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71.
Youjun Li Hao Zhou Fengzhi Li Siew Wee Chan Zhijie Lin Zhiyi Wei Zhou Yang Fusheng Guo Chun Jye Lim Wancai Xing Yuequan Shen Wanjin Hong Jiafu Long Mingjie Zhang 《Cell research》2015,25(7):801-817
The tumor suppressor Merlin/NF2 functions upstream of the core Hippo pathway kinases Lats1/2 and Mst1/2, as well as the nuclear E3 ubiquitin ligase CRL4DCAF1. Numerous mutations of Merlin have been identified in Neurofibromatosis type 2 and other cancer patients. Despite more than two decades of research, the upstream regulator of Merlin in the Hippo pathway remains unknown. Here we show by high-resolution crystal structures that the Lats1/2-binding site on the Merlin FERM domain is physically blocked by Merlin''s auto-inhibitory tail. Angiomotin binding releases the auto-inhibition and promotes Merlin''s binding to Lats1/2. Phosphorylation of Ser518 outside the Merlin''s auto-inhibitory tail does not obviously alter Merlin''s conformation, but instead prevents angiomotin from binding and thus inhibits Hippo pathway kinase activation. Cancer-causing mutations clustered in the angiomotin-binding domain impair angiomotin-mediated Merlin activation. Our findings reveal that angiomotin and Merlin respectively interface cortical actin filaments and core kinases in Hippo signaling, and allow construction of a complete Hippo signaling pathway. 相似文献
72.
73.
The aim of this study was to establish a basic manipulation protocol of preantral follicles for deriving developmentally competent oocytes. Primary, early and late secondary follicles retrieved from the ovaries of 14-day-old F1 (C57BL/6 x DBA2) female mice mechanically or enzymatically were cultured singly and in vitro growth of the follicles and maturation of intrafollicular oocytes were subsequently monitored. A mechanical method retrieved more (p < 0.0001) follicles (339 +/- 48 vs. 202 +/- 28) than an enzymatic method. However, the enzymatic method collected more singly isolated follicles that could be provided for subsequent culture (102 +/- 26 vs. 202 +/- 28). When an enzymatic method was employed, early and late secondary follicles required 9 and 6 days for reaching the maximal incidence of the pseudoantral stage. However, primary follicles were not possible to develop into the pseudoantral stage. The optimal duration of oocyte maturation from the onset of follicle culture was 7 days and 5-7 days for early and late secondary follicles, respectively. A general decrease in oocyte diameter (65.2-65.53 microm vs. 75 microm) and zona thickness (5.41-5.74 microm vs. 7.76 microm) was detected in in vitro-derived compared with in vivo-derived matured oocytes. Pronuclear formation was detected in 86-94% of mature oocytes after parthenogenetic activation and no significant difference was detected among groups. These results showed that preantral follicles retrieved by an enzymatic method underwent step-by-step growth in vitro, which could yield mature oocytes. 相似文献
74.
Lim KT Jang G Ko KH Lee WW Park HJ Kim JJ Kang SK Lee BC 《Animal reproduction science》2008,103(3-4):239-248
The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer. 相似文献
75.
Yeom JS Kim ES Lim KJ Oh JH Sohn MJ Yoo SB Kim E Bae I Jung YJ Park JW 《The Journal of parasitology》2008,94(6):1410-1414
The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse. 相似文献
76.
Li Z McNulty DE Marler KJ Lim L Hall C Annan RS Sacks DB 《The Journal of biological chemistry》2005,280(14):13871-13878
In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells. 相似文献
77.
Alternative splicing of mRNA allows many gene products with different functions to be produced from a single coding sequence. Exon skipping is the most commonly known alternative splicing mechanism. A comprehensive database of alternative splicing by exon skipping is made available for the human genome data. 1,229 human genes are identified to exhibit alternative splicing by exon skipping. Availability: http://sege.ntu.edu.sg/wester/ashes/. 相似文献
78.
Structural 3D motifs in RNA play an important role in the RNA stability and function. Previous studies have focused on the characterization and discovery of 3D motifs in RNA secondary and tertiary structures. However, statistical analyses of the distribution of 3D motifs along the RNA appear to be lacking. Herein, we present a novel strategy for evaluating the distribution of 3D motifs along the RNA chain and those motifs whose distributions are significantly non-random are identified. By applying it to the X-ray structure of the large ribosomal subunit from Haloarcula marismortui, helical motifs were found to cluster together along the chain and in the 3D structure, whereas the known tetraloops tend to be sequentially and spatially dispersed. That the distribution of key structural motifs such as tetraloops differ significantly from a random one suggests that our method could also be used to detect novel 3D motifs of any size in sufficiently long/large RNA structures. The motif distribution type can help in the prediction and design of 3D structures of large RNA molecules. 相似文献
79.
Jinsook Kim Christina Lilliehook Amanda Dudak Johannes Prox Paul Saftig Howard J. Federoff Seung T. Lim 《The Journal of biological chemistry》2010,285(30):22919-22926
Nectin-1 is known to undergo ectodomain shedding by α-secretase and subsequent proteolytic processing by γ-secretase. How secretase-mediated cleavage of nectin-1 is regulated in neuronal cells and how nectin-1 cleavage affects synaptic adhesion is poorly understood. We have investigated α-and γ-secretase-mediated processing of nectin-1 in primary cortical neurons and identified which protease acts as a α-secretase. We report here that NMDA receptor activation, but not stimulation of AMPA or metabotropic glutamate receptors, resulted in robust α- and γ-secretase cleavage of nectin-1 in mature cortical neurons. Cleavage of nectin-1 required influx of Ca2+ through the NMDA receptor, and activation of calmodulin, but was not dependent on calcium/calmodulin-dependent protein kinase II (CaMKII) activation. We found that ADAM10 is the major secretase responsible for nectin-1 ectodomain cleavage in neurons and the brain. These observations suggest that α- and γ-secretase processing of nectin-1 is a Ca2+/calmodulin-regulated event that occurs under conditions of activity-dependent synaptic plasticity and ADAM10 and γ-secretase are responsible for these cleavage events. 相似文献
80.
Yoon YS Yoon DS Lim IK Yoon SH Chung HY Rojo M Malka F Jou MJ Martinou JC Yoon G 《Journal of cellular physiology》2006,209(2):468-480
Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H2O2 (200 microM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF beta1- or H2O2-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DeltaTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence. 相似文献