The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression.     

A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Patterns of Endopolyploidy During Seedling Development in Cabbage (Brassica oleracea L.)

Flow cytometric analysis of nuclear DNA contents of somatictissues of cabbage (Brassica oleracea L.) has revealed extensiveendopolyploidization, resulting in tissues that comprise mixturesof cells with different DNA contents, ranging from 2C to 16C.Patterns of endopolyploidy are specific to each developmentalstage. Multiple polyploidy was not present in the embryos ofdry seeds. Rapid endoreduplication occurred in the radicle andthe hypocotyl of the embryos during seed germination. Furtherendoreduplication cycles were detected in all tissues exceptthose of the shoot tips. In five cabbage cultivars tested, seedlingscontained cells of four ploidy levels, corresponding to 2C,4C, 8C and 16C. Multiploidy may be an integral part of differentiationprograms in cabbage plants. The biological significance of endoreduplicationin cabbage plants is discussed. Copyright 2001 Annals of BotanyCompany Cabbage (Brassica oleracea L.), endopolyploidy, endoreduplication, flow cytometry     

The Lyb-3+5+ subset of B cells is not required for lupus-like autoantibody formation caused by graft-vs-host reaction

We asked the question whether or not the Lyb-3+5+ B cell subset, which is lacking in CBA/N immune defective mice, is required for the lupus-like autoantibody formation caused by graft-vs-host reaction (GVHR). (CBA/N X DBA/2)F1 male defective mice injected with DBA/2 T cells produced IgG autoantibodies to the same extent as did nondefective F1 mice suffering from GVHR. Although a very small number of DBA/2 B cells might have contaminated the T cell inocula, it was shown that these were B cells of the defective F1 mice that produced autoantibodies during the GVHR. This was demonstrated by detecting autoantibodies carrying an immunoglobulin allotype of the F1 recipient. Furthermore, the defective F1 male mice injected with CBA/N lymphoid cells, which were lacking Lyb-3+5+ B cells, also produced autoantibodies. Isotype analysis of antinuclear antibodies revealed that some of them belonged to IgG3 isotype. It was concluded that the ontogenically late-appearing B cell subset is not required for GVH autoimmunity.     

Demonstration of the presence of G-proteins in hepatic microsomal fraction

The presence of G-proteins in isolated hepatic microsomal vesicles is demonstrated. The G-proteins were identified by their capacity to be ADP-ribosylated by cholera and pertussis toxins. Cholera toxin identified 42 and 45 kDa proteins, corresponding to alpha s-1 and alpha s-2, respectively. Pertussis toxin identified a 40 kDa protein corresponding to alpha i. The microsomal G-proteins are identical to the corresponding G proteins of the plasma membrane, but are present in different proportions; the microsomes have considerably less alpha s proteins than the plasma membrane.     

Bioisostere of valtrate,anti-HIV principle by inhibition for nuclear export of Rev

Rational design by the MO calculation disclosed 5,6-dihydrovaltrate (2) as the bioisostere of valtrate (1), the Rev-export inhibitor with anti-HIV activity. The synthesis of 2 was accomplished by ingenious use of asymmetric Diels–Alder reaction and stereoselective epoxidation associated with the adjacent hydroxyl group. Because of similar biological potency to 1, the analog 2 should be recognized as a promising scaffold for new anti-HIV agents with an unprecedented mechanism of action, inhibition for nuclear export of Rev protein, in the conventional remedy.     

Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation.

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.