Orbital ordering and magnetic field effect in MnV2O4.

We studied the structural properties of an orbital-spin-coupled spinel oxide, MnV2O4, mainly by single-crystal x-ray diffraction measurement. It was found that a structural phase transition from cubic to tetragonal and ferrimagnetic ordering occur at the same temperature (Ts,TN=57 K). The structural phase transition was induced also by magnetic field above Ts. In addition, magnetic-field-induced alignment of tetragonal domains results in large magnetostriction below Ts. We also found that the structural phase transition is caused by the antiferro-type ordering of the V t2g orbitals.

Analysis of possible silencer elements of ovine interferon-tau gene.

During the peri-implantation period significant production of ovine interferon-tau (olFNtau) by the trophectoderm is detected in day 13-16 conceptuses, but its level rapidly declines thereafter. To understand molecular mechanisms by which oIFNtau gene expression is down-regulated, a variety of deletion constructs were prepared from upstream sequences of the oIFNtau gene and examined for possible silencer regions by using transient transfection into human choriocarcinoma, JEG3, cells. Two regions between -700 to -654 bases (distal region) and from -503 to -453 bases (proximal region) were found to be the possible negative regulatory regions. With probes prepared from these regions, gel mobility shift assay (GMSA) was then conducted. DNA-protein complexes were observed, but the gel shift pattern was different between nuclear extracts from days 14 (active oIFNtau production) and 20 (minute oIFNtau production) ovine trophoblasts. Day 20 nuclear extracts exhibited more band patterns than those of day 14; most notably the distal region between -692 and -668 bases exhibited the distinct band with nuclear extracts from day 20, but not from day 14 trophoblasts. In addition, the band patterns from day 20 trophoblast nuclear proteins were similar to those detected with JEG3 and HeLa cell nuclear extracts. Taken together, these observations suggest that the upstream sequences identified could serve as negative regulatory regions to which various nuclear factors bind, resulting in reduction of oIFNtau gene transcription.

Effects of PMA and transcription factors on ovine interferon-tau transactivation in various cell lines.

Interferon-tau (IFNtau) is produced by the trophectoderm of ruminant ungulates and its gene transactivation in vitro has so far been achieved only in human choriocarcinoma cells, JAR and JEG3. To examine if ovine IFNtau gene transactivation could be induced in cells other than JAR or JEG3 cells and its activation could be aided by the expression of a protooncogene(s), a transient transfection system was developed with the upstream region of ovine IFNtau gene that had been inserted into the chloramphenicol acetyltransferase (CAT) reporter plasmid (IFNtau-CAT). The effect of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on IFNtau-CAT transcriptional activity was examined in JEG3, human embryonic kidney (293), HeLa and Vero cells. Upon transfection and PMA treatment, ovine IFNtau gene was transactivated in two unrelated cell lines, JEG3 and 293 cells. Since IFNtau-CAT was not induced in HeLa or Vero cells, HeLa and JEG3 cells were further examined for their ability to support IFNtau-CAT transactivation in a co-transfection system. While the expression of c-myc, interferon regulatory factor 1 or 2 (IRF-1 or IRF-2) was not effective, CAT activity was strongly enhanced in both JEG3 and HeLa cells with the co-transfection of c-Jun or c-Jun plus c-Fos. These data suggest that ovine IFNtau gene transcription induced by PMA is not specific for trophoblast cells and a protooncogene, c-jun, is a downstream effector of PMA activated nuclear factors in its signal transduction cascade resulting in IFNtau gene transactivaion.

Identification of a functional transcriptional factor AP-1 site in the sheep interferon tau gene that mediates a response to PMA in JEG3 cells.

To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5'-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation.