Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.

MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.

No abstract

Maturation-promoting factor (MPF), which is functionally defined by Its ability to induce frog oocyte maturation independent ofprotein synthesis, is hypothesized to be the mitotic inducer in eukaryotic cells. Previous studies have demonstrated that the cdc2 protein kinase complex (p34c-cyclin) meets the criteria for MPF. In the present study, weshow that MPF activity in extracts of unfertilized Xenopus eggs can be resolved into three fractions by Q-Sepharose chromatography. Of the total MPF activity recovered, %20% was in the flow-through fraction that was accounted for by the cdc2 kinase complex, -40% was in the 0.2 M NaCI eluate, and the remaining =40% was in the 0. Almost 20 yr after its discovery, MPF was purified to near homogeneity from Xenopus and starfish oocytes (6, 7), although the recovery was low ( 1%). Significantly, as a result ofthis purification, one component ofthe purified MPF was discovered to be the 34-kDa gene product of cdc2 (p34cdc2) (7,8), which, based on genetic studies in yeast, is essential for entry into mitosis and encodes a highly conserved kinase (9). p34cdc2 is complexed with cyclin and is the major M phase-specific H1 kinase (10, 11). Consequently, MPF, originally defined as a biological activity, could now be associated with a specific molecule, namely p34cdc2-cyclin or the cdc2 kinase complex.In the present study, we addressed the question whether the cdc2 kinase complex is the only molecular species that meets the criteria for MPF. This question was raised for several reasons. For example, although Labbe et al. (7) showed that MPF copurified with H1 kinase in each of six successive chromatographic fractionations of starfish oocyte extract, Gerhart et al. (5) and Nguyen-Gia et al. (12) observed that MPF and histone kinase activities did not copurify during chromatography of Xenopus egg extract. In addition, we have shown that the mitosis-specific monoclonal antibody (mAb) MPM-2 inhibits Xenopus oocyte maturation and depletes MPF activity from both unfertilized Xenopus egg extract and mitotic HeLa cell extract (13) but does not recognize the cdc2 kinase complex (14). These facts suggested that MPF activity in the M phase extract might involve more than one factor and, thus, a more comprehensive investigation of MPF activity in M phase extract was required. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 11530The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.