ARF-BP1/Mule Is a Critical Mediator of the ARF Tumor Suppressor

SummaryAlthough the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status

Protective effect of midazolam against convulsion in neonatal rats via down-regulation of LC3 and Beclin-1 expression

Purpose: To investigate the effect of midazolam on growth of neurocytes in vitro and in neonatal rats. Methods: Neurocyte proliferation and activity of lactate dehydrogenase were assessed by MTT and lactate dehydrogenase assays, respectively. Western blotting was used to determine the effect of midazolam on LC3, Bax, p62 and Beclin-1 protein expressions. Results: The suppression of neurocyte proliferation byconvulsion was alleviated significantly (p < 0.05) by midazolum treatment. Exposure of convulsion model of neurocytes to midazolum suppressed LC3, Bax, p62 and Beclin-1 protein expression. Midazolum exposure of convulsion model of neurocytes suppressed LDH, caspase-3, caspase-8 and caspase-9 activities. The 3-MA (autophagy inhibitor) treatment also significantly (p < 0.05) promoted neurocyte viability after convulsion induction. In convulsion-induced neurocytes, 3-MA exposure suppressed expression of caspase-3/8/9, LC3, Bax, Beclin-1 and p62, while application of midazolum treatment to the rats with convulsion markedly decreased brain water content and neurocyte apoptosis (p < 0.05). Treatment with midazolum inhibited LC3, p62 and Beclin-1 expression in the rat model of convulsion. Conclusion: Midazolum promotes neurocyte proliferation and inhibits edema development via downregulation of autophagy. Therefore, midazolum can potentially be used for the treatment of convulsion, but further studies need to be carried out first. Keywords: Convulsion, Neurocytes, Caspase, Autophagy, Mitochondrial pathwa

Pathological phenotypes and in vivo DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella

Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction–modification (R–M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R–M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB–E, while restriction involves additional three genes dptF–H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R–M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification.National Natural Science Foundation (China) (Grant 31170085)National Natural Science Foundation (China) (Grant 31070058)Ministry of Science and Technology of the People's Republic of China (973 and 863 Programs)China Scholarship CouncilNational Science Foundation (U.S.) (Grant CHE-1019990)Shanghai Municipal Council of Science and Technology. Shanghai Pujiang Program (Grant 12PJD021

Evaluation of a Novel Biphasic Culture Medium for Recovery of Mycobacteria: A Multi-Center Study

on L-J slants. Automated liquid culture systems are expensive. A low-cost culturing medium capable of rapidly indicating the presence of mycobacteria is needed. The aim of this study was to develop and evaluate a novel biphasic culture medium for the recovery of mycobacteria from clinical sputum specimens from suspected pulmonary tuberculosis patients.<0.001).

Characterization of ARF-BP1/HUWE1 Interactions with CTCF, MYC, ARF and p53 in MYC-Driven B Cell Neoplasms

Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of ARF-BP1 resulted in elevated steady state levels of p53, growth arrest and apoptosis. Co-immunoprecipitation studies identified a multiprotein complex comprised of ARF-BP1, ARF, p53, MYC and the multifunctional DNA-binding factor, CTCF, which is involved in the transcriptional regulation of MYC, p53 and ARF. ARF-BP1 bound and ubiquitylated CTCF leading to its proteasomal degradation. ARF-BP1 and CTCF thus appear to be key cofactors linking the MYC proliferative and p53-ARF apoptotic pathways. In addition, ARF-BP1 could be a therapeutic target for MYC-driven B lineage neoplasms, even if p53 is inactive, with inhibition reducing the transcriptional activity of MYC for its target genes and stabilizing the apoptosis-promoting activities of p53

Electromagnetic Wave Theory and Applications

Contains table of contents for Section 3 and reports on four research projects.California Institute of Technology/Jet Propulsion Laboratory Agreement 959548National Aeronautics and Space Administration Grant NAGW-1617National Aeronautics and Space Administration Agreement 958461U.S. Navy - Office of Naval Research Grant N00014-89-J-1107U.S. Navy - Office of Naval Research Grant N00014-92-J-1616U.S. Navy - Office of Naval Research Grant N00014-92-J-4098Digital Equipment CorporationJoint Services Electronics Program Contract DAAL03-92-C-0001U.S. Navy - Office of Naval Research Agreement N00014-90-J-1002U.S. Navy - Office of Naval Research Agreement N00014-89-J-1019DEMACOU.S. Army Cold Regions Research and Engineering Laboratory Contract DACA89-93-K-0009U.S. Department of Transportation Agreement DTRS-57-92-C-00054TTD1Advanced Research Projects Agency/Consortium for Superconducting Electronics Contract MDA972-90-C-0021National Science Foundation Fellowship MIP 88-58764National Science Foundatio

ARF–NRF2: A new checkpoint for oxidative stress responses?

NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor which plays a major role in oxidative stress responses by regulating antioxidant gene expression. We have recently identified the ARF tumor suppressor as a key regulator of NRF2. ARF can significantly inhibit NRF2 transcriptional activities, and the ARF-NRF2 interaction may function as a novel checkpoint for oxidative stress responses

Analysis on Local Adhesion Differences of Hot Dip Galvanized Coating on Low-Alloy High-Strength Angle Steel

The reasons for the local adhesion differences of the hot-dip galvanized coating on high-strength angle steel were analyzed by means of scanning electron microscopy (SEM) and optical microscopy (OM)， and the microstructures of the coating and steel base in the good bonding area and poor bonding area of galvanized angle steel were compared. Results showed that: (1) The adhesion difference of the coating on steel substrate were closely related to the microstructure of the caoting. The coating area with good bonding strength was characterized with small column-like or lumpy ζ phase and discontinuous δ phase. When the coating suffering hammering， the hammered area was generally in the form of pits， and the alloy layer was extruded and deformed without cracks. The coating area with poor bonding strength consisted of a coarse columnar or bulky ζ phase and a continuous and thick δ layer， which would be stripped from the substrate interface when the coating suffering hammering. (2) Moreover， the surface microstructure of steel substrate had influence on the galvanized coating. During galvanizing， equilibrium structure was in favor of the diffusion of Fe， facilitating the growth of ζ and limiting the growth of δ， which was beneficial to the bonding between coating and substrate. However， the appearance of non-equilibrium structure could inhibit the diffusion of Fe， lower the growth of ζ and benefit to the growth of δ because of the solution strengthening，lattice distortion and internal residual stress，which was harmful for the bonding strength of hot-dip galvanized coating

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells

IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p

Abstract Background Long noncoding RNAs (LncRNAs) represent a class of widespread and diverse endogenous RNAs that can posttranscriptionally regulate gene expression through the interaction with RNA-binding proteins and micro RNAs (miRNAs). Here, we report that in breast carcinoma cells, the insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to lncRNA urethral carcinoma-associated 1 (UCA1) and suppresses the UCA1-induced invasive phenotype. Methods RT-qPCR and RNA sequence assays were used to investigate the expression of UCA1 and miRNAs in breast cancer cells in response to IMP1 expression. The role of IMP1-UCA1 interaction in cell invasion was demonstrated by transwell analysis through loss-of-function and gain-of-function effects. RNA pull-down and RNA binding protein immunoprecipitation (RIP) were performed to confirm the molecular interactions of IMP1-UCA1 and UCA1-miR-122-5p involved in breast cancer cells. Results In breast cancer cells, IMP1 interacts with UCA1 via the “ACACCC” motifs within UCA1 and destabilizes UCA1 through the recruitment of CCR4-NOT1 deadenylase complex. Meanwhile, binding of IMP1 prevents the association of miR-122-5p with UCA1, thereby shifting the availability of miR-122-5p from UCA1 to the target mRNAs and reducing the UCA1-mediated cell invasion. Accordingly, either IMP1 silencing or UCA1 overexpression resulted in reduced levels of free miR-122-5p within the cytoplasm, affecting miR-122-5p in regulating its target mRNAs. Conclusions Our study provides initial evidence that interaction between IMP1 and UCA1 enhances UCA1 decay and competes for miR-122-5p binding, leading to the liberation of miR-122-5p activity and the reduction of cell invasiveness