Interaction of cholesterol-crystallization-promoting proteins with vesicles.

In this study, the interaction of mucin and concanavalin A-binding proteins isolated from human bile with cholesterol/phospholipid vesicles was investigated. Using resonance energy transfer assays originally developed by Struck, Hoekstra and Pagano [(1981) Biochemistry 20, 4093-4099], no significant protein-induced fusion or aggregation of vesicles was demonstrated. Instead of fusion, these proteins induced destabilization of cholesterol/phospholipid vesicles, as monitored by release of entrapped carboxyfluorescein. A good correlation (rho = 0.81) was obtained between the extent of leakage and the nucleation-promoting activity of the concanavalin A-binding proteins. We conclude that aggregation or fusion of cholesterol/phospholipid vesicles is not an obligatory step in cholesterol crystallization. Biliary protein-induced crystallization seems to be preceded by vesicle disruption.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.

Sequence of exons and the flanking regions of human bilirubin-UDP-glucuronosyltransferase gene complex and identification of a genetic mutation in a patient with Crigler-Najjar syndrome, type I.

Crigler-Najjar syndrome, type I is a heterogeneous disorder that may result from mutations of various regions of the bilirubin-UDP-glucuronosyltransferase gene complex that encodes two bilirubin-UDP-glucuronosyltransferase isoforms and a phenol-UDP-glucuronosyltransferase isoform in the human liver. The two bilirubin-UDP-glucuronosyltransferase messenger RNAs and the phenol-UDP-glucuronosyltransferase messenger RNA have identical 3' regions derived from four consecutive exons. The 5' region of each messenger RNA is unique and is derived from distinct single exons. By screening a human genomic library with probes corresponding to various regions of the messenger RNAs, we have isolated five cosmid clones containing overlapping segments of this large gene complex that spans at least 84 kb of the human genome. To facilitate the amplification of each exon by polymerase chain reaction and their adjacent splice junctions, we have delineated the intron-exon boundaries of the four common region exons and the two single exons that encode the unique regions of the two bilirubin-UDP-glucuronosyltransferase isoforms and have described sequences of the regions flanking each exon. All exons encoding the two bilirubin-UDP-glucuronosyltransferase isoforms and their splice junctions were amplified from the DNA of two control subjects and a Crigler-Najjar syndrome, type I patient. The DNA from the Crigler-Najjar syndrome, type I patient revealed a point mutation in exon 3 (a common region exon) resulting in a stop codon. RNA blot showed that the two bilirubin-UDP-glucuronosyltransferase messenger RNAs in the liver of the Crigler-Najjar syndrome, type I patient were of normal length but were reduced in concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.

Use of 1H-NMR to determine the distribution of lecithin between the micellar and vesicular phases in model bile.

Biliary cholesterol/phospholipid vesicles play an important role in the pathogenesis of gallstone disease. A prerequisite for the study of the lipid composition and stability of these vesicles is a reliable method to quantify the amount of vesicular lipid. In the present report we show that NMR can be used to determine the distribution of biliary lecithin between the micellar and vesicular phases. The relatively large size of the vesicles leads to such a broadening of the lipid resonances that they are no longer visible in high resolution 1H-NMR spectra. Since micelles are much smaller, lipid present in the micellar phase does give rise to sharp peaks in 1H-NMR spectra. Micellar lecithin can easily be quantified in these spectra. The resonances of cholesterol are masked by the closely related bile acid that is present in a much higher concentration. By determining the difference between chemically and NMR estimated lecithin, the distribution of this phospholipid between the micellar phase and vesicular phase can be assessed. We have compared the results of NMR with gel permeation and density gradient ultracentrifugation. Using standard fractionation conditions, both gel permeation and density gradient ultracentrifugation lead to an underestimation of vesicular lecithin, the difference being minor at relatively high total lipid concentrations (10 g/dl) but large in diluted model bile. We conclude that 1H-NMR can be used to determine the distribution of lecithin in model bile.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical analysis of uridine diphosphate-glucuronosyltransferase in four patients with the Crigler-Najjar syndrome type I.

The functional heterogeneity of uridine diphosphate-glucuronosyltransferase (UDPGT) and its deficiency in human liver were investigated. The monoclonal antibody (MAb) WP1, which inhibits bilirubin and phenol-glucuronidating activity, was used to immunopurify UDPGTs from human liver. Purified UDPGTs were injected into mice to obtain new MAbs. Immunoblotting of microsomes with MAb HEB7 revealed at least three polypeptides in liver (56, 54, and 53 kD) and one in kidney (54 kD). In liver microsomes from four patients (A, B, C, and D) with Crigler-Najjar syndrome type I (CN type I), UDPGT activity towards bilirubin was undetectable (A, B, C, and D) and activity towards phenolic compounds and 5-hydroxytryptamine either reduced (A and B) or normal (C and D). UDPGT activity toward steroids was normal. Immunoblot studies revealed that the monoclonal antibody WP1 recognized two polypeptides (56 and 54 kD) in liver microsomes from patient A and none in patient B. With HEB7 no immunoreactive polypeptides were seen in these two patients. Patient C showed a normal banding pattern and in patient D only the 53-kD band showed decreased intensity. These findings suggest considerable heterogeneity with regard to the expression of UDPGT isoenzymes among CN type I patients.

Normal platelet aggregation in myotonic dystrophy.

In 18 patients with myotonic dystrophy, spontaneous aggregation and platelet aggregation induced by thrombin, adenosine diphosphate and epinephrine were compared with normal aggregation patterns. In 17 of the 18 patients the results were not significantly different from normal. In 1 patient spontaneous aggregation and hypersensitive platelets were found. These results are in disagreement with earlier reports on a specific hypersensitivity to epinephrine in myotonic dystrophy. Neither the clinical data (myotonia, paresis) nor the laboratory data (creatine kinase, myoglobin, immunoglobulin G) were correlated with the platelet aggregations.