The connexin31 (Cx31) gene, a member of the connexin multigene family, is expressed in a characteristic spatiotemporal pattern during placental development in rodents. To elucidate the trophoblast‐specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA‐less promoter region. Transcription is initiated in exon 1 from two transcription start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5′ flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF‐κB element, a CAAT‐box and E‐box/E‐box‐related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho‐1 and the mouse keratinocyte cell line Hel37, which both express Cx31, were chosen. Only constructs including exon 1 and the complete intron showed high activity in transient transfection experiments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any obvious changes in luciferase activity. However, deletion of 1.1 kb of the intron sequence downstream of the splice donor site resulted in the loss of promoter activity. The intron exhibits no enhancer activity for the gene; however, the mRNA stability was increased in the presence of the intron sequence. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.

To examine the role of cell-cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell-cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively. In vitro growth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by beta-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of beta-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell-cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects.

We have investigated the properties of gap junction channels of three human malignant trophoblast (choriocarcinoma) cell lines: BeWo, Jeg-3 and JAr, as well as in Jeg-3 cells stably transfected with rat connexin40 (Cx40). Reverse-transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis and immunostaining demonstrated expression of Cx40 in BeWo and JAr cell lines. JAr cells also expressed minor amounts of Cx43. Very low levels of Cx40 transcripts were revealed by RT-PCR in parental Jeg-3 cells, but Cx40 protein was not detected. To compare properties of endogenously and exogenously expressed Cx40 channels we have transfected Jeg-3 cells with rat Cx40. Recordings with dual whole-cell methods were used to determine the junctional conductance (g j) in the various cell lines and transfectants. Cx40 channels exogenously expressed in Jeg-3 cells demonstrated steep voltage sensitivity in the transjunctional voltage range of ±30 to ±40 mV and a unitary mainstate conductance of 175 pS, values which are similar to the data obtained from endogenously expressed Cx40 in BeWo cell pairs. In addition, greater driving forces resulted in a lower unitary conductance of about 30 pS, exclusively in BeWo cells. Between JAr cell pairs we determined a g j of 10 nS and unitary conductances were predominantly 100 and 152 pS. Voltage dependence was less sensitive in JAr cells compared to Cx40 transfectants and BeWo cells. Thus, coexpression of Cx43 and Cx40 leads to a macroscopic conductance with a mixture of properties expected for each connexin, whereas single-channel properties of each connexin type are maintained.

We have investigated the effect of pressure and magnetic field on the specific heat of CeCu2Si2 and CeNi2Ge2, two undoped compounds close to an antiferromagetic (afm) quantum critical point. In a CeCu2Si2 sample showing an afm phase at p=0, application of a pressure of p=0.7 GPa results in a replacement of this phase by superconductivity and to non-Fermi-liquid (NFL) behaviour in C/T. In CeNi2Ge2 the NFL behaviour present at p=0 is suppressed by either a magnetic field B=8 T or a pressure p=1.3 GPa. For both compounds, the NFL effects are consistent (as T→0) with C/T=γ0-αT1/2 as expected when TN→0.

Ce-based heavy-fermion (HF) metals behave as Kondo-lattice systems and can be classified with the aid of a single coupling parameter, ¦J ¦/W,where J < 0is the local exchange integral and W the conduction-band width. Depending on its actual composition, the exemplary material CeCu2Si2 chooses one out of two ground states: HF superconductivity and a new magnetic HF phase “A”. In a narrow composition range, these two phases are nearly degenerate and expell each other upon varying either the temperature or the external magnetic field. The one-parameter-scaling approach appears inapplicable to the U-based HF metals. For the exemplary material UPd2Al3, antiferromagnetic ordering between seemingly local 5f moments coexists, on a microscopic scale, with HF superconductivity. Whether this coexistence can be explained by assuming 5 f states localized on a tetravalent U-ion with non-magnetic crystal-field ground state remains to be shown. We discuss arguments which invokeitinerant 5 f states in the U-based HF metals to be distinguished from thelocalized 4f states in the Ce-based counterparts.

Many human tumors over-express erbB-2 and EGF receptors. The membrane localization of these receptor tyrosine kinases make them appropriate targets for directed tumor therapy. We have used recombinant DNA technology to produce single-chain antibody exotoxin A (scFv-ETA) fusion proteins which specifically bind the erbB-2 and EGF receptors. The scFv portion is composed of the heavy- and light-chain variable domains of monoclonal antibodies which recognize the extracellular portion of each receptor. We have previously described the anti-tumor activity of the bacterially produced scFv(FRP5)-ETA directed to the erbB-2 receptor. In this paper we describe the characteristics of scFv(225)-ETA, a protein which binds the EGF receptor. The bacterially produced recombinant protein binds to the receptor with high affinity and inhibits the in vitro growth of the EGF receptor over-expressing tumor cell lines A431 and MDA-MB468. Combination treatment with scFv-(FRP5)-ETA and scFv(225)-ETA led to an additive inhibitory effect on the in vitro growth of A431 cells. SKBR3 cells expressing low levels of EGF receptor but high levels of p185erbB-2 were not affected by scFv(225)-ETA treatment but were sensitive to scFv(FRP5)-ETA. Stimulation of SKBR3 cells and HCII RI#11 mouse mammary epithelial cells expressing the human erbB-2 with EGF led to an increase in scFv(FRP5)-ETA activity, showing that the EGF-induced activation of erbB-2 can potentiate the action of the erbB-2-directed toxin. Treatment of athymic nude mice with scFv(FRP5)-ETA and the combination of both scFv-ETA proteins led to the transient arrest of growth of established A431 tumors. scFv(225)-ETA treatment alone was the most effective, leading to tumor shrinkage during the course of treatment, whereas treatment with the parental monoclonal antibody 225 led to retarded tumor growth.