Cell Surface Of Hepatocytes Research Articles

Prediction of Total Hepatic Clearance by Combining Metabolism, Transport, and Permeability Data in the In Vitro–In Vivo Extrapolation Methods: Emphasis on an Apparent Fraction Unbound in Liver for Drugs

Poulin and coworkers recently proposed in three manuscripts (2012. J Pharm Sci 101:838-851; 2012. J Pharm Sci 101:4308-4326; and 2013. J Pharm Sci 102:in press) a novel in vitro-to-in vivo extrapolation (IVIVE) method for clearance (CL) involving estimation of apparent fraction unbound in liver (fu(liver)) based on albumin-facilitated hepatic uptake and correction of unbound drug according to the ionization fraction either side of the plasma membrane. This novel IVIVE method has improved the prediction accuracy, and, hence, reduced the bias in predicting hepatic metabolic CL referring to plasma kinetics of several acidic/neutral drugs in preclinical species and humans either based on microsomal or hepatocyte data. So far, the prediction performance of this novel IVIVE method has been assessed for metabolic CL only. Because CL might also be governed by transporters effect and/or permeation limitation in addition to metabolism, an extension of the proposed IVIVE method from metabolic data to transport and permeability data was necessary. Therefore, it was assumed that the concept should also work for multiple CL processes predictions because it is applicable as long as the drug gets to the hepatocyte cell surface. In this study, the proposed IVIVE method was assessed using a large database of predictions of total hepatic CL in rats and humans by combining in vitro hepatocyte data on metabolism, transport, and permeability. The proposed IVIVE method is similarly effective in minimizing average prediction bias and improves accuracy unlike other IVIVE methods. Overall, the present study confirms the utility of the novel IVIVE method for predicting total hepatic CL of drugs under in vivo conditions either by considering metabolism data only or combining metabolism with transporter and permeability data. This study will facilitate the predictions of total hepatic CL in physiologically based pharmacokinetics (PBPK) model.

Sugar functionalised PEGA surfaces support metabolically active hepatocytes

We demonstrate sugar functionalised hydrogel surface coatings which enable the retention of hepatocyte-specific cell function during in vitro culture. The materials were designed to exploit a unique characteristic of hepatocyte biology, with β-galactose moieties displayed to allow cellular adhesion via the specific asialoglycoprotein receptors (ASGP-R) expressed on hepatocyte cell surfaces. Polyethylene glycol acrylamide hydrogel (PEGA) was modified with a galactose containing ligand, lactobionic acid (LA) with D-glucuronic acid (GlcA) used as a non-ASGP-R binding control. FT-IR analysis of the resultant gels revealed that the sugars had been covalently incorporated into the material. Fluorescence labelling was used to quantify the degree of saccharide integration. Cell culture experiments showed that hepatocytes attached preferentially to sugar containing gel coatings, when compared to non-functionalised PEGA controls. It was observed that cells on the LA and GlcA containing surfaces were more metabolically active, compared to controls, and proliferated to a monolayer by day 7 in culture and maintained some characteristics of hepatocyte functionality, such as urea synthesis over the course of 7 days. Despite these observations, further investigations using flow cytometry and RT-PCR, proved that cell attachment was unlikely to involve an ASGP-R mediated mechanism.

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Conserved regions from Plasmodium falciparum MSP11 specifically interact with host cells and have a potential role during merozoite invasion of red blood cells

Despite significant global efforts, a completely effective vaccine against Plasmodium falciparum, the species responsible for the most serious form of malaria, has not been yet obtained. One of the most promising approaches consists in combining chemically synthesized minimal subunits of parasite proteins involved in host cell invasion, which has led to the identification of peptides with high binding activity (named HABPs) to hepatocyte and red blood cell (RBC) surface receptors in a large number of sporozoite and merozoite proteins, respectively. Among these proteins is the merozoite surface protein 11 (MSP11), which shares important structural and immunological features with the antimalarial vaccine candidates MSP1, MSP3, and MSP6. In this study, 20-mer-long synthetic peptides spanning the complete sequence of MSP11 were assessed for their ability to bind specifically to RBCs. Two HABPs with high ability to inhibit invasion of RBCs in vitro were identified (namely HABPs 33595 and 33606). HABP-RBC bindings were characterized by means of saturation assays and Hill analysis, finding cooperative interactions of high affinity for both HABPs (n(H) of 1.5 and 1.2, K(d) of 800 and 600 nM for HABPs 33595 and 33606, respectively). The nature of the possible RBC receptors for MSP11 HABPs was studied in binding assays to enzyme-treated RBCs and cross-linking assays, finding that both HABPs use mainly a sialic acid-dependent receptor. An analysis of the immunological, structural and polymorphic characteristics of MSP11 HABPs supports including these peptides in further studies with the aim of designing a fully effective protection-inducing vaccine against malaria.

Inhibition of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) secretion by prodomain overexpression or pefabloc treatment as measured by a luciferase assay

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protease that acts as a regulator of circulating plasma cholesterol levels by enhancing the endocytosis of the low density lipoprotein receptor (LDL‐R) at the cell surface of hepatocytes. In this study, we show that overexpression of PCSK9's prodomain reduces the secretion of its mature form, as measured by a newly developed PCSK9‐luciferase assay. Similarly, treatment of cells with the non‐specific permeable serine protease inhibitor pefabloc decreases the secretion of mature PCSK9. We show by overexpression and immunoprecipitation that the prodomain is interchangeable on the catalytic domain of PCSK9. Medium from PCSK9‐transfected CHO cells overexpressing the prodomain or treated by pefabloc leads to enhanced surface LDL‐R expression in hepatic cells in contrast to medium from PCSK9‐transfected CHO cells only, demonstrating the functional consequences of both modes of inhibition.

114 DIFFERENTIAL HEPATIC MICRORNA EXPRESSION PROFILES ASSOCIATED WITH INTERFERON ALPHA TREATMENT RESPONSE IN PATIENTS WITH CHRONIC HEPATITIS C

antiviral strategies. The tight junction protein claudin-1 (CLDN1) has been shown to be essential for HCV entry. Using genetic immunization, we produced six monoclonal antibodies efficiently inhibiting HCV infection by targeting host entry factor CLDN1. Monoclonal anti-CLDN1 antibodies bound to human CLDN1 on the cell surface of primary human hepatocytes and Huh7.5.1 with high affinity. Competition and binding studies demonstrated that antibodies target conformational epitopes of the first extracellular loop. Using an infectious HCV cell culture system we demonstrate that monoclonal anti-CLDN1 antibodies efficiently inhibit HCV Jc1-Luc and Con1-Luc infection of Huh7.5.1 cells in a dose-dependent manner. Anti-CLDN1 antibodies efficiently cross-neutralize infection of primary human hepatocytes by HCV pseudotypes (HCVpp) derived from genotypes 1–6. In patients with chronic HCV infection, anti-CLDN1 antibodies broadly crossinhibit entry of HCVpp bearing envelope glycoproteins of the viral quasispecies population. Furthermore, anti-CLDN1 antibodies markedly inhibit entry of HCV escape variants isolated from six patients undergoing liver transplantation which were resistant to autologous host responses. In conclusion, these results suggest that targeting HCV entry factor CLDN1 using monoclonal anti-CLDN1 antibodies constitutes a novel antiviral approach to prevent primary HCV infection, such as after liver transplantation and might also restrain virus spread in chronically infected patients. I.F and S.K. contributed equally to this work.

Mechanism of LDL binding and release probed by structure-based mutagenesis of the LDL receptor

The LDL receptor (LDL-R) mediates cholesterol metabolism in humans by binding and internalizing cholesterol transported by LDL. Several different molecular mechanisms have been proposed for the binding of LDL to LDL-R at neutral plasma pH and for its release at acidic endosomal pH. The crystal structure of LDL-R at acidic pH shows that the receptor folds back on itself in a closed form, obscuring parts of the ligand binding domain with the epidermal growth factor (EGF)-precursor homology domain. We have used a structure-based site-directed mutagenesis approach to examine 12 residues in the extracellular domain of LDL-R for their effect on LDL binding and release. Our studies show that the interface between the ligand binding domain and the EGF-precursor homology domain seen at acidic pH buries residues mediating both LDL binding and release. Our results are consistent with an alternative model of LDL-R whereby multiple modules of the extracellular domain interact with LDL at neutral pH, concurrently positioning key residues so that at acidic pH the LDL-R:LDL interactions become unfavorable, triggering release. After LDL release, the closed form of LDL-R may target its return to the cell surface.

Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

The human sodium-dependent vitamin C transporter-2 (hSVCT2) plays an important role in cellular accumulation of ascorbic acid in liver cells. However, little is known about the molecular determinants that direct hSVCT2 to the cell surface in hepatocytes. We addressed this issue using live cell imaging methods to resolve the distribution and trafficking of truncated or mutated hSVCT2 constructs in a cellular model of human hepatocytes, HepG2 cells. Whereas a full-length hSVCT2-yellow fluorescent protein (YFP) fusion protein was functionally expressed at the cell surface in HepG2 cells, serial truncation and mutation analysis demonstrated an essential role for both NH(2)- and COOH-terminal sequence(s) for cell surface expression and function. Video-rate confocal imaging showed evidence of dynamic hSVCT2-YFP containing intracellular trafficking vesicles, the motility of which was impaired following disruption of microtubules using nocodazole. However, in a HepG2 cell line stably expressing hSVCT2-YFP at the cell surface, plasma membrane levels of hSVCT2 were unaffected by inhibition of microtubule-associated motor proteins; rather, surface expression of hSVCT2-YFP was increased following treatment with myosin inhibitors. Together, these results show that 1) both NH(2)- and COOH-terminal sequences are essential for proper localization of hSVCT2, 2) cell surface delivery is dependent on intact microtubules, and 3) peripheral microfilaments regulate insertion and retrieval of hSVCT2 into the plasma membrane.

Cell-surface arylsulfatase A and B on sinusoidal endothelial cells, hepatocytes, and Kupffer cells in mammalian livers

Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.

Interacting signals in the control of hepcidin expression

The amount of iron in the plasma is determined by the regulated release of iron from most body cells, but macrophages, intestinal enterocytes and hepatocytes play a particularly important role in this process. This cellular iron efflux is modulated by the liver-derived peptide hepcidin, and this peptide is now regarded as the central regulator of body iron homeostasis. Hepcidin expression is influenced by systemic stimuli such as iron stores, the rate of erythropoiesis, inflammation, hypoxia and oxidative stress. These stimuli control hepcidin levels by acting through hepatocyte cell surface proteins including HFE, transferrin receptor 2, hemojuvelin, TMPRSS6 and the IL-6R. The surface proteins activate various cell signal transduction pathways, including the BMP-SMAD, JAK-STAT and HIF1 pathways, to alter transcription of HAMP, the gene which encodes hepcidin. It is becoming increasingly apparent that various stimuli can signal through multiple pathways to regulate hepcidin expression, and the interplay between positive and negative stimuli is critical in determining the net hepcidin level. The BMP-SMAD pathway appears to be particularly important and disruption of this pathway will abrogate the response of hepcidin to many stimuli.

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

The hepatic uptake of VLDL in lrp−ldlr−/−vldlr−/− mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.

Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal sequence of the P450IID6 molecule that could explain a translocation of the molecule to the luminal side of the ER, allowing its expression on the cell surface. These results indicate that, in all likelihood, P450IID6 molecule is not present on the cell surface of normal rat and human hepatocytes. Other mechanisms than antibody-mediated cell lysis directed against membrane P450IID6 antigenic determinants must be found to account for the destruction of hepatocytes observed in this disease.

Intracellular Recycling and Iron Donating Ability of Transferrin Receptor 2α Protein Expressed in Transferrin Receptor-Deficient Chinese Hamster Ovary Cells.

[Introduction] Transferrin receptor 2 (TfR2), a homologue of the transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like the classical TfR1, TfR2α is a type II membrane protein expressing mainly in the hepatocytes as homodimer on their cell surface, but the β form lacks intracellular and transmembrane portions and therefore is likely to be an intracellular protein. Although the main physiological functions of these two isoforms are still not fully understood, TfR2α binds transferrin (Tf), and is therefore thought likely to be involved in cellular iron metabolism. Disruption of TfR2 leads to hemochromatosis, implying that the function of TfR2 might be a regulation of iron metabolism. However, TfR1 have remarkably high binding affinity to diferric Tf and only little expression on the cell surface of hepatocytes so that TfR1 must be fully saturated under physiological conditions. Besides, TfR2 is upregulated in some conditions in that iron accumulation was observed, for instance in the liver of the patients of hepatitis C, indicating that TfR2 might also function as the iron donator for the hepatocytes. The aim of the present study was to investigate the interactions of TfR2α with Tf, and to elucidate whether TfR2α have the ability of iron donation to the cells.[Methods] To investigate the functional properties of TfR2α, we expressed TfR2α protein with FLAG-tagging in transferrin receptor-deficient Chinese hamster ovary (CHO) cells (TRVb). The stably transfected cells expressing TfR2α-FLAG were applied for the 125I -labeled Tf (125I -Tf) binding study at 4 °C. The cells were applied for Tf and iron uptake study using 125I -Tf and 59Fe -loaded at 37 °C. To determine the fate of Tf internalized into the cells, the release of internalized Tf were then investigated. Degradation of released Tf was also investigated by precipitation using trichloroacetic acid/phosphotungustic acid.[Results] The association constant for binding of 125I-Tf to TfR2α was calculated to be 5.6 × 106 M−1 from non-linear least squares curve fitting to a saturable binding isotherm, which is about 50 times lower than that of TfR1. Although CHO cells showed a receptor-independent non-specific association with Tf at 37 °C, we observed cell-associated Tf persisting after acid-washing in TfR2α overexpressing cells, confirming that the existence of Tf internalization via TfR2α. After internalization, efflux of Tf, without substantial degradation, is also confirmed. Thus, Tf internalized by TfR2α, and presumably recycles, like TfR1 pathway. Overexpressed TfR2α protein was also shown to mediate iron uptake although its rate of iron donation is slower than TfR1.[Discussion and Conclusions] TfR2α binds to Tf although its affinity for Tf is low, and TfR2α possesses iron donating ability to the cells when TfR2α is over-expressed in our study. Although TfR2α has been reported to be involved in iron sensing and regulation of hepcidin expression, the involvement of the Tf binding and iron donating properties of TfR2α should be considered when the pathophysiology of iron metabolism is investigated, especially in the conditions that TfR2α is upregulated, for instance in hepatitis C.

Host cell responses induced by hepatitis C virus binding†

Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

Mapping the heparin-binding domain of human hepatic lipase

Human hepatic lipase (HL) is known to bind to the cell surface of hepatocytes and the sinusoidal endothelium of the liver. In each case, it appears that the enzyme remains associated with the cell surface through an ionic interaction with heparan sulfate proteoglycans. However, it remains unclear as to which residues are responsible for this critical function of the enzyme. In the present study, we have used a systematic approach to map the heparin-binding regions of human HL by utilizing peptide arrays spanning the complete sequence of the mature protein. Following probing with biotin–heparin, six peptides spanning residues 301–320 and 465–476 were identified as regions binding to heparin. Probing of an additional array containing these six parent peptides and a comprehensive series of mutant peptides identified two putative HL heparin-binding domains. The first was composed of residues R310, K312, K314, and R315 at the distal N-terminal domain and the second was composed of residues R473, K474, and R476 at the C-terminal end of the protein.

Histidylated Lipid-modified Sendai Viral Envelopes Mediate Enhanced Membrane Fusion and Potentiate Targeted Gene Delivery

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.

Functional Dissection of an AP-2 β2 Appendage-binding Sequence within the Autosomal Recessive Hypercholesterolemia Protein

The autosomal recessive hypercholesterolemia (ARH) protein plays a critical role in regulating plasma low density lipoprotein (LDL) levels. Inherited defects in ARH lead to a hypercholesterolemia that closely phenocopies that caused by a defective LDL receptor. The elevated serum LDL-cholesterol levels typical of ARH patients and the pronounced accumulation of the LDL receptor at the cell surface of hepatocytes in ARH-null mice argue that ARH operates by promoting the internalization of the LDL receptor within clathrin-coated vesicles. ARH contains an amino-terminal phosphotyrosine-binding domain that associates physically with the LDL receptor internalization sequence and with phosphoinositides. The carboxyl-terminal half of ARH contains a clathrin-binding sequence and a separate AP-2 adaptor binding region providing a plausible mechanism for how ARH can act as an endocytic adaptor or CLASP (clathrin-associated sorting protein) to couple LDL receptors with the clathrin machinery. Because the interaction with AP-2 is highly selective for the independently folded appendage domain of the beta2 subunit, we have characterized the ARH beta2 appendage-binding sequence in detail. Unlike the known alpha appendage-binding motifs, ARH requires an extensive sequence tract to bind the beta appendage with comparably high affinity. A minimal 16-residue sequence functions autonomously and depends upon ARH residues Asp253, Phe259, Leu262, and Arg266. We suggested that biased beta subunit engagement by ARH and the only other beta2 appendage selective adaptor, beta-arrestin, promotes efficient incorporation of this mechanistically distinct subset of CLASPs into clathrin-coated buds.

Leukocyte–hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD)

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.

Gene Expression Profiling of Alcoholic Liver Disease in the Baboon ( Papio hamadryas) and Human Liver

The molecular pathogenesis of alcoholic liver disease (ALD) is not well understood. Gene expression profiling has the potential to identify new pathways and altered molecules in ALD. Gene expression profiles of ALD in a baboon model and humans were compared using DNA arrays. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used for downstream analysis of array results. cDNA array analysis revealed differential expression of several novel genes and pathways in addition to genes known to be involved in ALD pathogenesis. Overall gene expression profiles were similar in both species, with a majority of genes involved with fibrogenesis and xenobiotic metabolism, as well as inflammation, oxidant stress, and cell signaling. Genes associated with stellate cell activation (collagens, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinase) were up-regulated in humans. Decreased expression of several metallothioneins was unexpected. Fourteen molecules related to the annexin family were up-regulated, including annexin A1 and A2. Immunofluorescence revealed a marked overexpression of annexin A2 in proliferating bile duct cells, hepatocyte cell surface, and selective co-localization with CD14-positive cells in human ALD. The gene expression profile of ALD is dominated by alcohol metabolism and inflammation and differs from other liver diseases. Annexins may play a role in the progression of fibrosis in ALD.