Marinobufagenin inhibits proliferation and migration of cytotrophoblast and CHO cells.

Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid that is involved in the inhibition of the sodium pump Na(+)/K(+)-ATPase. Increased plasma levels of MBG have been reported in patients with volume expansion-mediated hypertension and preeclampsia. We have recently demonstrated that MBG impairs both the proliferation and growth factor-induced migration of human first trimester cytotrophoblast (CTB) cells, crucial for proper placental development. However, the intracellular signaling mechanisms regulating the MBG-induced impairment of CTB differentiation, migration and invasion are unknown. The human extravillous CTB cell line SGHPL-4 was utilized for this study. The phosphorylation of MAP kinase protein ERK1/2 was evaluated by Cellular Activation of Signaling ELISA (CASE) in control CTB cells and those treated with MBG. MBG at concentrations of 10 and 100nM inhibited CTB cell proliferation, migration and invasion (60%, 50% and 50%, respectively). MBG also caused a significant decrease in the phosphorylation of ERK1/2. In addition, MBG decreased proliferation, migration, and ERK1/2 activity in another motile cell line, CHO cells. Another sodium pump inhibitor, ouabain, similarly decreased proliferation and ERK1/2 activity in CTB and CHO cells. These data suggest that the changes observed in cell function may be mediated by inhibition of Na(+)/K(+)-ATPase. We demonstrate that the MBG-induced impairment of CTB cell proliferation, migration and invasion is associated with decreased ERK1/2 activity which may be mediated by inhibition of Na(+)/K(+)-ATPase.

Self-seal reagent: evaporation control for molecular histology procedures without chambers, clips or fingernail polish.

Sensitive nucleic acid based detection methods such as in situ PCR, in situ RT-PCR and PRINS have great potential in the areas of developmental biology, pathogenesis and diagnostics. However, control of evaporation from in situ reactions is critical to ensure reliable data. Self-Seal Reagent, a component added directly to the in situ reaction mixture, effectively controls evaporation during in situ procedures by creating an evaporation-limiting barrier around the periphery of a standard cover glass as the reaction proceeds. At the end of the procedure, the cover glass is easily removed by soaking in an aqueous solution. A model is presented for how Self-Seal Reagent controls evaporation while maintaining reagent concentrations. Self-Seal Reagent is shown to be effective in the detection of HIV sequences in cells by in situ PCR.

Liver-directed gene transfer in non-human primates.

To develop a primate model for liver-directed gene therapy, we studied several gene transfer vehicles and routes in eight rhesus monkeys (Macaca mulatta). For this purpose, we used first-generation, replication-deficient adenoviral vectors carrying the Escherichia coli lacZ gene (Ad.CMVlacZ) or a lacZ-containing plasmid (pCMV beta) with lipofectamine for transfection. The reporter gene construct was infused into either the portal vasculature, common bile duct, or saphenous vein. Adenovirus-mediated gene transfer via the portal vein resulted in expression of lacZ in over 70% of hepatocytes by days 3-7, but was accompanied by acute hepatitis. Adenovirus-mediated gene transfer via the common bile duct resulted in lacZ expression in less than 10% of hepatocytes and was accompanied by portal inflammation. The animals mounted a significant immune response, as demonstrated by adenoviral antigen-induced T-cell proliferation and production of neutralizing anti-adenovirus antibodies and antibodies to E. coli beta-galactosidase (beta-Gal). Activation of the immune response was associated with rapid decrease of the reporter gene by days 13-21. Lipofectamine-mediated gene transfer was inefficient, and no lacZ expression in the liver was detected. To limit the host immune response, 4 animals were immunosuppressed by cyclophosphamide/prednisone and then infused with the Ad.CMVlacZ via the portal vein or the saphenous vein. The monkeys showed sustained expression of lacZ for up to 35 days with no evidence of inflammation. The primates transduced via the saphenous vein showed a level of beta-Gal expression in the liver similar to that of the portal vein-infused animals. In conclusion, adenovirus-mediated gene transfer to non-human primate livers via the portal vein or saphenous vein is efficient, but it results in transient expression and is accompanied by an immune response to both vector and transgene products and acute hepatitis, whereas lipofectamine-mediated transfer is inefficient. Manipulation of the host immune response may expand potential applications of adenoviral vectors for liver-directed gene transfer.

Conservation of hepatitis C virus 5' untranslated sequences in hepatocellular carcinoma and the surrounding liver.

Persistent infection by hepatitis C virus is a major risk factor for the development of hepatocellular carcinoma, but the mechanism of hepatocarcinogenesis is unknown. To study the association of hepatitis C virus with hepatocellular carcinoma, we sequenced part of the 5' untranslated region of hepatitis C virus from the tumor tissue and the surrounding nontumorous liver of three patients with hepatocellular carcinoma. No sequence differences between tumor-derived and liver-derived hepatitis C virus isolates were detected. The conservation of the 5' untranslated region of hepatitis C virus--not only in infected hepatocytes, but also in neoplastic cells--suggests that the regulatory elements at the 5' terminus of the viral genome play an important role in the pathobiology of hepatitis C virus.

Tissue- and development-specific expression of HBGF-1 mRNA.

The gene for heparin-binding growth factor-1 (HBGF-1) encodes a 15.5-18 kDa polypeptide that affects the proliferation and differentiation of a broad range of mammalian cells and is widely distributed among normal adult tissues. In this study, we show that normal tissues of the adult rat express HBGF-1 transcripts in one of three patterns: a 4.4 kb mRNA was the predominant HBGF-1 transcript in brain, heart and lung; a 1.4 kb mRNA was the predominant transcript in the liver; approximately equal levels of the 1.4 and 4.4 kb mRNAs were found in the kidney. HBGF-1 expression was localized in two tissues: central nervous system expression of HBGF-1 was significantly higher in the brain stem compared to the cerebrum and cerebellum; renal expression of HBGF-1 was significantly higher in the medulla compared to the cortex. Analysis of the postnatal changes in HBGF-1 expression using the newborn rat kidney revealed that the level of HBGF-1 mRNA is low at birth and does not rise to adult levels until the seventh postnatal day. These findings demonstrate that HBGF-1 expression is specific for tissue type and stage of development.

"Staircase" closure of lower lip defects.

Attention is called to a previously described method of closing certain common surgical defects of the lower lip. Advancement flaps are used from one or both sides, resulting in a staircase configuration. The advantages, disadvantages, and applications of the method are described.