Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis.

BACKGROUND & AIMS: Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS: BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS: Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS: The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: role of divalent metals in the dimerization and phosphorylation of enzyme I.

The function of divalent metal ions (Mg2+ and Mn2+) in the dimerization and phosphorylation of enzyme I has been studied. Only a dimeric form of the enzyme can be phosphorylated [Misset, O., Brouwer, M., & Robillard, G. T. (1980) Biochemistry 19, 883--890; Hoving, H., Lolkema, J. S., & Robillard, G. T. (1981) Biochemistry 20, 87--93]. Kinetic studies of phosphoryl-group exchange between phosphoenolpyruvate and pyruvate and measurements of initial enzyme I phosphorylation rates revealed that a divalent metal ion must be bound to the enzyme to render the dimer active. Mn2+ binding experiments by means of electron paramagnetic resonance showed binding of at least one Mn2+ per unphosphorylated dimer with a binding constant comparable to the activation constant found in the kinetic studies and a 10-fold tighter binding of only one Mn2+ per phosphorylated dimer. Gel filtration experiments provided evidence that divalent metals produce about a 10-fold stabilization of the dimers, in addition to their effect on the specific dimer activity. The stability of the dimer was also strongly dependent on salts such as LiCl, NaCl, KCl, and a series of tetraalkylammonium chlorides. The relative effects of these salts suggest that hydrophobic interactions possibly play a significant role in enzyme I dimerization.

Group-specific auto-immune antibodies directed to granulocytes as a cause of chronic benign neutropenia in infants.

Chronic benign neutropenia of infancy is a disease which develops a few months after birth and which is characterized by a severe selective neutropenia, accompanied by benign but persisting infections. The cause of the disease is still unknown. The sera from 5 such patients were tested for the presence of granulocyte antibodies as a possible cause of the disease. For the detection of these antibodies immunofluorescence, agglutination and granulocytotoxicity were used. All sera contained antibodies which reacted both with the neutrophils of one or both parents of the patient and a part of a panel of unrelated donors. From the reaction patterns against the panel we could identify the specificity of three sera. Two sera were directed to the neutrophil-specific antigen NA2, and the third one reacted with a hitherto not yet recognized neutrophil-specific alloantigen which we called NE1. In 4 patients we could confirm the autoimmune character of the disease by demonstrating the antibody on the patients' own granulocytes. These results suggest that autoimmunity may be the cause of many cases of benign infantile neutropenia.