The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4-BSLF1-BBLF2/3 complex

The Epstein-Barr virus (EBV)-encoded replication proteins that account for the basic reactions at the replication fork are thought to be the EBV Pol holoenzyme, consisting of the BALF5 Pol catalytic and the BMRF1 Pol accessory subunits, the putative helicase-primase complex, comprising the BBLF4, BSLF1, and BBLF2/3 proteins, and the BALF2 single-stranded DNA-binding protein. Immunoprecipitation analyses using anti-BSLF1 or anti-BBLF2/3 protein-specific antibody with clarified lysates of B95-8 cells in a viral productive cycle suggested that the EBV Pol holoenzyme physically interacts with the BBLF4-BSLF1-BBLF2/3 complex to form a large complex. Although the complex was stable in 500 mM NaCl and 1% NP-40, the BALF5 protein became dissociated in the presence of 0.1% sodium dodecyl sulfate. Experiments using lysates from insect cells superinfected with combinations of recombinant baculoviruses capable of expressing each of viral replication proteins showed that not the BMRF1 Pol accessory subunit but rather the BALF5 Pol catalytic subunit directly interacts with the BBLF4-BSLF1-BBLF2/3 complex. Furthermore, double infection with pairs of recombinant viruses revealed that each component of the BBLF4-BSLF1-BBLF2/3 complex makes contact with the BALF5 Pol catalytic subunit. The interactions of the EBV DNA polymerase with the EBV putative helicase-primase complex warrant particular attention because they are thought to coordinate leading- and lagging-strand DNA synthesis at the replication fork.     

Methods of synthesis of glycosyl fluorides

In recent years glycosyl fluorides have been utilized as versatile sugar donors in the synthesis of natural products and carbohydrates. This paper provides an update on the advances made in the preparation of glycosyl fluorides during the last decade (1988-1998).     

Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation. To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes. Our results were as follows. (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis. (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain. (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells. (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1. (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus. (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay. Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1. These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1.     

Gene pathway analysis of the mechanism by which the Rho-associated kinase inhibitor Y-27632 inhibits apoptosis in isolated thawed human embryonic stem cells

Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1β, TGF-β, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1β, TGF-β, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-β receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.     

Leu309 plays a critical role in the encapsulation of substrate protein into the internal cavity of GroEL

In the crystal structure of the native GroEL.GroES.substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL.GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K).substrate complex failed to bind GroES to produce a stable GroEL(L309K).GroES.substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K).GroES.substrate complex was stabilized with BeF(x), but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu(309)), appears to play a critical role in encapsulation of the substrate.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.