Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats.

Gene therapy for inherited disorders might cause an immune response to the therapeutic protein. A solution would be to introduce the gene in the fetal or neonatal period, which should lead to tolerization. Lentiviral vectors mediate long-term gene expression, and are well suited for gene therapy early in development.

Selection of tumour specific promoters for adenoviral gene therapy of cholangiocarcinoma.

Background/aims: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector.

The role of proximal-enhancer elements in the glucocorticoid regulation of carbamoylphosphate synthetase gene transcription from the upstream response unit.

As part of the urea cycle, carbamoylphosphate synthetase (CPS) converts toxic ammonia resulting from amino-acid catabolism into urea. Liver-specific and glucocorticoid-dependent expression of the gene involves a distal enhancer, a promoter-proximal enhancer, and the minimal promoter itself. When challenged with glucocorticoids, the glucocorticoid-responsive unit (GRU) in the distal enhancer of the carbamoylphosphate-synthetase gene can only activate gene expression if, in addition to the minimal promoter, the proximal enhancer is present.

The type 4 subfamily of P-type ATPases, putative aminophospholipid translocases with a role in human disease.

The maintenance of phospholipid asymmetry in membrane bilayers is a paradigm in cell biology. However, the mechanisms and proteins involved in phospholipid translocation are still poorly understood. Members of the type 4 subfamily of P-type ATPases have been implicated in the translocation of phospholipids from the outer to the inner leaflet of membrane bilayers.

Gene therapy for barrett's esophagus: adenoviral gene transfer in different intestinal models.

Adenoviral gene therapy could potentially be used for treatment of patients with a Barrett's esophagus. In order to study the feasibility of this approach it is important to study adenoviral intestinal transduction both in vitro and in vivo. In the present study, we used differentiating Caco-2 cells, closed intestinal loops and a Barrett's esophagus rat model to test transduction of adenoviruses expressing green fluorescent protein.

AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle.

Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading.

The RL-ET-14 cell line mediates expression of glutamine synthetase through the upstream enhancer/promoter region.

Background/aims: The expression of glutamine synthetase (GS) in the mammalian liver is confined to the hepatocytes surrounding the central vein and can be induced in cultures of periportal hepatocytes by co-cultivation with the rat-liver epithelial cell line RL-ET-14. We exploited these observations to identify the regulatory regions of the GS gene and the responsible signal-transduction pathway that mediates this effect.

Methods: Fetal hepatocytes of wild-type or GS-transgenic mice were co-cultured with RL-ET-14 cells to induce GS expression.

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter.

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline.

Effect of arginine deficiency on arginine-dependent post-translational protein modifications in mice.

Transgenic mice that overexpress arginase-I in their small-intestinal enterocytes suffer from a pronounced, but selective decrease in circulating arginine levels during the suckling period, resulting in impaired growth and development of hair, muscle and immune system. In the present study, we tested the hypothesis that the arginine-deficiency phenotype is caused by arginine-specific post-translational modifications, namely, an increase in the degree of mono-ADP-ribosylation of proteins because of reduced competition by free arginine residues and/or an increase in protein-tyrosine nitration because of an increased O2- production by NO synthases in the presence of limiting amounts of arginine. Arginine ADP-ribosylation and tyrosine nitration of proteins in the affected organs were assayed by Western blot analysis, using specific anti-ADP-ribosylarginine and protein-nitrotyrosine antisera.

Diosgenin-induced biliary cholesterol secretion in mice requires Abcg8.

The plant sterol diosgenin has been shown to stimulate biliary cholesterol secretion in mice without affecting the expression of the adenosine triphosphate-binding cassette transporter heterodimer Abcg5/g8. The aim of this study was to investigate the mechanism of diosgenin-induced cholesterol hypersecretion and to identify the genes involved. Surprisingly, despite its lack of effect on Abcg5/g8 expression in wild-type mice, diosgenin did not stimulate biliary cholesterol secretion in mice deficient for Abcg8.

Kupffer cells and not liver sinusoidal endothelial cells prevent lentiviral transduction of hepatocytes.

Lentiviral vectors can stably transduce dividing and nondividing cells in vivo and are best suited to long-term correction of inherited liver diseases. Intraportal administration of lentiviral vectors expressing green fluorescent protein (Lenti-GFP) in mice resulted in a higher transduction of nonparenchymal cells than hepatocytes (7.32 +/- 3.

Mechanisms of glucocorticoid signalling.

It has become increasingly clear that glucocorticoid signalling not only comprises the binding of the glucocorticoid receptor (GR) to its response element (GRE), but also involves indirect regulation glucocorticoid-responsive genes by regulating or interacting with other transcription factors. In addition, they can directly regulate gene expression by binding to negative glucocorticoid response elements (nGREs), to simple GREs, to GREs, or to GREs and GRE half sites (GRE1/2s) that are part of a regulatory unit. A response unit allows a higher level of glucocorticoid induction than simple GREs and, in addition, allows the integration of tissue-specific information with the glucocorticoid response.

Hepatic response to right ventricular pressure overload.

Background & Aims: Modifying the afferent blood supply to the liver does not change the zonal expression pattern of hepatic enzymes in the rat.

Methods: We used pulmonary trunk banding (PTB) to study the effect of an efferent hindrance of blood flow on hepatic architecture and zonation of gene expression.

Results: Most PTB rats developed right ventricular hypertrophy and congestive heart failure.

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence.

The ins and outs of reverse cholesterol transport.

It is generally assumed that HDL is the obligate transport vehicle for 'reverse cholesterol transport', the pathway for removal of excess cholesterol from peripheral tissues via the liver into bile and subsequent excretion via the feces. During the last few years, intensive research has generated exciting new data on the separate processes involved in reverse cholesterol transport. Many 'new' proteins, particularly members of the ABC transporter and nuclear receptor subfamilies, that mediate or influence cholesterol fluxes have been identified and characterized.

ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis.

ApoAV has been discovered recently as a novel modifier of triglyceride (TG) metabolism, but the pathways involved are currently unknown. To gain insight into the function of apoAV, adenovirus-mediated gene transfer of murine apoa5 to C57Bl/6 mice was employed. The injection of low doses of Ad-apoa5 (1-5 x 10(8) plaqueforming units/mouse) dose-dependently reduced plasma very low density lipoprotein (VLDL)-TG levels.

Stereological measurement of porto-central gradients in gene expression in mouse liver.

The liver is thought to consist of lobules, numerous repeating, randomly oriented units. Within these lobules, genes are expressed in gradients along the porto-central axis, which spans the distance between portal and central veins. We have developed a robust stereological method to map all points in an image to their position on this porto-central axis.

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters.

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein (ABCB4), and sterolin 1 and 2 (ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species.

Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer.

Bilirubin is glucuronidated by bilirubin UDP-glucuronyltransferase (UGT1A1) before biliary excretion. Because bilirubin is toxic, patients with Crigler-Najjar type I (CN), who have no UGT1A1 activity, suffer severe brain damage early in childhood. The Gunn rat is the model for CN type 1.

Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineage.

To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of alpha-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules.

Induction of insolubility by herpes simplex virus VP22 precludes intercellular trafficking of N-terminal Apoptin-VP22 fusion proteins.

The herpes simplex virus protein VP22 has the intriguing ability to deliver proteins from an expressing cell to neighboring cells. Fusion of VP22 to Apoptin, a protein that induces apoptosis in tumor cells but not in normal cells, might enhance the delivery of Apoptin. To analyze this hypothesis two fusion proteins of VP22 and full-length Apoptin were constructed, namely VP22-VP3 and VP3-VP22, and their apoptosis-inducing ability and intercellular spreading behavior were analyzed by transfection in tumor cells.

Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice.

Background/aim: Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models.

Methods: Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.

One step further towards real high-throughput functional genomics.

In a recent paper by Michiels et al. an important step was made towards genuine high throughput functional genomics. The authors produced an arrayed adenoviral library containing >120000 cDNAs isolated from human placenta.

Upstream and intronic regulatory sequences interact in the activation of the glutamine synthetase promoter.

Glutamine synthetase (GS) is expressed at high levels in subsets of cells in some tissues and at low levels in all cells of other tissues, suggesting that the GS gene is surrounded by multiple regulatory elements. We searched for such elements in the 2.5-kb upstream region and in the 2.