemia (HI) and provided neuronal progenitors to the infarct area, but only few new neurons could survive under an unfavorable microenvironment and lack of appropriate trophic support. Recently, neural stem / progenitor cells were isolated from the infarct area in adult mouse brain and elucidated its character as a neural stem / progenitor cell. However, it is still unclear whether the infarct area in neonatal brain has neural stem / progenitor cells as same feature as adult brain. Therefore, the present study was conducted to investigate whether neural stem / progenitor cells could be isolated from infarct area in neonatal rat brain, and to clear its feature. Methods HI brain injury was induced in 7-days-old rat pups by the left common carotid artery occlusion followed by 120 minutes exposure to 8% oxygen. Three days after HI, the brain is removed and sliced at 2 mm thickness, and then six columns are punched out from various infarct areas for cell culture using neurosphere method. The cell clusters are analyzed by immunocytochemistry and mRNA expression for neuron, astrocyte and oligodendrocyte. Also, the cell clusters are investigated the ability of induction for neuron, astrocyte and oligodendrocyte. Results The numbers of neurospere-like cell clusters from infarct areas at 3 days after HI are dramatically increased compared with those from sham control animals. Four and 30 days incubation of the cell clusters provided various types of cells positive for BrdU, Nestin, NG2, β III tubulin, GFAP, O4, Vimentin or Iba1. These cell clusters expressed mRNA such as Nanog, Sox2, Oct3/4 and Rex1 as same as ES cell. Also these cell clusters could be differentiated into neurons, astrocytes or oligodendrocytes, and expressed mRNA such as Nestin, βIIItubulin, GFAP and MBP. Conclusion We have isolated injury-induced neural stem / progenitor cells form ischemic area after neonatal HI. This cell has a potential to use as a source for new neurons driving CNS repair after neonatal HI.

Mutations in the COL4A5 gene in Alport syndrome: A possible mutation in primordial germ cells. Using a combination of gene amplification with single strand conformation polymorphisms analysis and sequencing, we examined the COL4A5 gene in 37 patients with Alport syndrome. In patient A8, a single base insertion was noted at codon 1,597 tyrosine in exon 49. The premature terminal signal appeared and 89 amino acids (approximately one-third) of the non-collagenous domain were lost. The mutation was present in the mother, hence she is heterozygous. In patient A12, the nucleotide changed from C to T at codon 1,679 glutamine in exon 51, which created a termination codon, and 7 amino acids at the carboxyl terminus were lost. Gene tracking using peripheral leukocytes revealed that the parents did not carry the mutant allele, while the sister was heterozygous. DNA samples from hair roots and skin fibroblasts of the mother were normal and immunological examination of the epidermis of the mother indicated that the α5(IV) chain was normally expressed. As these results suggest that somatic cells of the mother do not carry the mutant allele, the primordial germ cells possibly carry a fresh mutation in the mother of patient A12.

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last step of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for defects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA in cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.

A patient with maple syrup urine disease (MSUD) associated with a E1 beta subunit deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1 beta subunit protein was identified by analysis of cDNA and genomic DNA by polymerase chain reaction. Total RNA isolated from lymphoblastoid cells was transcribed into cDNA and amplified using a set of primers located within exon 3 and exon 9 of the E1 beta gene. Agarose gel electrophoresis of cDNA amplification products revealed two shortened bands as well as a faint band of normal size. Nucleotide sequencing of the shortened cDNA amplification products showed that sequences corresponding to exon 5 and both exons 5 and 6 were absent. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the E1 beta gene revealed a single base substitution from G to T of the invariant GT dinucleotides at 5' splice site of the intron 5. Analysis of family members using primer-specified restriction map modification showed that the patient is homozygous for this mutation. We postulate that this mutation leads to the skipping of either exon 5 or both exons 5 and 6, thus producing two shortened E1 beta mRNA. The percentage of normal and two shortened transcripts was estimated to be 9, 71 and 20%, respectively. To our best knowledge, this is the first documented example of exon skipping in the E1 beta gene as the cause of MSUD and the novel mutation of the invariant G at the 5' splice site which results in two alternatively spliced mRNA due to the skipping of the preceding exon as well as both preceding and following exon.

昨年スプライシング異常と思われる2症例を本学会で報告したが，今回はこれらの患者について，さらにmRNAレベルでの解析を行った。まず患者末梢白血球よりmRNAを抽出し，逆転写酵素を用いてcDNAを作製した。このcDNAを鋳型にnested PCR法を用いて変異部近傍領域を増幅し，塩基配列決定を行った。症例1ではエクソン内のcryptical siteを利用し，異常なスプライシングをおこしていることがわかった。一方，症例2ではスプライシング異常を認めなかった。そのため症例2についてはさらに培養皮膚線維芽細胞のmRNAを解析した。線維芽細胞は末梢白血球と異なり，変異部に隣接するエクソンのスキッピングであることがわかった。mRNAの発現には組織特異性があると考えられた。

Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto 860 (T. M., R. H., K. K., S . K., F. E., 1. M. ); Clinical Laboratory of Kumamoto City Medical Association, Kurnamoro 860 (E. K. ) ; Department of Pediatrics, Sapporo Medical College, Sapporo 060 (K. 0.); Department of Pediatrics, Toyohashi Municipal Hospital, Toyohhi 440 (Y. S.) ; Department of Obstetrics and Gynecology, Japanese Red Cross Nagoya First Hospitd, Nagoya 467 (K. I. ); Department of Pediatrics, lapan Red Cross Medical Center, Tokyo 1.50 ( H . Y . ) ; Department of Pediatrics, Medical School, Nagoya City University, Nagoya 467 (I. K.); Diagnostic Science Department Shionogi and Co., Ltd., Osaka 566 (S.K.); Human Genome Center, The institute of

VLCAD deficiency is an autosomal recessive disorder caused by a defect of fatty acid oxidation. The phenotype is classified into three clinical forms on the basis of the onset of symptoms: a severe form with neonatal onset; a milder form with childhood onset; and a late-onset form. The neonatal form is the most common, and has a higher mortality rate than the others. We report the case of a newborn infant with VLCAD deficiency who developed ventricular fibrillation, which was successfully treated by intensive care, but who suddenly died after a respiratory syncytial virus infection. Early institution of i.v. glucose treatment and active immunization with vaccine, such as palivizumab (anti-RSV mAb), may be important to reduce the frequency and severity of life-threatening episodes.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out-of-frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in-frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino-mediated exon skipping, which changes out-of-frame deletions to in-frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon-skipping event in cells of DMD patients before the clinical trial.Fibroblasts isolated from four DMD patients were induced to differentiate into the myogenic lineage by infection with Ad.CAGMyoD. The cells were then transfected with two types of morpholino. The exon-skipping event was analyzed on reverse transcription-polymerase chain reaction.Morpholino B30, which is located at the splicing enhancer of exon 51 of the DMD gene, yielded the desired exon 51-skipping event in all deletion patterns of cells tested. Morpholino I25, which is located at the exon donor, induced two different exon-skipping patterns, which are total or partial exon 51-skipping events. According to the sequence analysis, the unexpected unskipped regions were the 95 bp section and the 188 bp section of exon 51, showing that the cryptic splicing donor was newly produced with I25. Unfortunately, these cryptic splicing donors gave rise to out-of-frame patterns. Based on these in vitro results, B30 would presumably be an effective therapy. Interestingly, the cocktail of B30 and I25 appeared to yield a more efficient exon 51-skipping event.An in vitro system was developed that could easily screen the effectiveness of antisense sequences and identify good candidates for therapy with morpholino.

Identification of a single base insertion in the COL4A5 gene in Alport syndrome. We identified a novel mutation in the COL4A5 gene of a Japanese patient with Alport syndrome. A combination of in vitro amplification of the exons with single strand conformation polymorphisms (SSCP) analysis suggested the presence of a mutation in exon 48. Sequencing of the amplified DNA revealed a single base (T) insertion which was between nucleotides T 4750 and G 4751 within the methionine 1516. This mutation caused a shift in the reading frame of nine amino acids and introduced a premature termination signal that would be expected to lack about two-thirds of the noncollagenous (NCI) domain. This mutation may interfere with type IV collagen assembly leading to increased permeability and play a causative role in the glomerular basement membrane abnormality of this patient with typical Alport syndrome. Gene tracking by restriction enzyme NlaIII digestion revealed that the patient's mother is heterozygous whereas the patient's brother and one sister are normal, albeit they have hematuria and proteinuria. Without gene analysis, they would have been misdiagnosed. We propose that the diagnosis of Alport syndrome should be made on the basis of both clinical phenotypes and molecular defects.

Maple syrup urine disease (MSUD) is an autosomal recessive disease caused by a deficiency in subunits of the branched-chain alpha-keto-acid dehydrogenase complex (BCKDH). To characterize the mutations present in five patients with MSUD (four classic and one intermediate), three-step analyses were established: (1), identification of the involved subunit by complementation analysis using three different cell lines derived from homozygotes having E1 alpha, E1 beta or the E2 mutant gene; (2), screening for a mutation site in cDNA of the corresponding subunit by RT-PCR-SSCP and (3), mutant analysis by sequencing the amplified cDNA fragment. Four single-base missense mutations, R115W, Q146K [corrected], A209T and I282T, were detected in the E1 alpha subunit. A single-base missense mutation H156R and three frame-shift mutations to generate stop codons downstream, including an 11-bp deletion of the tandem repeat in exon 1, a single-base (T) deletion and a single-base (G) insertion, were identified in the E1 beta subunit gene. All except one (11-bp deletion in E1 beta (Nobukuni, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y., Endo, F., Yoshioka, A. and Matsuda, I. (1991) J. Clin. Invest. 87, 1862-1866)) were novel mutations. The sites of amino-acid substitution were all conserved in other species. Thus, mutations causing MSUD are heterogenous.

Carbamyl phosphate synthetase I (CPS I; EC6,3,4,16) is an autosomal recessive disorder characterized by hyperammonemia. We studied the molecular bases of CPS I deficiency in a newborn Japanese girl with consanguineous parents. Northern and Western blots revealed a marked decrease in CPS I mRNA and enzyme protein but with a size similar to that of the control, respectively. Sequencing of the patient's cDNA revealed a nine-nucleotide deletion at position 832-840. Sequencing analysis of the genomic DNA revealed a G to C transversion at position 840, the last nucleotide of an exon in the splice donor site. This substitution altered the consensus sequence of the splice donor site and the newly cryptical donor site in the exon caused the 9-bp in-frame deletion. This report seems to be the first complete definition of CPS I deficiency, at the molecular level.

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

Fabry's disease is an X-linked lysosomal storage disorder resulting from alpha-galactosidase A deficiency. Although ischemic stroke is recognized as an important manifestation of Fabry's disease, hemorrhagic stroke is considered to be rare. Here, we report our recent clinical experience with three hemizygous male patients with Fabry's disease who developed cerebral hemorrhage. One patient had classic type Fabry's disease with p.Ala37Val mutation and others had cerebrovascular variant with p.Glu66Gln mutation. Degeneration of the cerebral small arteries secondary to deposition of glycosphingolipids and aging, in addition to hypertension and antiplatelet/anticoagulant agents, are considered to be contributing factors for hemorrhage. Fabry's disease is frequently associated with not only ischemic but also hemorrhagic stroke, especially in elderly patients.