Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

Transmission of human T-cell leukemia virus type 1 to mice

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10(5) spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.     

Interaction of actin and ADP with the head domain of smooth muscle myosin: implications for strain-dependent ADP release in smooth muscle

Transient kinetic methods were used to study interactions between actin, MgADP, and smooth muscle (chicken gizzard) myosin subfragment 1 (smS1). The equilibrium dissociation constant (Kd) of actin for smS1 was 3.5 nM, tighter than that of skeletal S1 (skS1). Actin binding to smS1 was weakened 5-fold by saturation with ADP compared to 30-60-fold for skS1. The Kd of ADP for smS1 was increased from 1.2 to 5 microM by actin, whereas for skS1 values increased from 2 to 100 microM. Thus, coupling between ADP and actin binding is weaker for smS1. Previous studies show that release of ADP from actin.smS1.ADP produces a tilt of the regulatory domain [Whittaker, M., Wilson-Kubalek, E. M., Smith, J. E., Faust, L., Milligan, R. A., and Sweeney, H. L. (1995) Nature 378, 748-751]. This result was confirmed by independent structural methods; tilting was absent for skS1, and the Kd for ADP was in agreement with the values measured here [Gollub, J., Cremo, C. R., and Cooke, R. (1996) Nat. Struct. Biol. 3, 796-802; Poole, K. I. V., Lorenz, M., Ellison, P., Evans, G., Rosenbaum, G., Boesecke, P., Holmes, K. C., and Cremo, C. R. (1997) J. Muscle Res. Cell Motility 18, 264]. We discuss tilting upon ADP release with respect to our measurements, previous measurements with skS1, and nucleotide concentrations in smooth muscle. We propose that these data suggest a strain-dependent ADP release mechanism that may be accentuated in smooth muscles.     

A new regulatory element that augments the Tax-dependent enhancer of human T-cell leukemia virus type 1 and cloning of cDNAs encoding its binding proteins

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) trans activates the 21-bp enhancer of HTLV-1. A sequence of more than two copies of the 21-bp enhancer is efficiently activated by Tax, but one copy is not activated extensively. Another sequence (TRE-2, positions -163 to -117) adjacent to the 21-bp enhancer in the long terminal repeat of HTLV-1 can enhance a single copy of the 21-bp enhancer activity in trans activation by Tax. This sequence contains motifs related to the Ets- and NF-kappa B-binding sequences, but mutations at these sites indicated that neither is responsive to cooperation with the 21-bp enhancer. A deletion mutation of TRE-2 identified 25 bases at positions -158 to -134 (TRE-2S) as an essential sequence, and TRE-2S was sufficient to give maximum cooperation with one copy of the 21-bp enhancer in trans activation by Tax protein. Using TRE-2S as a probe, we screened a cDNA library of HUT102 cells by the Southwestern (DNA-protein) procedure and isolated two cDNA clones, THP-1 and -2. These two clones encode TRE-2S-binding proteins, and they differ by only an extra 17 amino acids in THP-2. Both THP proteins contain five zinc finger motifs which are strikingly similar to those of the GLI family, an amplified gene product in glyoma cells. The binding site of THP-1 and -2 was GAACCACCCA in TRE-2S, which is highly homologous to the GLI-binding site. These results suggest that binding of THP to TRE-2S may be involved in cooperation with one copy of the 21-bp enhancer in responding to Tax trans activation.     

Glial-cell-line-derived neurotrophic factor is required for bud initiation from ureteric epithelium

The shapes of different organs can be explained largely by two fundamental characteristics of their epithelial rudiments - the pattern of branching and the rate of proliferation. Glial-cell-line-derived neurotrophic factor (GDNF) has recently been implicated in the development of metanephric ureteric epithelium (Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E. J., Huang, S. P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996). Nature 382, 73-76; Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996). Nature 382, 70-73; Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Proc. Nat. Acad. Sci. USA 93, 10657-10661). We have analysed the target cells of GDNF and the manner in which it controls ureteric development, and have compared it with other growth factors that have been associated with the regulation of branching morphogenesis, namely hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGFbeta1). We show that GDNF binds directly to the tips of ureteric bud branches, and that it has the ability to promote primary ureteric buds from various segments of Wolffian duct and to attract ureteric branches towards the source of GDNF. It increases cell adhesion, but is not obviously mitogenic for ureteric cells. The data indicate that GDNF is required primarily for bud initiation. Comparison of GDNF, HGF and TGFbeta1 suggests that the latter act later than GDNF, and may represent a partially redundant set of mesenchyme-derived growth factors that control ureteric development. Thus, GDNF is the first defined inducer in the embryonic metanephric kidney.