Notch signaling in normal and disease States: possible therapies related to glycosylation.

The Notch signaling pathway is involved in a wide variety of highly conserved developmental processes in mammals. Importantly, mutations of the Notch protein and components of its signaling pathway have been implicated in an array of human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL, Alagille Syndrome, Spondylocostal Dysostosis). In mammals, Notch becomes activated upon binding of its extracellular domain to ligands (Delta and Jagged/Serrate) that are present on the surface of apposed cells. The extracellular domain of Notch contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats. Many of these EGF repeats are modified at evolutionarily-conserved consensus sites by an unusual form of O-glycosylation called O-fucose. Work from several groups indicates that O-fucosylation plays an important role in ligand mediated Notch signaling. Recent evidence also suggests that the enzyme responsible for addition of O-fucose to Notch, protein O-fucosyltransferase-1 (POFUT1), may serve a quality control function in the endoplasmic reticulum. Additionally, some of the O-fucose moieties are further elongated by the action of members of the Fringe family of beta-1,3-N-acetylglucosaminyltransferases. The alteration in O-fucose saccharide structure caused by Fringe modulates the response of Notch to its ligands. Thus, glycosylation serves an important role in regulating Notch activity. This review focuses on the role of glycosylation in the normal functioning of the Notch pathway. As well, potential roles for glycosylation in Notch-related human diseases, and possible roles for therapeutic targeting of POFUT1 and Fringe in Notch-related human diseases, are discussed.

Hypertransaminasemia in childhood as a marker of genetic liver disorders.

BACKGROUND: The widespread use of routine biochemical assays has led to increased incidental findings of hypertransaminasemia. We aimed to evaluate the prevalence of different causes of raised aminotransferase levels in children referred to a university department of pediatrics. METHODS: We investigated 425 consecutive children (age range, 1-18 years) with isolated hypertransaminasemia. All patients had raised aminotransferase levels on at least two occasions in the last month before observation. Cases due to major hepatotropic viruses were excluded. RESULTS: During the first 6 months of observation, 259 children showed normalized liver enzymes. Among the remaining 166 patients with hypertransaminasemia lasting for more than 6 months, 75 had obesity-related liver disease; 51, genetic disorders; 7, autoimmune hepatitis; 5, cholelithiasis; 3, choledochal cyst; and 3, celiac disease. Among the 51 children with genetic disorders, 18 had Wilson disease; 14, muscular dystrophy; 4, alpha-1-antitrypsin deficiency; 4, Alagille syndrome; 4, hereditary fructose intolerance; 3, glycogen storage disease (glycogenosis IX); 2, ornithine transcarbamylase deficiency; and 2, Shwachman's syndrome. In 22 children, the hypertransaminasemia persisted for more than 6 months in the absence of a known cause. CONCLUSIONS: Genetic disease accounted for 12% of cases of isolated hypertransaminasemia observed in a tertiary pediatric department. A high level of suspicion is desirable for an early diagnosis of these disorders, which may present with isolated hypertransaminasemia and absence of typical clinical signs.

Lipoprotein pattern and plasma lecithin cholesterol acyl transferase activity in children with Alagille syndrome.

Alagille syndrome is frequently associated with hyperlipidemia and xanthoma. The aim of the study was to assess the lipid profile (plasma lipoproteins, apolipoproteins (apo)) and lecithin cholesterol acyl transferase (LCAT) activity, with and without treatment with cholestyramine in Alagille syndrome. Five children (mean age = 6 +/- 4 years) with Alagille syndrome were studied at two different times while receiving no treatment, and while receiving cholestyramine. They were compared with 12 normal controls, who were not different from patients for age and sex. In Alagille syndrome, total serum cholesterol, triglycerides and phospholipids were elevated compared with the controls (P < 0.008). VLDL-cholesterol, LDL-cholesterol, HDL-triglycerides, LDL-triglycerides and VLDL-phospholipids were higher, whereas HDL-cholesterol was lower than controls (P < 0.03). Apo B, CIII, E and lipoprotein particles Lp AI were higher (P < 0.001), whereas Lp AI:AII was lower than controls (P < 0.03). Lipoprotein-X was present in the 5 children with Alagille syndrome and explained in part the elevation of plasma cholesterol, phospholipids, and apo CIII. LCAT activity was decreased (P < 0.01) and might cause some abnormalities of HDL with lower cholesterol, higher triglycerides, apo E and apo CIII contents than controls, and abnormalities of VLDL and LDL with higher cholesterol, triglycerides, phospholipids and apo B contents than controls. Some of the risk factors of atherosclerosis were found in Alagille syndrome, namely high levels of plasma cholesterol, LDL cholesterol, apo B, apo B/apo AI. Treatment with cholestyramine resulted in a few modifications to the lipid profile, while lipoprotein-X and the decrease of LCAT activity persisted.

Ozzy, a Jag1 vestibular mouse mutant, displays characteristics of Alagille syndrome.

The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T-->A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.

Fecal elastase 1, serum amylase and lipase levels in children with cholestasis.

BACKGROUND/AIM: The pancreatic functions of children with cholestatic liver diseases were unclear. Due to anatomic vicinity and common ontogenic origin, hepatobiliary disorders of infancy may also affect pancreatic function. The aim of the study was to evaluate the exocrine pancreatic function and common pancreatic function tests in children with cholestatic disorders. METHODS: In 40 children with cholestasis, fecal elastase 1 (FE1) concentrations were measured. Serum amylase and lipase values were tested. The diagnoses included 32 patients with extrahepatic cholestasis (biliary atresia (BA) and choledochal cyst), and 8 patients with intrahepatic cholestasis (progressive familial intrahepatic cholestasis and Alagille syndrome). None had renal insufficiency or clinical symptoms/signs of acute pancreatitis. RESULTS: All the patients had normal FE1 (>200 microg/g). Nineteen percent (7/37) had elevated serum amylase levels (>100 U/l). Thirty-two percent (12/37) had elevated serum lipase levels above the normal (>120 U/l). Seventy-three percent (8/11) of BA patients with bilirubin >2 mg/dl had elevated serum lipase levels compared to 18% (3/17) with bilirubin < or = 2 mg/dl (p = 0.0036). None had detectable pancreatic abnormality on ultrasonography and magnetic resonance images. CONCLUSIONS: None of the cholestatic children in this study had exocrine pancreatic insufficiency as detected by FE1. Hyperamylasemia and/or hyperlipasemia were frequently found. In children with BA, those with impaired biliary excretion tended to have elevated serum pancreatic enzymes as compared with those who had no jaundice. A decreased hepatic metabolism may be the cause.

Superoxide dismutase activity in children with chronic liver diseases.

BACKGROUND/AIMS: Liver disease in infancy has multiple etiologies. As reactive oxygen intermediates are involved in several types of tissue damage, we have investigated whether different forms of liver disease in infancy are associated with increased free radical generation, using an indirect approach in which superoxide dismutase (a free radical scavenger) activity is determined in the liver tissue. METHODS: A total of 48 liver biopsies performed at diagnosis were evaluated retrospectively. Nine infants had biliary atresia, eight Alagille syndrome, seven alantitrypsin deficiency and 12 cryptogenic hepatitis. As controls we studied 12 biopsies with normal histology obtained from seven children with portal vein thrombosis and five children who underwent biopsy for management reason but had no liver disease. Superoxide dismutase activity in liver biopsy specimens was measured using the cytochrome C method by spectrophotometry and expressed as U SOD/mg protein. RESULTS: Superoxide dismutase activity was significantly increased in biliary atresia (1.25 +/- 0.56 U SOD/mg protein, p<0.0001) and Alagille syndrome (1.31 +/- 0.56 U SOD/mg protein, p<0.0001) as compared with al-antitrypsin deficiency (0.75 +/- 0.3 U SOD/mg protein), neonatal hepatitis (0.72 +/- 0.37 U. SOD/mg protein) and normal controls (0.4 +/- 0.7 U. SOD/mg protein). The highest level of SOD activity was found, however, in control children with portal vein thrombosis (2.09 +/- 0.96 U SOD/mg protein; p<0.0001 as compared to the other groups). CONCLUSION: Superoxide dismutase, a key enzyme in free radical protection, is increased significantly in the liver tissue of infants with cholestatic liver disease due to bile duct damage and in children with portal vein thrombosis, suggesting that products of free radical reactions are involved in the pathogenesis of these disorders.

Further studies on aminopeptidase-M in blood in children with cholestatic liver diseases and viral hepatitis.

Aim of this study was to determine and further characterize the serum aminopeptidase-M in children with liver diseases. Based on our new assay, we have shown two fractions of the enzyme. Activity of the first fraction is expressed in undiluted serum at pH adjusted from 8.5 (pH of storaged serum) to 7.4. Activity of the second fraction (cryptic activity) appears in the serum (pH 7.4) as a result of dilution and/or addition of aniline naphthalene sulfonic acid. In children with Alagille syndrome, extrahepatic biliary duct atresia, Byler's disease, and acute hepatitis due to hepatitis B virus infection, activities of both fractions are highly elevated as compared to healthy children or those with chronic viral hepatitis. Moreover, serum aminopeptidase-M seems to reflect other aspects of the pathological process than those reflected by the alanine aminotransferase and gamma-glutamyltranspeptidase. Due to increased activity and broad substrate specificity, the enzyme seems to be also a cofactor of cholestasis and hepatitis.

Neutral endopeptidase activity is not elevated in serum in children with cholestatic liver disease: a unique role of aminopeptidase-m in sequential hydrolysis of peptides.

The aim of this study was to determine whether neutral endopeptidase activity is elevated in serum in children with cholestatic liver disease (Alagille syndrome), and whether the enzyme cooperates with the serum aminopeptidase-M in degradation of peptides. Our data suggest that neutral endopeptidase activity remains at a very low level,.undetectable with the assays we have applied, both in the serum from healthy children and those with cholestasis. In contrast, the serum aminopeptidase-M activity is highly increased in cholestasis. We have demonstrated that aminopeptidase-M alone is capable of sequential and complete hydrolysis of enkephalins and low-molecular-weight, nonspecific peptides. The rate of release of free amino acids from both exogenous or endogenous substrate peptides was statistically significantly higher in serum in children with cholestasis compared with healthy children (P < 0.05). Substance P (Ki = 2.5 micromol/liter) and bradykinin (Ki = 27 micromol/liter) were shown to be potent inhibitors of the serum aminopeptidase-M. We postulate that aminopeptidase-M, except when regulating the activity of bioactive peptides, may serve as a scavenger of short, nonspecific peptides in the circulation. Our data seem to provide new insights for further studies on the role of serum peptidases both in physiology and pathophysiology.