Discoloration Improvement by Mechanically-Milled Binary Oxides as Radiopacifier for Mineral Trioxide Aggregates.

Mineral trioxide aggregates (MTA) have been widely used in endodontic treatments, but after some time, patients suffer tooth discoloration due to the use of bismuth oxide (Bi2O3) as a radiopacifier. Replacement of Bi2O3 with high energy ball-milled single (zirconia ZrO2; hafnia, HfO2; or tantalum pentoxide, Ta2O5) or binary oxide powder was attempted, and corresponding discoloration improvement was investigated in the present study. Bi2O3-free MTA is expected to exhibit superior discoloration. The radiopacity, diametral tensile strength, and discoloration of MTA-like cements prepared from the as-milled powder were investigated. Experimental results showed that MTA-like cements prepared using Ta2O5 exhibited a slightly higher radiopacity than that of HfO2 but had a much higher radiopacity than ZrO2. Milling treatment (30 min to 3 h) did not affect the radiopacities significantly. These MTA-like cements exhibited superior color stability (all measured ΔE00 < 1.0) without any perceptible differences after UV irradiation. MTA-like cements prepared using ZrO2 exhibited the best color stability but the lowest radiopacity, which can be improved by introducing binary oxide. Among the investigated samples, MTA-like cement using (ZrO2)50(Ta2O5)50 exhibited excellent color stability and the best overall performance with a radiopacity of 3.25 mmAl and a diametral tensile strength of 4.39 MPa.

Novel function and intracellular localization of methionine adenosyltransferase 2beta splicing variants.

Human methionine adenosyltransferase 2beta (MAT2beta) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2beta variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.

DNA-PKcs regulates a single-stranded DNA endonuclease activity of Artemis.

Human nuclease Artemis belongs to the metallo-beta-lactamase protein family. It acquires double-stranded DNA endonuclease activity in the presence of DNA-PKcs. This double-stranded DNA endonuclease activity is critical for opening DNA hairpins in V(D)J recombination and is thought to be important for processing overhangs during the nonhomologous DNA end joining (NHEJ) process. Here we show that purified human Artemis exhibits single-stranded DNA endonuclease activity. This activity is proportional to the amount of highly purified Artemis from a gel filtration column. The activity is stimulated by DNA-PKcs and modulated by purified antibodies raised against Artemis. Moreover, the divalent cation-dependence and sequence-dependence of this single-stranded endonuclease activity is the same as the double-stranded DNA endonuclease activity of Artemis:DNA-PKcs. These findings further expand the range of DNA substrates upon which Artemis and Artemis:DNA-PKcs can act. The findings are discussed in the context of NHEJ.

Identification of galectin-3-binding protein as a factor secreted by tumor cells that stimulates interleukin-6 expression in the bone marrow stroma.

We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications.

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.

Heptad repeats regulate protein phosphatase 2a recruitment to I-kappaB kinase gamma/NF-kappaB essential modulator and are targeted by human T-lymphotropic virus type 1 tax.

The switching on-and-off of I-kappaB kinase (IKK) and NF-kappaB occurs rapidly after signaling. How activated IKK becomes down-regulated is not well understood. Here we show that following tumor necrosis factor-alpha stimulation, protein phosphatase 2A (PP2A) association with IKK is increased. A heptad repeat in IKKgamma, helix 2 (HLX2), mediates PP2A recruitment. Two other heptad repeats downstream of HLX2, termed coiled-coil region 2 (CCR2) and leucine zipper (LZ), bind HLX2 and negatively regulate HLX2 interaction with PP2A. HTLV-1 transactivator Tax also binds HLX2, and this interaction is enhanced by CCR2 but reduced by LZ. In the presence of Tax, PP2A-IKKgamma binding is greatly strengthened. Interestingly, peptides spanning CCR2 and/or LZ disrupt IKKgamma-Tax and IKKgamma-PP2A interactions and potently inhibit NF-kappaB activation by Tax and tumor necrosis factor-alpha. We propose that when IKK is resting, HLX2, CCR2, and LZ form a helical bundle in which HLX2 is sequestered. The HLX2-CCR2-LZ bundle becomes unfolded by signal-induced modifications of IKKgamma or after Tax binding. In this conformation, IKK becomes activated. IKKgamma then recruits PP2A via the exposed HLX2 domain for rapid down-regulation of IKK. Tax-PP2A interaction, however, renders PP2A inactive, thus maintaining Tax-PP2A-IKK in an active state. Finally, CCR2 and LZ possibly inhibit IKK activation by stabilizing the HLX2-CCR2-LZ bundle.

DNA-PKcs dependence of Artemis endonucleolytic activity, differences between hairpins and 5' or 3' overhangs.

During V(D)J recombination, the RAG proteins create DNA hairpins at the V, D, or J coding ends, and the structure-specific nuclease Artemis is essential to open these hairpins prior to joining. Artemis also is an endonuclease for 5' and 3' overhangs at many DNA double strand breaks caused by ionizing radiation, and Artemis functions as part of the nonhomologous DNA end joining pathway in repairing these. All of these activities require activation of the Artemis protein by interaction with and phosphorylation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In this study, we have identified a region of the Artemis protein involved in the interaction with DNA-PKcs. Furthermore, the biochemical and functional analyses of C-terminally truncated Artemis variants indicate that the hair-pin opening and DNA overhang endonucleolytic features of Artemis are triggered by DNA-PKcs in two modes. First, autoinhibition mediated by the C-terminal tail of Artemis is relieved by phosphorylation of this tail by DNA-PKcs. Thus, C-terminally truncated Artemis derivatives imitate DNA-PKcs-activated wild type Artemis protein and exhibit intrinsic hairpin opening activity. Second, DNA-PKcs may optimally configure 5' and 3' overhang substrates for the endonucleolytic function of Artemis.

FLI-1 functionally interacts with PIASxalpha, a member of the PIAS E3 SUMO ligase family.

FLI-1 is a transcription factor of the ETS family that is involved in several developmental processes and that becomes oncogenic when overexpressed or mutated. As the functional regulators of FLI-1 are largely unknown, we performed a yeast two-hybrid screen with FLI-1 and identified the SUMO E3 ligase PIASxalpha/ARIP3 as a novel in vitro and in vivo binding partner of FLI-1. This interaction involved the ETS domain of FLI-1 and required the integrity of the SAP domain of PIASxalpha/ARIP3. SUMO-1 and Ubc9, the ubiquitin carrier protein component in the sumoylation pathway, were also identified as interactors of FLI-1. Both PIASxalpha/ARIP3 and the closely related PIASxbeta isoform specifically enhanced sumoylation of FLI-1 at Lys(67), located in its N-terminal activation domain. PIASxalpha/ARIP3 relocalized the normally nuclear but diffusely distributed FLI-1 protein to PIASxalpha nuclear bodies and repressed FLI-1 transcriptional activation as assessed using different ETS-binding site-dependent promoters and different cell systems. PIASxalpha repressive activity was independent of sumoylation and did not result from inhibition of FLI-1 DNA-binding activity. Analysis of the properties of a series of ARIP3 mutants showed that the repressive properties of PIASxalpha/ARIP3 require its physical interaction with FLI-1, identifying PIASxalpha as a novel corepressor of FLI-1.

InsPecT: identification of posttranslationally modified peptides from tandem mass spectra.

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.

Tumor necrosis factor alpha-dependent drug resistance to purine and pyrimidine analogues in human colon tumor cells mediated through IKK.

Development of drug resistance in cancer is one of the main challenges in chemotherapy, and many mechanisms are still unknown. In this study, we show that tumor necrosis factor alpha (TNFalpha) increases postdrug survival from 5-fluoro-2'-deoxyuridine (FdUrd) in two human colon tumor cell lines. This resulted in the development of drug-resistant cells in a TNFalpha-dependent manner. Interestingly, although the drug-resistant cells were selected using FdUrd, they are also resistant to a number of other antimetabolites in the DNA synthesis pathway in a TNFalpha-dependent manner. Only in the drug-resistant cells (p35-colo201) TNFalpha treatment resulted in G(0)-G(1) arrest but not in the parental colo201 and other cell types. Blocking TNFalpha-induced cell cycle arrest sensitized drug-resistant cells to FdUrd. TNFalpha-induced cell cycle arrest required IKK. IKK inhibition by a small molecule inhibitor or by the knockdown of IKKalpha, IKKbeta, or RelA/p65 using siRNA, but not the inhibition of JNK, MEK, p38, or caspase-8 pathways, blocked TNFalpha-induced G(0)-G(1) arrest and restored sensitivity to FdUrd of drug-resistant cells. TNFalpha reduced the transcripts and protein levels of phosphorylated retinoblastoma protein (Rb), Rb, E2F1, and Cdk4 only in drug-resistant p35-colo201 cells. This effect of TNFalpha was reversed by IKK inhibitor, suggesting that TNFalpha-induced cell cycle arrest is probably due to the reduction of Rb, E2F1, and Cdk4. Taken together, this study shows that, in vitro, TNFalpha-induced cell cycle arrest through IKK can provide a mechanism for the development of drug resistance to anti-cancer drugs, purine and pyrimidine analogues.