Absorption, distribution, and biliary excretion of cafestol, a potent cholesterol-elevating compound in unfiltered coffees, in mice.

Cafestol is a diterpene present in unfiltered coffees. It is the most potent cholesterol-elevating compound present in the human diet. However, the precise mechanisms underlying this effect are still unclear. In contrast, cafestol is also known as a hepatoprotective compound, which is likely to be related to the induction of glutathione biosynthesis and conjugation. In the present study, we investigated whole-body distribution, biliary excretion, and portal bioavailability of cafestol in mice. First, dissection was used to study distribution. Five hours after an oral dose with (3)H-labeled cafestol, most activity was found in small intestine, liver, and bile. These results were confirmed by quantitative whole-body autoradiography in a time course study, which also showed elimination of all radioactivity within 48 h after administration. Next, radiolabeled cafestol was dosed intravenously to bile duct-cannulated mice. Five hours after the dose 20% of the radioactivity was found in bile. Bile contained several metabolites but no parent compound. After intestinal administration of radioactive cafestol to portal vein-cannulated mice, cafestol was shown to be rapidly absorbed into the portal vein as the parent compound, a glucuronide, and an unidentified metabolite. From the presence of a glucuronide in bile that can be deconjugated by a bacterial enzyme and the prolonged absorption of parent compound from the gastrointestinal tract, we hypothesized that cafestol undergoes enterohepatic cycling. Together with our earlier observation that epoxidation of the furan ring occurs in liver, these findings merit further research on the process of accumulation of this coffee ingredient in liver and intestinal tract.

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. A model for fetal or neonatal gene therapy is the inherited disorder of bilirubin metabolism, Crigler-Najjar disease (CN). The absence of bilirubin UDP-glucoronyltransferase (UGT1A1) activity in CN patients causes high serum levels of unconjugated bilirubin and brain damage in infancy. CN is attractive for the development of gene therapy because the mutant Gunn rat closely mimics the human disease. Injection of UGT1A1 lentiviral vectors corrected the hyperbilirubinemia for more than a year in rats injected as fetuses and for up to 18 weeks in rats injected the day of birth. UGT1A1 gene transfer was confirmed by the presence of bilirubin glucuronides in bile. All animals injected with UGT1A1 lentiviral vectors developed antibodies to UGT1A1. Animals injected with green fluorescent protein (GFP) lentiviral vectors did not develop antibodies to GFP. Our results indicate that fetal and neonatal gene therapy with immunogenic proteins such as UGT1A1 does not necessarily lead to tolerization.

CR-EST: a resource for crop ESTs.

The crop expressed sequence tag database, CR-EST (http://pgrc.ipk-gatersleben.de/cr-est/), is a publicly available online resource providing access to sequence, classification, clustering and annotation data of crop EST projects. CR-EST currently holds more than 200,000 sequences derived from 41 cDNA libraries of four species: barley, wheat, pea and potato. The barley section comprises approximately one-third of all publicly available ESTs. CR-EST deploys an automatic EST preparation pipeline that includes the identification of chimeric clones in order to transparently display the data quality. Sequences are clustered in species-specific projects to currently generate a non-redundant set of approximately 22,600 consensus sequences and approximately 17,200 singletons, which form the basis of the provided set of unigenes. A web application allows the user to compute BLAST alignments of query sequences against the CR-EST database, query data from Gene Ontology and metabolic pathway annotations and query sequence similarities from stored BLAST results. CR-EST also features interactive JAVA-based tools, allowing the visualization of open reading frames and the explorative analysis of Gene Ontology mappings applied to ESTs.