As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5 or 3 nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies ϳ500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.The Hepatitis C virus (HCV) is a distinct member of the family Flaviviridae, comprising a group of enveloped viruses to which the flaviviruses, with the prototype Yellow fever virus, and the animal-pathogenic pestiviruses like Classical swine fever virus and Bovine viral diarrhea virus (BVDV) belong (41). These viruses have in common a single-stranded RNA genome of positive polarity carrying one long open reading frame (ORF) that is flanked at the 5Ј and 3Ј ends by nontranslated regions (NTRs). In HCV, the genome has a length of ϳ9,600 nucleotides and encodes a ϳ3,000-amino-acid-long polyprotein carrying the structural proteins in the amino-terminal quarter and the nonstructural (NS) proteins in the remainder (for reviews, see references 4 and 43).During and after translation, the polyprotein is cleaved in the structural region by host cell enzymes, and the NS proteins are cleaved by two viral proteinases, giving rise to at least 10 different products. These are arranged from the amino to the carboxy terminus as follows: core (C)-envelope protein 1 (E1)-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (22, 23, 54). The amino-terminal products C, E1, and E2 are the major constituents of the virus particle, and they are released from the polyprotein precursor by host cell signal peptidases (23). The function of the small hydrophobic polypeptide p7 is unknown. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase, responsible for polyprotein cleavage at the NS2/3 junction (20, 24). NS3 carries two enzymatic activities residing in two well-defined globular domains (62): a chymotrypsin-like serine proteinase spanning the ϳ180 aminoterminal NS3 residues, and nucleoside triphosphatase (NTPase)/helicase activ...

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6–12 weeks), second (ST, 22–24 weeks) and third (TT, 32–38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

The Glypican 3 (Gpc3) gene is expressed abundantly in the fetal liver, is inactive in the normal adult liver, and is frequently reactivated in hepatocellular carcinoma (HCC). This reactivation in HCC has led to considerable interest in Gpc3 as a diagnostic tumor marker and its possible role in tumorigenesis. Despite this interest, the basis for Gpc3 regulation is poorly understood. On the basis of the similarities between Gpc3 and alpha-fetoprotein expression in the liver, we reasoned that common factors might regulate these 2 genes. Here we identify zinc fingers and homeoboxes 2 (Zhx2) as a regulator of Gpc3. Mouse strainspecific differences in adult liver Gpc3 messenger RNA levels and transgenic mouse studies indicate that Zhx2 represses Gpc3 expression in the adult liver. We also demonstrate that Gpc3 is activated in the regenerating liver following a carbon tetrachloride treatment and that the level of Gpc3 induction is controlled by alpha-fetoprotein regulator 2 (Afr2). Conclusion: We show that Zhx2 acts as a repressor of Gpc3 in the adult liver, and this raises the interesting possibility that Zhx2 might also be involved in Gpc3 reactivation in HCC. We also show that Gpc3 is activated in the regenerating liver in an Afr2-dependent manner. Zhx2 and Afr2 represent the first known regulators of Gpc3. (HEPATOLOGY 2007;46:1541-1547

Liver function is crucial for maintaining metabolic homeostasis in mammals. Numerous genes must be properly regulated for the liver to develop and perform a variety of activities. Several recent gene-knockout studies in mice have clarified the roles of GATA6, HNF4alpha, and Foxa1/Foxa2 in early stages of liver formation. After the liver forms, transcriptional changes continue to occur; during the perinatal period, certain genes such as alpha-fetoprotein and H19 are silenced, others are activated, and position-dependent (or zonal) regulation is established. Zhx2 was recently identified as one factor involved in postnatal repression of alpha-fetoprotein and other genes. Furthermore, several studies indicate that negative regulation is involved in the zonal control of glutamine synthetase. Finally, exciting new evidence indicates that signaling through the Wnt/beta-catenin pathway is also involved in zonal regulation in the adult liver.

IntroductionPreeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE.MethodsSerum samples were collected at 32–36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG–3 cells under 1% and 8% O2 for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured.ResultsThe protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-β-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG–3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation.ConclusionsPlacental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls.