Shinji Iijima

There are two transcription start sites in the growth-associated protein 43 (GAP-43) promoter, and several repressive elements have been reported in the control region. But the repressive effects have been analyzed only for the distal transcription start site. Among the repressive elements reported, we found that modulator I repressed GAP-43 gene expression from the proximal promoter in non-neuronal cells. We also found a novel stimulative element immediately downstream of modulator I.

α-Fetoprotein (AFP) is a fetal serum protein abundant in fetal liver whose expression is downregulated in adult liver. The promoter–proximal region of the AFP gene contains two binding sites for CCAAT/enhancer-binding protein α (C/EBPα), two for hepatocyte nuclear factor-1α (HNF-1α) and one for nuclear factor-1 (NF-1). Luciferase reporter assays showed that a combination of C/EBPα, HNF-1α, NF-1 and coactivator p300 gave the maximal activity but the BRG1 and BRM, ATPase subunit of the chromatin remodeling complex SWI/SNF, repressed the transactivation of the AFP gene by these transcription factors. Deletion analyses of C/EBPα binding sites suggested that C/EBPα recruited SWI/SNF complex and caused the repression. This repression may also play important roles in the downregulation of the AFP gene in adult hepatocytes.

Summary: In this study, we investigated the costimulatory activity of l‐selectin in primary mouse T cells. Proliferation induced by immobilized anti‐CD3 antibody was enhanced by immobilized anti‐l‐selectin antibody. In contrast to the anti‐CD28 antibody, anti‐l‐selectin antibody did not enhance interleukin‐2 (IL‐2) expression. One of the cyclin‐dependent kinase (cdk) inhibitors, p27, was reduced by costimulation with anti‐l‐selectin antibody, as with anti‐CD28 antibody, suggesting that the enhancement of T‐cell proliferation is the result of a reduced p27 level. Since anti‐l‐selectin antibody enhanced the activation of extracellular signal‐regulated protein kinase (ERK) induced by anti‐CD3 antibody, ERK plays an important role in signal integration during costimulation. These results suggest that the mechanism of T‐cell costimulation is at least partially different between CD28 and l‐selectin, although the two mechanisms share a common downstream event, a reduction of p27 level, as a critical biochemical event in the cell cycle progression of T cells.

We report here the generation of transgenic chickens using a retroviral vector for the production of recombinant proteins. It was found that the transgene expression was suppressed when a Moloney murine leukemia virus-based retroviral vector was injected into chicken embryos at the blastodermal stage. When a concentrated viral solution was injected into the heart of developing embryos after 50 to 60 h of incubation, transgene expression was observed throughout the embryo, including the gonads. For practical production, a retroviral vector encoding an expression cassette of antiprion single-chain Fv fused with the Fc region of human immunoglobulin G1 (scFv-Fc) was injected into chicken embryos. The birds that hatched stably produced scFv-Fc in their serum and eggs at high levels (∼5.6 mg/ml). We obtained transgenic progeny from a transgenic chicken generated with this procedure. The transgene was stably integrated into the chromosomes of transgenic progeny. The transgenic progeny also expressed scFv-Fc in the serum and eggs.

The expression, activation and involvement in growth regulation of a small GTPase, RhoA, were examined in rat primary hepatocyte cultures. Hepatocytes freshly isolated from liver expressed RhoA protein at high levels. The total level of RhoA protein in the cells decreased markedly within a day in monolayer cultures. Thereafter, RhoA expression recovered as cell-cell attachment occurred during the culture. On the other hand, the level of the active form of RhoA decreased as the culture proceeded. Ca²⁺ depletion in the medium to disrupt cadherin engagement triggered RhoA activation without de novo protein synthesis, indicating cadherin engagement regulates RhoA activation in hepatocytes. Hepatocyte growth stimulation by HGF was enhanced by Ca²⁺ depletion or introduction of a constitutively active form of RhoA. The Clostridium botulinum C3 enzyme inhibited hepatocyte growth with stimulation by HGF. These results suggest that RhoA has a crucial role in hepatocyte growth control.

To get immortalized endothelial cells, human endothelial cells were transfected with a temperature-sensitive simian virus 40 DNA. The majority of transfected cells showed a prolonged life span and temperature-dependent cell growth. One of the clones, #5-1, grew either at 33 or 37 degrees C and could be cultured more than 110 generations. The cells expressed several endothelial cell specific characteristics.