Search

To elucidate the role of complement-mediated uptake in Leishmania major infection in vivo, transgenic BALB/c mice that express the cobra venom factor (CVF) under control of the alpha1-antitrypsin promoter were infected. CVF expression in these mice leads to a continuous activation and subsequent consumption of complement C3 in the serum. In contrast to susceptible non-transgenic BALB/c mice, CVF-transgenic mice are highly resistant to L. major infection and show a significantly reduced parasite dissemination. Transient depletion of C3 in wild-type BALB/c mice delays progression of lesions for some days. Both CVF-transgenic and non-transgenic mice exhibit similar T cell responses upon infection. However, in CVF-transgenic mice, no infiltration of neutrophils, which were the prominent infiltrating cells at the site of infection in normal susceptible mice, could be detected. We conclude that C3 cleavage is required for the attraction of neutrophils that participate in parasite dissemination.

The orphan hepatic nuclear factor (HNF) HNF4alpha is of pivotal importance for liver development and hepatocellular differentiation and plays an essential role in a regulatory circuitry to control a wide range of metabolic processes. It also targets genes in other organs, including pancreas, kidney, intestine, and colon; promotes expression of an epithelial phenotype; triggers de novo formation of functional tight junctions; and contributes to epithelial cell polarity. In particular, HNF4alpha dysfunction leads to metabolic disorders, including diabetes. We used the chromatin immunoprecipitation (ChIP) cloning procedure and a bioinformatic approach to search for candidate genes associated with impaired liver, pancreas, and kidney function. We identified two novel targets regulated by HNF4alpha, which participate in the control, at least in part, in cell-cycle regulation and are members of the mitogen-activated kinase pathway. In multiple ChIP assays, ribosomal S6 kinase 4 (RSK4) and p21-activated kinase 5 (PAK5) were confirmed, and in vitro binding of HNF4alpha was evidenced by electrophoretic mobility shift assays (EMSA) using oligonucleotides, which harbor novel binding sites. We also used EMSA to probe for binding sites in promoters of HNF1alpha, apolipoprotein B, alpha1-antitrypsin, and angiotensinogen. We further studied RSK4 and PAK5 kinase expression in streptozotocin-induced diabetic rat kidney and brain and observed significant repression of HNF4alpha, RSK4, and PAK5 as determined by quantitative real-time reverse transcriptase-polymerase chain reaction. RSK4 and PAK5 may provide a molecular rationale for late-stage complications in disease, and further studies are warranted to explore these targets for the treatment of diabetic nephro- and neuropathy, frequently seen in patients with HNF4alpha dysfunction.

2,3-Butanedione monoxime (BDM) is a reversible myosin AT-Pase inhibitor with phosphatase-like activity. It is being evaluated for its therapeutic benefit in organ preservation in addition to its use to prompt cardioplegic arrest. Its effects on transcriptional regulation of ion channels and Ca(2+)-handling proteins, surprisingly, are basically unknown. We therefore studied expression of genes coding for ion channels and Ca(2+)-handling proteins in explanted hearts and cultures of Ca(2+)-tolerant cardiomyocytes. In addition, we studied the effect of daily treatment with 250 mg of BDM/kg of body weight for up to 72 h. Furthermore, Ca(2+)-tolerant cardiomyocytes were isolated and cultured in the presence of 15 mM BDM and harvested 24 or 72 h after dosing. It is noteworthy that a modest to highly significant increase in transcript level of ion channels, ion exchangers, Ca(2+)-binding proteins, and cytoskeletal proteins was observed after treatment of rats with BDM. Likewise, with cultures of cardiomyocytes, statistically significantly increased transcript levels of potassium and sodium ion channels as well as some ion exchangers (PMCA2 and 4) were noted, even though expression of genes coding for Ca(2+)-binding and cytoskeletal proteins was repressed. This preponderance of transcriptional up-regulation of cardiac-specific genes suggests a mechanism of action whereby unilateral dephosphorylation of coded proteins resulted in a feedback loop of regulation (e.g., transcriptional activation of coding genes).

Polychlorinated biphenyls (PCBs) are persistent environmental chemicals that accumulate at the apex of food chains. Several scientific committees support its designation as a human carcinogen, even though the precise mechanism of carcinogenesis remains controversial. In view of its uncertain ability to cause DNA damage in human liver, we investigated the effects of Aroclor 1254, Aroclor 1016, and 4-chlorobiphenyl (4-CB) on DNA adduct formation in cultures of primary human hepatocytes from five donors. Based on (32)P-postlabeling assays, we detected DNA adducts in native human liver as well as untreated, i.e., control cultures of human hepatocytes. Treatment of human hepatocytes with Aroclor 1016 and Aroclor 1254 resulted in four-fold (NP1) and seven-fold (butanol) increases in DNA adduct formation. Further, two and six new adduct spots were detected by multidirectional thin-layer chromatography after nuclease P1 and butanol enrichment. Treatment of human hepatocyte cultures with 4-CB led to 209 adducts per 10(9) nucleotides at the 60 microM concentration, and we show metabolically activated PCBs to be more efficient in the production of DNA-binding species compared with higher chlorinated PCB mixtures. Our study is therefore highly suggestive for a link between PCB exposure and DNA insult in human hepatocytes., (Copyright 2003 Elsevier Science (USA))

Numerous studies have established the pivotal role of liver-enriched transcription factors in organ development and cellular function, and there is conclusive evidence for transcription factors to act in concert in liver-specific gene expression. During organ development and in progenitor cells the timely expression of certain transcription factors is necessary for cellular differentiation, and there is overwhelming evidence for hierarchical and cooperative principles in a networked environment of transcription factors. The search for molecular switches that control stem cell imprinting and liver-specific functions has lead to the discovery of many interactions between such different molecules as transcription factors, coactivators, corepressors, enzymes, DNA, and RNA. Many of these interactions either repress or activate liver-specific gene expression. It thus can be demonstrated that specific mutational changes in liver-enriched transcription factors lead to altered intermolecular interactions with the consequence of human disease. This review provides an overview of our current knowledge about liver-enriched transcription factors and their role in liver function and development. We review the basic principles of gene transcription, the role of liver-enriched transcription factors in liver gene regulation, and the classification of transcription factors by their DNA-binding domains.