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Kinetic properties of the purified a, fi, and y subspecies of protein kinase C (PKC) to respond to diacylglycerol, phosphatidylserine (PtdSer), and Ca2l were reinvestigated in the presence of several fatty acids. Although responses of these enzyme subspecies to the lipids slightly differed from one another, the reaction velocity of these subspecies was significantly enhanced by synergistic action of diacylglycerol and a cis-unsaturated fatty acid. Arachidonic, oleic, linoleic, linolenic, and docosahexaenoic acids were active in this role, whereas saturated fatty acids such as palmitic and stearic acids were inactive. Elaidic acid was also inactive. In the presence of both PtdSer and diacylglycerol, the cis-unsaturated fatty acids increased further an apparent affinity of PKC to Ca2' and allowed the enzyme to exhibit almost full activation at nearly basal levels of Ca21 concentration. The concentration of fatty acid giving rise to the maximum activation of enzyme was -20-50 ,uM. The result presented herein implies that the receptor-mediated release of unsaturated fatty acids from phospholipids may take part, in synergy with diacylglycerol, in the activation of PKC even when the Ca2+ concentration is low. A possibility arises, then, that the activation of PKC is an integral part of the signal-induced degradation cascade of various membrane phospholipids, which is initiated by the actions of phospholipase C and phospholipase A2.The hydrolysis of phosphatidylinositol, particularly its 4,5-bisphosphate, catalyzed by phospholipase C is generally accepted to be crucially important to initiate signal transduction for eliciting cellular responses (1-3). Recent studies have suggested that receptor-mediated hydrolysis of phosphatidylcholine may also be involved in transmembrane signaling (for a review, see refs. 4 and 5). In fact, it is becoming clear that both phospholipase A2 (6) and phospholipase D (7-11; see ref. 4 for additional references) are activated in a signaldependent manner.Early reports from this laboratory (12, 13) have described that diacylglycerol produced in membranes activates protein kinase C (PKC) in the presence of Ca2' and phospholipids, especially phosphatidylserine (PtdSer). Kinetic analysis has shown that diacylglycerol increases an apparent affinity of the enzyme for Ca2' and PtdSer and thereby activates PKC in the micromolar range of Ca2+ concentrations (13). Subsequent studies in several laboratories (14-21) have found that, in the absence of PtdSer, unsaturated fatty acids such as arachidonic and oleic acids may activate PKC to various degrees, most efficiently activating the y subspecies, and a potential role ofunsaturated fatty acids as second messengers has been postulated. More recently, synergistic action of fatty acids and diacylglycerol for the activation of PKC has been reported (refs. 22-25; also S. G. Chen and K. Murakami, personal communication). Studies on the interaction of fatty acids with diacylglycerol and Ca2l have revealed, however, conflicting results. In some studie...

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3+ CD69+ T cells and a higher production of TNF-α from spleen cells. When arthritis is induced, Cry1−/−Cry2−/− mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-α, IL-1β and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1−/−Cry2−/− mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti–TNF-α Ab significantly reduced the severity and halted the progression of the arthritis of Cry1−/−Cry2−/− mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1−/−Cry2−/− mice significantly reduced the trans activation of TNF-α gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.

So far, only a few of the interactions between the ≈30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 Å resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed β-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 β-propeller and the other one from the β-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic α-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 β-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 β-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 β-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82•Nup159•Nup116 complex.assembly | evolutionary conservation | nucleo-cytoplasmic transport | site-directed mutagenesis | X-ray crystallography F ollowing the discovery of the nuclear pore complex (NPC) in the 1950s, numerous electron microscopic studies since have refined our understanding of its architecture to a resolution in the upper single-digit nanometer range (1). Collectively, these studies showed that NPCs are embedded in ≈100 nm wide circular openings, resulting from a circumscribed fusion of the double membrane of the nuclear envelope. The NPC consists of a symmetric central core and asymmetric filament-like structures that project to the nucleoplasmic and cytoplasmic sides. The core of the NPC displays a twofold axis of symmetry in the plane of the membrane and an eightfold rotational symmetry in the nucleocytoplasmic direction. As observed by cryoelectron tomography, the NPC can undergo huge structural changes (2) that remain to be characterized at the atomic level.Beginning in the 1980s, biochemical and genetic analyses of the NPC and its surrounding pore membrane domain of the nuclear envelope has yielded an inventory consisting of ≈30 distinct NPC proteins termed nups (for nucleoporins) and of three distinct integral membrane proteins termed poms (for pore membrane proteins) that anchor the NPC. "Asymmetric" nups make up the filament-like structures...

ABSTRACT2-Lysophosphatidylcholine (lysoPtdCho), a product of the hydrolysis of phosphatidylcholine catalyzed by phospholipase A2, greatly potentiates the activation of human resting T lymphocytes that is induced by a membranepermeant diacylglycerol plus a calcium ionophore, as determined by the expression of the a subunit of the interleukin 2 receptor and thymidine incorporation into DNA. LysoPtdCho per se is inactive unless both diacylglycerol and a calcium ionophore are present. This effect of lysoPtdCho is also observed when diacylglycerol is replaced by a tumor-promoting phorbol ester. Other lysophosphatides including lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid are inert except for lysophosphatidylethanolamine, which is far less effective than IysoPtdCho. Tracer experiments with radioactive choline indicate that, when T lymphocytes are stimulated with an antigenic signal, lysoPtdCho is indeed produced in a time-dependent fashion, although the concentration of this lysophospholipid accumulated remains to be quantitated. It suggests that phospholipase A2 is directly involved in the signal transduction pathway through protein kinase C to induce long-term cellular responses.A single dose of a tumor-promoting phorbol ester, together with a calcium ionophore, can induce T-lymphocyte activation, as determined by secretion of interleukin 2, expression of the a subunit of the interleukin 2 receptor (IL-2Ra), and incorporation of thymidine into DNA (for a review, see ref.1). On the other hand, a single dose of a membrane-permeant diacylglycerol (DAG) is normally insufficient to induce cell activation due to its rapid metabolism within the cell, and it is known that sustained activation of protein kinase C (PKC) by a large dose or repeated doses of a membrane-permeant DAG is essential to induce activation of T lymphocytes (2, 3). The formation of DAG from receptor-mediated hydrolysis of inositol phospholipids is, however, normally transient, and recent evidence strongly suggests that, at relatively later phases in cellular responses, DAG is produced from signalinduced breakdown of phosphatidylcholine (PtdCho), which is initiated presumably by the activation of phospholipase D (for reviews, see refs. 4 and 5). Previous reports from this laboratory (6, 7) suggest that cis-unsaturated fatty acids greatly enhance PKC activation when DAG is available. It has also been briefly described that 2-lysophosphatidylcholine (lysoPtdCho), the other product of receptor-mediated hydrolysis of PtdCho by phospholipase A2, synergizes with a membrane-permeant DAG to activate human resting T lymphocytes (8). In fact, activation of both phospholipase C and phospholipase A2 by a single agonist has been reported by Axelrod and coworkers (9). The receptor-mediated degradation of various membrane phospholipids, therefore, may be important in causing cellular responses such as cell proliferation and differentiation. The studies presented herein were undertaken to explore further the detailed kinects of the lysoPtd...

Although a single dose of phorbol 12-myristate 13-acetate (PMA) allowed HL-60 cells to differentiate to macrophages, a single dose of membrane-permeant diacylglycerol (DAG), 1,2-dioctanoylglycerol (1,2-DiC8), was normally isufcient to differentiate these cells. These cells metabolized 1,2-DiCs very rapidly, and 1,2-DiC8 available to protein kinase C (PKC) activation was removed from the incubation medium at a rate proportional to cell density. However, increasing the duration of exposure of HL-60 cells to this DAG either by its repeated addition or by decreasing the cell density greatly enhanced their differentiation to macrophages as Measured by CD11b expression. During this differentiation induced by DAG, neither measurable translocation nor depletion (down-regulation) of PKC was observed. When the cells were exposed to PMA, on the other hand, some PKC subspecies were instantaneously translocated to membranes and subsequently disappeared very quickly, whereas the a-subspecies was decreased to the level of w6O% of the resting cell, but thereafter its activity was maintained at a nearly constant level in membranes. After -4 hr, the PKC subspecies, once depleted, reappeared gradually in the membrane fraction.The results suggest that sa activation of PKC is essential to differentiation of HL-60 cells to macrophages, and depletion of the enzyme is not needed. Perhaps translocation of PKC represents an extreme state of the active form of the enzyme, which may result from PMA action, and the a-subspecies presumably-plays a key role in HL-60 cell differentiation.The HL-60 cell line is frequently used as a model system for investigation of the mechanism of cell differentiation, since retinoic acid and several other chemicals lead this cell to differentiate to a granulocyte, whereas phorbol 12-myristate

After introducing biological DMARDs, increase of ccfDNA at 8 weeks was associated with improvement of disease activities. Compared with biomarkers reported, ccfDNA is able to predict the early therapeutic effects of biological DMARDs in RA patients.