Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.     

Histone folds mediate selective heterodimerization of yeast TAF(II)25 with TFIID components yTAF(II)47 and yTAF(II)65 and with SAGA component ySPT7

We show that the yeast TFIID (yTFIID) component yTAF(II)47 contains a histone fold domain (HFD) with homology to that previously described for hTAF(II)135. Complementation in vivo indicates that the yTAF(II)47 HFD is necessary and sufficient for vegetative growth. Mutation of highly conserved residues in the alpha1 helix of the yTAF(II)47 HFD results in a temperature-sensitive phenotype which can be suppressed by overexpression of yTAF(II)25, as well as by yTAF(II)40, yTAF(II)19, and yTAF(II)60. In yeast two-hybrid and bacterial coexpression assays, the yTAF(II)47 HFD selectively heterodimerizes with yTAF(II)25, which we show contains an HFD with homology to the hTAF(II)28 family We additionally demonstrate that yTAF(II)65 contains a functional HFD which also selectively heterodimerizes with yTAF(II)25. These results reveal the existence of two novel histone-like pairs in yTFIID. The physical and genetic interactions described here show that the histone-like yTAF(II)s are organized in at least two substructures within TFIID rather than in a single octamer-like structure as previously suggested. Furthermore, our results indicate that ySPT7 has an HFD homologous to that of yTAF(II)47 which selectively heterodimerizes with yTAF(II)25, defining a novel histone-like pair in the SAGA complex.     

Therapeutic Strategies for Localized Prostate Cancer II: Perineal Prostatectomy, X-Rays, Protons, Neutrons, and Combination Brachytherapy

Application of improved imaging, diagnostic, and computer techniques is beginning to have an impact on the management of localized prostate cancer. It is possible to perform a range of surgical and radiation procedures with less morbidity than in the past. The changes in therapy for patients with localized disease derive from better knowledge of anatomy for invasive procedures and optimization of virtual planning for noninvasive methods. Perineal prostatectomy and combinations of beam and seed radiation offer both patient and physician reasonable therapeutic options.     

On the age of the hominid fossils at the Sima de los Huesos, Sierra de Atapuerca, Spain: paleomagnetic evidence

We report new paleomagnetic data for the Middle Pleistocene hominid-bearing strata in the Sima de los Huesos, North Spain. Sediments (brown muds with human and bear fossils and the underlying sterile clayey and sandy unit) preserve both normal and reversed magnetic components. The sterile unit has exclusively reversed magnetization, dating back to the Matuyama Chron, and thus is Lower Pleistocene in age. The overlying fossiliferous muds have a dominant normal magnetization that overprints a partially resolved reversed magnetization. These data are compatible with one of the reversal events that occurred during the Brunhes Chron. Combined with the existing U-series dates and evidence from the macro- and microfauna, these paleomagnetic results suggest an age of the hominid fossils between 325 to 205 ka, whereas the underlying sand and silts are older than 780 ka.