Characterization of the Alu-rich 5'-flanking region of the human prothrombin-encoding gene: identification of a positive cis-acting element that regulates liver-specific expression.

The nt sequence of 6127 bp of sequence upstream of the human prothrombin-encoding gene (F2) has been determined. Since we previously characterized 417 bp of DNA immediately upstream from the transcription start point (tsp), 6544 bp of continuous flanking sequence are known. Eleven Alu repeat sequences present in this region comprise 45% of the sequence; other repetitive sequences were identified by searching GenBank. The tsp was found to be heterogeneous by exon mapping and primer extension analysis. To localize the cis-acting sequences responsible for the liver-specific expression of F2, hybrid cat genes were constructed with various lengths of F2 5'-flanking region cloned upstream from a promoterless cat gene. After transfection into HepG2 and HeLa cells, it was inferred that the region between nt -1101 and -798 was required for synthesis in HepG2 cells; no synthesis was observed using these constructs in HeLa cells. Two sequences for known liver-specific or regulatory cis-acting sequences were identified in this region.

An investigation of optimal gold particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy.

We investigated the optimal gold particle size for use with polarized incident light (epi polarization) microscopy with immunogold immunohistological preparation in both immunogold indirect (IGS) and silver-enhanced immunogold-silver staining (IGSS) techniques. A range of gold particle sizes from 5 nm-40 nm was used along with tissue of known immunoreactivity with a well-characterized primary monoclonal antibody. Checkerboard titrations were carried out for each technique and for each particle size. The preparations were viewed using a standard polarized incident light microscope and assessed in a semi-quantitative manner. Adequate visualization of gold particles was achieved using the indirect staining method only with a particle size of 40 nm. With silver enhancement (IGSS), particles of all sizes were clearly seen. However, 5-nm particles were considered optimal for this method because of reduced background staining, high titration of antisera possible, and crisp localization of the visual signal.

Differential effects of warfarin on the intracellular processing of vitamin K-dependent proteins.

The vitamin K-dependent carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues occurs during the endoplasmic reticulum processing of a limited number of proteins. The fate of the under-gamma-carboxylated proteins during protein processing was studied. When human hepatoma (HepG2) cells were grown in the presence of warfarin, under-gamma-carboxylated prothrombin was secreted into the medium. In contrast, prothrombin secretion from a rat hepatoma (H-35) cell line was blocked by warfarin, and intracellular forms which were retained were degraded. When rat prothrombin (rFII) was stably transfected into warfarin treated HepG2 cells, endogenous human prothrombin (hFII) was secreted in an under-gamma-carboxylated form, while rFII accumulated intracellularly. These data indicate that retention and degradation of under-gamma-carboxylated prothrombin by human hepatocytes is related to a structural difference in rFII and hFII. When rFII and hFII were transfected into a warfarin treated transformed human embryonic kidney cell line (293), both proteins were secreted in an under-gamma-carboxylated form and intracellular retention was not observed. However, the secretion of rFII was greatly diminished. Cellular retention of under-gamma-carboxylated forms is therefore tissue specific, but degradation is not.

The human prothrombin gene: transcriptional regulation in HepG2 cells.

The human prothrombin gene is expressed predominantly in hepatocytes. Previous work indicated that this tissue specificity is transcriptionally regulated. In order to identify the cis-acting regulatory elements in the 5' flanking region of the human prothrombin gene which may direct the expression of prothrombin in hepatocytes, a series of hybrid plasmids were constructed linking portions of the 5' flanking region of the human prothrombin gene to the bacterial chloramphenicol acetyltransferase gene. Expression of these hybrid plasmids was examined in calcium phosphate-mediated transient transfections of HepG2 cells, a human hepatoblastoma cell line which expresses prothrombin, and HeLa cells, an adenocarcinoma cell line which does not express detectable amounts of prothrombin. Both the prothrombin promoter and an upstream regulatory region containing sequence homologous to the hepatocyte nuclear factor 1 (HNF-1) binding site (nucleotides -919 to -790 relative to the prothrombin transcription initiation site) were required for expression in HepG2 cells. The upstream region also exhibited non-tissue-specific enhancer activity. Gel mobility shift assays confirmed cell-type-specific differences in the protein-DNA interactions between proteins in HepG2 or HeLa nuclear extracts and either the promoter region or the upstream regulatory region of the gene.

Polarized incident light microscopical enhancement of immunogold and immunogold-silver preparations: its role in immunohistology.

Polarized incident light microscopy (PILM) is a recognized method of visualization of bound colloidal gold in cytological immunocytochemical preparations. This study investigates its role in the assessment of histological sections using both indirect immunogold and immunogold-silver staining methods. With dark field or bright field illumination this technique was found to be advantageous and allowed easy detection staining at low magnification and accurate detection of low levels of stain product. We advocate this technique as a valuable microscopical enhancement method for immunogold immunohistology.

Structural features of the kringle domain determine the intracellular degradation of under-gamma-carboxylated prothrombin: studies of chimeric rat/human prothrombin.

Vitamin K antagonists such as warfarin inhibit the vitamin K-dependent gamma-glutamyl carboxylation during protein processing and block the secretion of under-gamma-carboxylated prothrombin (FII) in the rat but not in the human or bovine. Under-gamma-carboxylated prothrombin is also secreted from warfarin-treated human (HepG2) cell cultures but is degraded in the endoplasmic reticulum in warfarin-treated rat (H-35) cell cultures. This differential response to warfarin has been shown to be determined by the structural difference in the proteins rather than by the origin of the cell line. When recombinant rat prothrombin (rFII) and human prothrombin (hFII) were expressed in a transformed human kidney cell line (HEK293), secretion of rFII but not hFII was drastically decreased in response to warfarin. To determine the structural signal required for this differential response, chimeric cDNAs with the propeptide/Gla domains, kringle domain, and serine protease domain exchanged between rFII and hFII were generated (FIIRHH and FIIHRR, FIIRRH and FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin-treated HEK293 cells and HepG2 cells. The presence of the hFII kringle domain changed the stability of rFII to that of hFII, and the rFII kringle domain changed the stability of hFII to that of rFII. The kringle domain therefore is critical in determining the metabolic fate of under-gamma-carboxylated prothrombin precursors during processing. Prothrombin contains two kringle structures, and expression of additional rFII/hFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two kringles plays a more important role in the recognition process.

Gilbert syndrome accelerates development of neonatal jaundice.

OBJECTIVE: Gilbert Syndrome (GS), associated with unconjugated hyperbilirubinemia and decreased bilirubin UDP-glucuronosyltransferase activity, is usually diagnosed after puberty. The role of GS in neonatal jaundice is unknown. This study tested the hypothesis that a recently identified molecular marker for GS (a TA insertion in the promoter of UGT1A, the gene encoding bilirubin UDP-glucuronosyltransferase) is associated with neonatal jaundice. STUDY DESIGN: Transcutaneous jaundice index was measured shortly after birth and daily for the first week of life in 151 healthy infants. Genomic DNA was isolated from blood or buccal brushings, and the UGT1A promoter was amplified by the polymerase chain reaction to yield 90 (A[TA]6TAA, normal) or 92 (A[TA]7TAA, GS) base pair products. Statistical analysis used Kruskal-Wallis, Wilcoxon, and Fisher's exact tests. RESULTS: Nineteen (13%) subjects were homozygous for the A(TA)7TAA polymorphism associated with GS. The A(TA)7TAA homozygotes had a greater increase in jaundice index during the first 2 days of life than heterozygotes or A(TA)6TAA homozygotes. CONCLUSION: Although peak jaundice levels did not differ among groups, newborn infants with the molecular marker for GS have an accelerated increase in neonatal jaundice during the first 2 days of life.

Antenatal diagnosis of choledochal cyst.

We report five infants in whom antenatal diagnosis of choledochal cyst was established by ultrasonography, and we review the seven previously reported cases. All but one infant had cystic dilatation of the common bile duct (type 1 cysts), and all infants were diagnosed during the second or third trimester. Eight of 12 infants (67%) developed jaundice in the first few days of life, but only 25% had a palpable abdominal mass. Seven of nine infants (78%) demonstrated complete obstruction of the distal common bile duct on intraoperative cholangiography. Liver histology was available for six patients. Five of six had cirrhosis or fibrosis with bile duct proliferation. All of the infants with fibrosis or cirrhosis had distal common bile duct obstruction. Despite liver biopsy findings of extensive fibrosis plus ascites with failure to thrive in one of our patients, all five patients demonstrated clinical and biochemical improvement following surgical excision and porto- or choledochoenterostomy. All were free of symptoms by 6 months of age. Congenital choledochal cyst should be considered in the differential diagnosis of any sonolucent abdominal mass of the fetus. Neonates with distal common bile duct obstruction and fibrosis in association with presumed choledochal cyst should have prompt surgical exploration, intraoperative cholangiography, and close postoperative follow-up. The long-term outcome with prompt surgical correction is excellent.