Galactose-terminated Glycoproteins Research Articles

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins Arezki Azzi,1,2y Tao Wang,1y Dao-Wei Zhu, Yong-Shui Zou, Wang-Yi Liu, and Sheng-Xiang Lin* 1 Structural Biology Platform, Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes of Biological Sciences (SIBS), Chinese Academy of Sciences, China 2 Laboratory of Oncology and Molecular Endocrinology, CHUL Research Center (CHUQ) and Laval University, Quebec, Canada

Clinical application of TcGSA

Asialoglycoprotein receptor (ASGP-R) is a hepatic cell surface receptor specific for galactose-terminated glycoprotein. The 99mTc-labeled asialoglycoprotein analog, TcGSA (galactosyl-human serum albumin-diethylenetriamine-pentaacetic acid) has been applied to human hepatic receptor imaging. This method is unique and provides information that is totally independent of the ICG test or Child-Turcotte Score. However, simple parameters, HH15 or LHL15 are no better than conventional Child-Turcotte Score. Parameters obtained from kinetic modeling were useful in detecting liver cirrhosis or postoperative liver failure. They provide quantitative data on functioning liver mass. Considering the moderate cost and widespread availability of gamma cameras, a TcGSA test should be performed on patients whose liver function are not adequately estimated by an ICG test or a Child-Turcotte score.

C-src Tyrosine Kinase Is Associated with the Asialoglycoprotein Receptor in Human Hepatoma Cells

The asialoglycoprotein (ASGP) receptor is expressed on hepatocytes and liver-derived cell lines and is responsible for the endocytosis of galactose-terminal glycoproteins via the coated pit pathway. Prior data showed that tyrosine kinase activity plays an important role in this endocytic process, though the critical kinase(s) responsible for this effect are unknown. We have detected a 60-kDa protein which coprecipitates with ASGP receptor in detergent-solubilized lysates of HepG2 cells. This protein autophosphorylates and binds radioactive ATP. It comigrates with authentic pp60 c-src and is recognized by a specific anti-src monoclonal antibody. The kinase associated with the ASGP receptor retains the ability to phosphorylate exogenous substrates on tyrosine. In conclusion, the tyrosine kinase c-src associates with the ASGP receptor, a protein of the coated pit pathway of endocytosis.

Mannose-receptor-mediated clearance of lysosomal α-mannosidase in scavenger endothelium of cod endocardium

Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod ( Gadus morhua) . Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal α-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle. Asialo–orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only. In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12°C in a serum free medium. Cod AEC endocytosed mannose-terminated glycoproteins ( 125I-α-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal α-mannosidase), whereas 125I-asialo–orosomucoid was not recognised. Uptake of radiolabelled mannose-terminated ligands was inhibited 80–100% in the presence of excess amounts of mannan, invertase, d-mannose, l-fucose or EGTA. Our results suggest that the cod endocardial endothelial cells express a specific Ca 2+-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells.

Crystal Structure of the Carbohydrate Recognition Domain of the H1 Subunit of the Asialoglycoprotein Receptor

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes. Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 Å resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not α-helical, while in tetranectin they form an α-helical coiled-coil.

Nodular regenerative hyperplasia of the liver: SPECT evaluation with Tc-99m stannous colloid and Tc-99m neoglycoalbumin

The authors describe a case of nodular regenerative hyperplasia of the liver and its findings on liver SPECT with Tc-99m radiocolloid and Tc-99m neoglycoalbumin. A 22-year-old woman with systemic lupus erythematosus was hospitalized for intractable thrombocytopenia. A diagnostic work-up including CT revealed multiple liver nodules and portal hypertension with splenomegaly, suggesting hypersplenism as a cause of the thrombocytopenia. Liver SPECT with Kupffer cell–oriented Tc-99m stannous colloid and hepatocyte-oriented Tc-99m neoglycoalbumin showed increased uptake in the liver nodules, indicating the presence of Kupffer cells and hepatocytes. The patient underwent splenectomy and wedge liver biopsy. Pathologic analysis of the liver specimen revealed many hyperplastic nodules and sinusoidal congestion without cirrhosis. Based on these histopathologic features, the diagnosis of nodular regenerative hyperplasia of the liver was made. FIGURE 1Fig. 1: (A) CT shows no appreciable liver nodules. Contrast-enhanced liver nodules are demonstrated in (B) the arterial-dominant and (C) the portal-dominant phases of the sequential contrast-enhanced liver CT. (D) CT during arterioportography shows many contrast-enhanced liver nodules. The largest nodule is seen in the left medial segment. These findings indicate that these nodules had arterial and portal blood supplies. Although most nodules were described to be hypodense as seen on plain CT (1), all nodules in this case are isodense.FIGURE 2Fig. 2: Planar (A) anterior and (B) posterior images with Tc-99m stannous colloid show a heterogeneous distribution and focal increased uptake. Splenomegaly with increased splenic uptake is also noted. According to previous reports, patchy liver uptake, focal photopenic areas with focal patchy uptake within the nodule, and photon-deficient defects were described as the findings of nodular regenerative hyperplasia on radiocolloid liver scintigraphy (2,3).FIGURE 3Fig. 3: Transaxial SPECT images with Tc-99m stannous colloid show focal increased uptake in the liver. The obvious focal uptake (arrow) corresponds to the nodule seen in the left medial segment on the contrast-enhanced CT. Because Smith (3) has raised the possibility of this finding, the focal increased uptake could be related to hyperplasia of Kupffer cells and to possible heterogeneous blood flow (4).FIGURE 4Fig. 4: Transaxial SPECT images with Tc-99m neoglycoalbumin, which is a specific ligand for the asialoglycoprotein (galactose-terminated glycoprotein) receptor on the surface of the hepatocyte (5), show focal increased uptake in the same areas as in the Tc-99m stannous colloid SPECT. The obvious focal uptake (arrow) corresponds to the nodule seen in the left medial segment on Tc-99m stannous colloid SPECT. Because the presence of both Kupffer cells and virtually normal hepatocytes in the nodules were shown, these SPECT findings suggested that the hepatic nodules could have a benign character, although it was difficult to recognize small nodules and confluent nodules on the SPECT images. Multiple benign liver nodules associated with systemic lupus erythematosus and portal hypertension raised the possibility of nodular regenerative hyperplasia of the liver. The patient underwent splenectomy to treat the thrombocytopenia, and a wedge liver biopsy was performed. Histopathologic examination of the liver specimen revealed many noncirrhotic hyperplastic liver nodules without perinodular fibrosis, the sizes of which ranged from 1 mm to 2 cm in diameter. Microscopic examination showed that the the nodules consisted of hyperplastic normal hepatocytes. Perivascular fibrosis was also seen. However, fibrous septa or infiltration of the inflammatory cells, which were suggestive of cirrhosis, were not seen. The diagnosis of nodular regenerative hyperplasia of the liver was made based on these histopathologic findings (4).

Quantitative analysis of scatter- and attenuation-compensated dynamic single-photon emission tomography for functional hepatic imaging with a receptor-binding radiopharmaceutical.

A new method for quantitative liver study was developed using the tracer technetium-99m diethylene triamine penta-acetic acid-galactosyl human serum albumin (99mTc-GSA), an analog ligand of the asialoglycoprotein receptor, which is a hepatocyte surface receptor specific for galactose-terminated glycoproteins. For quantitative dynamic single-photon emission tomographic (SPET) studies, attenuation compensation using transmission computed tomography (TCT) and the triple energy window (TEW) scatter compensation method were evaluated. As the TCT source, we used an uncollimated multi-tube source with the TEW scatter compensation method. To verify the accuracy of cross-calibrated SPET values as compared with measured radioactivities, we performed SPET of a cylindrical water pool phantom which contains seven hot rods filled with different concentrations of 99mTc activities, simulating the scan conditions in human studies. The results of the phantom studies showed good linearity and accuracy of the SPET values, with R2=0.993 and a regression line of y=0.941x+5.48. From the analysis of a kinetic model based on a one-compartment model, focussing on the initial stage of several minutes after 99mTc-GSA injection and taking the physiological expression presented in a three-compartment analysis into account, we introduced the Rutland equation (Patlak plot) in the 99mTc-GSA study by which the overall and regional effective hepatic blood flow (EHBF) and hepatic blood pool volume were determined. Preliminary clinical evaluations were performed for four normal male subjects (23-35 years of age) and one patient. Forty sequential 30-s dynamic SPET acquisitions were obtained for a period of 20 min following the intravenous injection of 99mTc-GSA with venous blood sampling at 10 min. After scatter compensation, the SPET images were reconstructed with attenuation compensation using an attenuation map obtained from TCT. The average normal value for the total EHBF was 468+/-83 ml/min and that for the hepatic blood pool volume, 777+/-123 ml. Functional images of the distribution of regional values of EHBF (ml/min/voxel) and hepatic blood pool volume (ml/voxel) were also generated corresponding to the original SPET images. The EHBF images showed regional liver function, higher in the right lobe than the left lobe in the normal cases, and the heptic blood pool volume images showed the distribution of intensified high values along major vascular structures. Receptor imaging with 99mTc-GSA using the Rutland method and dynamic SPET with scatter and attenuation compensation is an effective technique that allows the evaluation of total and regional hepatic functional parameters (EHBF, hepatic blood pool) in vivo.

The Major Subunit of the Asialoglycoprotein Receptor Is Expressed on the Hepatocellular Surface in Mice Lacking the Minor Receptor Subunit

The mammalian asialoglycoprotein receptor (ASGPR) is located on the sinusoidal membrane of hepatocytes where it binds and endocytoses galactose-terminated glycoproteins (asialoglycoproteins). ASGPR is composed of two highly homologous subunits, termed hepatic lectin 1 and 2. Despite numerous studies the contribution of both subunits to biosynthesis and functional activity of ASGPR in vivo has remained controversial. Mice lacking the murine hepatic lectin (MHL)-2 subunit are viable and fertile without obvious phenotypic abnormalities. In the absence of MHL-2, knockout mice express MHL-1 protein at reduced levels. Here, we examine the intracellular fate and function of this remaining subunit. The results show that MHL-1 reaches the hepatocellular surface in knockout mice but is unable to effectively remove any one of three different radiolabeled ligands within 30 min. A small but detectable residual ligand clearance in knockout mice at 4 h is apparently not mediated by remaining MHL-1. Serum concentrations of galactose-terminating glycoproteins are not elevated in these ASGPR-deficient mice. However, competitive in vitro degradation experiments suggest that other endogenous ASGPR ligands, the nature of which remain to be determined, accumulate in serum of knockout animals.

In vitro fusion of endocytic vesicles: effects of reagents that alter endosomal pH.

Ricin, a plant toxin that binds to galactose-terminated glycoproteins and glycolipids on the cell surface, is internalized into endosomes before reaching the cytosol where it exerts its toxic activity. Fusion of early endosomes containing ricin or transferrin was demonstrated by using postnuclear supernatant fractions from K-562 cells. For both ligands, fusion depended on time, temperature, and ATP and was blocked by preincubation with N-ethylmaleimide. Some reagents that increase endosomal pH, the ionophores monensin and nigericin and the weak base chloroquine, stimulated the rate of fusion. However, bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, did not alter the rate of fusion. Moreover, it reduced or eliminated stimulation caused by monensin, nigericin, or chloroquine. Thus, the increased rate of fusion did not correlate with the higher lumenal pH of the endosome. The results suggest instead that fusion was stimulated by reagents that promoted accumulation of cations within the vesicles.

Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNAin vitro

We have shown that antisense oligonucleotides can be targeted to hepatitis B virus (HBV)-infected cells, resulting in specific inhibition of viral protein synthesis and replication in vitro. The targeting system was based on the internalization of DNA complexes by highly selective receptors for galactose-terminal glycoproteins, asialoglycoproteins, on the surface of hepatocytes. Our objective in this study was to determine whether antisense DNA could be targeted to hepatocytes to prevent subsequent infection by HBV. A 21-mer phosphorothioate-linked oligo DNA complementary to the HBV polyadenylation signal and 5'-upstream sequences was complexed to a targetable DNA carrier consisting of asialoglycoprotein coupled to polylysine. Pretreatment of Huh7, asialoglycoprotein receptor (+) cells, with antisense complexes before lipofection with an HBV-plasmid at a level of 6.5 x 10(6) copies of plasmid per cell inhibited the amount of newly synthesized, core-associated viral DNA in Huh7 cells to undetectable levels, less than 0.1 pg, as assessed by quantitative polymerase chain reaction (PCR). Hepatitis B viral RNA transcripts were decreased by 60% compared with controls as detected by RNase protection assays, and HBV surface antigen (HBsAg) accumulation was inhibited by 97%. The inhibition lasted for 6 days and was dose dependent. Controls consisting of antisense alone and a random oligo complex showed no significant effect on any of the parameters under identical conditions. We conclude that preexposure of cells to targeted complexed antisense DNA can substantially block viral gene expression and viral replication after transfection of HBV DNA.

Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNA in vitro

We have shown that antisense oligonucleotides can be targeted to hepatitis B virus (HBV)-infected cells, resulting in specific inhibition of viral protein synthesis and replication in vitro. The targeting system was based on the internalization of DNA complexes by highly selective receptors for galactose-terminal glycoproteins, asialoglycoproteins, on the surface of hepatocytes. Our objective in this study was to determine whether antisense DNA could be targeted to hepatocytes to prevent subsequent infection by HBV. A 21-mer phosphorothioate-linked oligo DNA complementary to the HBV polyadenylation signal and 5'-upstream sequences was complexed to a targetable DNA carrier consisting of asialoglycoprotein coupled to polylysine. Pretreatment of Huh7, asialoglycoprotein receptor (+) cells, with antisense complexes before lipofection with an HBV-plasmid at a level of 6.5 x 106 copies of plasmid per cell inhibited the amount of newly synthesized, core-associated viral DNA in Huh7 cells to undetectable levels, less than 0.1 pg, as assessed by quantitative polymerase chain reaction (PCR). Hepatitis B viral RNA transcripts were decreased by 60% compared with controls as detected by RNase protection assays, and HBV surface antigen (HBsAg) accumulation was inhibited by 97%. The inhibition lasted for 6 days and was dose dependent. Controls consisting of antisense alone and a random oligo complex showed no significant effect on any of the parameters under identical conditions. We conclude that preexposure of cells to targeted complexed antisense DNA can substantially block viral gene expression and viral replication after transfection of HBV DNA. (Hepatology 1996 Jun;23(6):1297-303)

Correction

We have shown that antisense oligonucleotides can be targeted to hepatitis B virus (HBV)-infected cells, resulting in specific inhibition of viral protein synthesis and replication in vitro. The targeting system was based on the internalization of DNA complexes by highly selective receptors for galactose-terminal glycoproteins, asialoglycoproteins, on the surface of hepatocytes. Our objective in this study was to determine whether antisense DNA could be targeted to hepatocytes to prevent subsequent infection by HBV. A 21-mer phosphorothioate-linked oligo DNA complementary to the HBV polyadenylation signal and 5'-upstream sequences was complexed to a targetable DNA carrier consisting of asialoglycoprotein coupled to polylysine. Pretreatment of Huh7, asialoglycoprotein receptor (+) cells, with antisense complexes before lipofection with an HBV-plasmid at a level of 6.5 x 106 copies of plasmid per cell inhibited the amount of newly synthesized, core-associated viral DNA in Huh7 cells to undetectable levels, less than 0.1 pg, as assessed by quantitative polymerase chain reaction (PCR). Hepatitis B viral RNA transcripts were decreased by 60% compared with controls as detected by RNase protection assays, and HBV surface antigen (HBsAg) accumulation was inhibited by 97%. The inhibition lasted for 6 days and was dose dependent. Controls consisting of antisense alone and a random oligo complex showed no significant effect on any of the parameters under identical conditions. We conclude that preexposure of cells to targeted complexed antisense DNA can substantially block viral gene expression and viral replication after transfection of HBV DNA. (Hepatology 1996 Jun;23(6):1297-303)

Estimation of remnant liver function before hepatectomy by means of technetium-99m-diethylenetriamine-pentaacetic acid galactosyl human albumin.

To improve the results of hepatectomy in cirrhotic patients, the likely reserve function of the liver was evaluated before surgery. Asialoglycoprotein receptor (ASGP-R) is a hepatic cell surface receptor specific for galactose-terminated glycoproteins. Technetium-99m-diethylene triamine pentaacetic acid-galactosyl human serum albumin (99mTc-GSA) is a newly developed analog ligand to ASPG-R. The probable functional reserve of the remnant liver after hepatectomy was estimated preoperatively as the hepatic binding protein (HBP) concentration specific for ASGP-R on the hepatocellular membrane of the remnant liver. This estimate was based on the effective liver volume rate, obtained by the uptake of 99mTc-GSA. In all, 3 normal volunteers, 3 patients with chronic hepatitis (CH), 9 patients with liver cirrhosis (LC), 2 patients with hepatic cystadenoma, 3 patients with hepatocellular carcinoma (HCC) associated with CH, and 21 HCC patients with LC were studied. The mean value +/- SD obtained for HBP in normal volunteers (three cases) and in patients with mild (four cases), moderate (two cases), and severe liver damage (five cases) were 0.74 +/- 0.03 microM, 0.43 +/- 0.042 microM, 0.31 +/- 0.05 microM, and 0.20 +/- 0.05 microM, respectively. Most of the cases in which the preoperative HBP of the remnant liver was above 0.22 microM had a good postoperative course irrespective of the type of hepatectomy. On the other hand, in subjects with a remnant liver HBP of between 0.22 and 0.11 microM, postoperative severe liver dysfunction occurred in about 50% of cases. In all cases with a remnant liver HBP below 0.1 microM, the prognosis was very poor, indicating that hepatectomy should be avoided. The HBP concentration detected by the 99mTc-GSA study is a very sensitive indicator of changes in the hepatic functional reserve, and the HBP value for the functional reserve of the remnant liver is extremely useful for estimating the liver function before and after hepatectomy.

Quantitative assessment of hepatocellular function throughin vivo radioreceptor imaging with technetium 99m galactosyl human serum albumin

Technetium 99m diethylenetriaminepentaacetic acid-galactosyl human serum albumin is a newly developed analog ligand to asialoglycoprotein receptor, which is a hepatic cell surface receptor specific for galactose-terminated glycoproteins. Hepatic functional imaging, which yields estimates of asialoglycoprotein receptor concentration, was performed after intravenous injection of 3 mg technetium 99m diethylenetriaminepentaacetic acid-galactosyl human serum albumin. A total of 75 human subjects were studied: 6 controls without liver diseases, 51 patients with chronic liver diseases and 18 patients with acute liver diseases. In chronic liver disease the asialoglycoprotein receptor concentration significantly correlated with the clinical severity based on the criteria of the Liver Cancer Study Group of Japan (rs = -0.890, p = 0.0001). Good correlations between the asialoglycoprotein receptor concentration and conventional liver function tests were also observed. In acute liver disease the asialoglycoprotein receptor concentration correlated well with the normotest (r = 0.796, p = 0.0001), prothrombin time (r = 0.701, p = 0.0002) and total serum bilirubin (r = -0.642, p = 0.0007). We conclude that the parameter, asialoglycoprotein receptor concentration, obtained from the kinetic analysis of technetium 99m diethylenetriaminepentaacetic acid-galactosyl human serum albumin time-activity data, is a sensitive measure of functioning hepatocyte mass in acute and chronic liver disease.

Determination of mouse major asialoglycoprotein receptor cDNA sequence

Hepatic lectins (asialoglycoprotein receptors) specifically recognize galactose-terminated glycoproteins and mediate endocytosis of these molecules. We now report the cloning and sequence of a cDNA encoding murine asialoglycoprotein receptor. It shows high homology with rat and human major receptor forms designated RHL-1 and HHL-1, respectively. They have many conserved regions, such as a transmembrane region, carbohydrate additional region and carbohydrate related region. According to the homology analysis, we concluded that the clone encodes the mouse major asialoglycoprotein receptor (MHL-1).

In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells.

The plasma clearance and the interaction of high (HMW) and low (LMW) molecular weight single-chain urokinase-type plasminogen activator (scu-PA) with rat liver cells was determined. 125I-Labeled HMW- and LMW-scu-PA were rapidly cleared from plasma with a half-life of 0.45 min and a maximal liver uptake of 55% of the injected dose. Liver uptake of scu-PA was mediated by parenchymal cells. Excess of unlabeled HMW-scu-PA reduced the liver uptake of 125I-HMW-scu-PA strongly. In vivo liver degradation of scu-PA was reduced by inhibitors of the lysosomal pathway. A high affinity binding site (Kd 45 nM, Bmax 1.7 pmol/mg cell protein) for both HMW- and LMW-scu-PA was determined on isolated parenchymal liver cells. Cross-competition binding studies showed that LMW- and HMW-scu-PA bind to the same site. Tissue-type plasminogen activator, mannose- or galactose-terminated glycoproteins did not affect the scu-PA binding to parenchymal liver cells. It is concluded that LMW- and HMW-scu-PA are taken up in the liver by a common, newly identified recognition site on parenchymal liver cells and are subsequently degraded in the lysosomes. It is suggested that this site is important for the regulation of the turnover of scu-PA.

Functional hepatic imaging with receptor-binding radiopharmaceutical: clinical potential as a measure of functioning hepatocyte mass.

Asialoglycoprotein receptor (ASGP-R) is a hepatic cell surface receptor specific for galactose-terminated glycoproteins. Technetium-99m diethylenetriaminepentaacetic acid-galactosyl human serum albumin (TcGSA) is a newly developed analog ligand to ASGP-R. Fourteen human subjects were studied: three normal volunteers, one with chronic hepatitis, 6 with liver cirrhosis, and 4 with hepatocellular carcinoma associated with liver cirrhosis. The receptor index parameter (LHL15), was obtained from the liver and heart time-activity data as the ratio of radioactivity of the liver over that of the liver plus heart at 15 min after intravenous injection of 1 mg of TcGSA. Means +/- standard deviations of LHL15 in normal volunteers (3 cases), patients with mild (4 cases), moderate (2 cases), and severe liver damage (5 cases) were 0.933 +/- 0.006, 0.789 +/- 0.045, 0.723 +/- 0.033, and 0.488 +/- 0.094, respectively. The difference between the mean values of each group was statistically significant (P less than 0.05). LHL15 correlated well with classical indicators for hepatic functional capacity such as serum albumin level, serum bilirubin level, prothrombin time, ICG R15 or Child-Turcotte criteria score. Our preliminary experiences of high correlations of TcGSA functional imaging data with clinical data suggest that the dynamic data using this receptor-binding radiopharmaceutical provides invaluable information with regard to liver function, and thus, the TcGSA study is potentially a noninvasive practical tool to measure functioning hepatocyte mass.

Delivery systems for gene therapy

Introduction of foreign genes into mammalian cells in vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cells in vivo, are technically difficult or lack cell specificity. We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstrated in vitro using asialoglycoprotein receptor (+) and (-) cell lines. Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy. The data indicate that a targetable gene delivery system can permit in vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.

Reduced Levels of Galactose-Terminated Glycoproteins in Rat Serum during Perinatal Development

Asialoglycoprotein receptor is an abundant protein localized in the sinusoidal domain of the hepatocyte plasma membrane. Its principle function is the clearance of serum glycoproteins which include several acute phase reactant proteins that have lost their terminal sialic acid residue. It has been reported that asialoglycoprotein receptor is nearly absent in mammalian fetal liver but rises to adult levels during the early postpartum period. The hypothesis to be tested was to determine whether defective glycoproteins, those lacking terminal sialic acid residues, are accumulated in fetal serum as a result of reduced receptor content. It was found that low levels of asialoglycoprotein receptor in 18-day-old fetal liver correlated to a threefold higher level of total asialoglycoproteins in fetal serum than adult serum as determined by a modified Western blot protocol employing radioiodinated ricinus communis agglutinin, a galactose-binding lectin. As hepatic asialoglycoprotein receptor accumulated at the time of birth, the elevated level of serum asialoglycoproteins decreased to adult values in an opposite manner. This study indicates that the hepatic asialoglycoprotein receptor plays an important role in reducing the amount of circulating defective glycoproteins at the time of birth.

Mouse asialoglycoprotein receptor cDNA sequence: Conservation of receptor genes during mammalian evolution

The asialoglycoprotein receptor internalizes galactose-terminated glycoproteins into mammalian hepatocytes for degradation in lysosomes. We report the cloning and sequencing of one murine asialoglycoprotein receptor cDNA which exhibits homology with rat and human receptor forms. Conserved regions may correlate with functional domains. The membrane-bound M (mouse) HL polypeptide does not contain a cleavable N-terminal signal sequence and is probably anchored to the membrane via an internal insertion sequence.