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Sample records for los genes apc

  1. Increased biomass production and glycogen accumulation in apcE gene deleted Synechocystis sp. PCC 6803

    PubMed Central

    2014-01-01

    The effect of phycobilisome antenna-truncation in the cyanobacterium Synechocystis sp. PCC 6803 on biomass production and glycogen accumulation have not yet been fully clarified. To investigate these effects here, the apcE gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of Synechocystis sp. PCC 6803. Biomass production of the apcE-deleted strain under photoautotrophic and atmospheric air conditions was 1.6 times higher than that of strain PCC 6803 (1.32 ± 0.01 versus 0.84 ± 0.07 g cell-dry weight L−1, respectively) after 15 days of cultivation. In addition, the glycogen content of the apcE-deleted strain (24.2 ± 0.7%) was also higher than that of strain PCC 6803 (11.1 ± 0.3%). Together, these results demonstrate that antenna truncation by deleting the apcE gene was effective for increasing biomass production and glycogen accumulation under photoautotrophic and atmospheric air conditions in Synechocystis sp. PCC 6803. PMID:24949254

  2. ApcMin, A Mutation in the Murine Apc Gene, Predisposes to Mammary Carcinomas and Focal Alveolar Hyperplasias

    NASA Astrophysics Data System (ADS)

    Moser, Amy Rapaich; Mattes, Ellen M.; Dove, William F.; Lindstrom, Mary J.; Haag, Jill D.; Gould, Michael N.

    1993-10-01

    ApcMin (Min, multiple intestinal neoplasia) is a point mutation in the murine homolog of the APC gene. Min/+ mice develop multiple intestinal adenomas, as do humans carrying germ-line mutations in APC. Female mice carrying Min are also prone to develop mammary tumors. Min/+ mammary glands are more sensitive to chemical carcinogenesis than are +/+ mammary glands. Transplantation of mammary cells from Min/+ or +/+ donors into +/+ hosts demonstrates that the propensity to develop mammary tumors is intrinsic to the Min/+ mammary cells. Long-term grafts of Min/+ mammary glands also gave rise to focal alveolar hyperplasias, indicating that the presence of the Min mutation also has a role in the development of these lesions.

  3. Rapid detection of translation-terminating mutations at the adenomatous polyposis coli (APC) gene by direct protein truncation test

    SciTech Connect

    Van Der Luut, R.; Khan, P.M.; Van Leeuwen, C.; Tops, C.; Roest, P.; Den Dunnen, J. )

    1994-03-01

    Familial adenomatous polyposis (FAP) is usually associated with protein truncating mutations in the adenomatous polyposis coli (APC) gene. The APC mutations are known to play a major role in colorectal carcinogensis. For the identification of protein truncating mutations of the APC gene, the authors developed a rapid, sensitive, and direct screening procedure. The technique is based on the in vitro transcription and translation of the genomic PCR products and is called the protein truncation test. Samples of DNA from individual FAP patients, members of a FAP family, colorectal tumors, and colorectal tumor-derived cell lines were used to show the effectiveness of this method. 9 refs., 2 figs.

  4. Severe Gardner syndrome in families with mutations restricted to a specific region of the APC gene

    SciTech Connect

    Davies, D.R.; Armstrong, J.G.; Thakker, N.

    1995-11-01

    Familial adenomatous polyposis (FAP) is associated with a number of extraintestinal manifestations, which include osteomas, epidermoid cysts, and desmoid tumors, often referred to as {open_quotes}Gardner syndrome.{close_quotes} Recent studies have suggested that some of the phenotypic features of FAP are dependent on the position of the mutation within the APC gene. In particular, the correlation between congenital hypertrophy of the retinal pigment epithelium (CHRPE) and APC genotype indicates that affected families may be divided into distinct groups. We have investigated the association between the dento-osseous features of GS on dental panoramic radiographs (DPRs) and APC genotype in a regional cohort of FAP families. DPRs were performed on 84 affected individuals from 36 families, and the dento-osseous features of FAP were quantified by a weighted scoring system. Significant DPR abnormalities were present in 69% of affected individuals. The APC gene mutation was identified in 27 of these families, and for statistical analysis these were subdivided into three groups. Group 1 comprised 18 affected individuals from seven families with mutations 5{prime} of exon 9; these families (except one) did not express CHRPE. Groups 2 comprised 38 individuals from 16 families with mutations between exon 9 and codon 1444, all of whom expressed CHRPE. Group 3 comprised 11 individuals from four families with mutations 3{prime} of codon 1444, none of whom expressed CHRPE. Families with mutations 3{prime} of codon 1444 had significantly more lesions on DPRs (P < .001) and appeared to have a higher incidence of desmoid tumors. These results suggest that severity of some of the features of Gardner syndrome may correlate with genotype in FAP. 32 refs., 2 figs., 2 tabs.

  5. Molecular genetic analysis of exons 1 to 6 of the APC gene in non-polyposis familial colorectal cancer.

    PubMed

    Joyce, J A; Froggatt, N J; Davies, R; Evans, D G; Trembath, R; Barton, D E; Maher, E R

    1995-12-01

    Familial adenomatous polyposis coli is caused by constitutional mutations in the APC gene. The hallmark of familial adenomatous polyposis coli is the presence of numerous (> 100) colorectal polyps, but mutations in the 5' end of the APC gene have been associated with familial colorectal cancer without florid polyposis. Although familial adenomatous polyposis coli accounts for only a minority of familial colorectal cancer cases, we hypothesised that APC mutations which were not associated with florid polyposis might make a significant contribution to nonpolyposis familial colorectal cancer. To investigate this possibility, we analysed 40 unrelated patients with familial colorectal cancer without classical familial adenomatous polyposis coli for mutations in exons 1 to 6 (codons 1 to 243) of the APC gene. No mutations were detected, but a C-->T polymorphism at nucleotide 333 (Arg-->Trp at codon 99) was identified. No 5' APC mutations were detected in two patients with desmoid tumours and a family history of colorectal cancer and polyps. We conclude that mutations in exons 1 to 6 of the APC gene are infrequent in patients with familial colorectal cancer who do not have many colorectal polyps. PMID:8835324

  6. Antitumor Molecular Mechanism of Chlorogenic Acid on Inducting Genes GSK-3 β and APC and Inhibiting Gene β -Catenin.

    PubMed

    Xu, Ruoshi; Kang, Qiumei; Ren, Jie; Li, Zukun; Xu, Xiaoping

    2013-01-01

    Objective. Inhibiting gene β -catenin and inducting genes GSK-3 β and APC, promoting the tumor cell apoptosis in Wnt pathway, by chlorogenic acid were discussed (CGA). Method. The different genes were scanned by the 4∗44K mouse microarray chips. The effect of the three genes was confirmed by RT-PCR technique with CGA dosage of 5, 10, and 20 mg/kg. Result. The expression of GSK-3 β and APC was upregulated in group of 20 mg/kg dosage (P < 0.05) and the expression of β -catenin was downregulated in the same dosage (P < 0.05). Conclusion. The results infer that the multimeric protein complex of β -catenin could be increased by CGA upregulated genes GSK-3 β and APC, which could inhibit the free β -catenin into the nucleus to connect with TCF. So the transcriptional expression of the target genes will be cut to abnormal cell proliferation. It is probably one of the ways that can stop the tumor increase by CGA. PMID:23844319

  7. Hereditary desmoid disease due to a frameshift mutation at codon 1924 of the APC gene.

    PubMed Central

    Eccles, D. M.; van der Luijt, R.; Breukel, C.; Bullman, H.; Bunyan, D.; Fisher, A.; Barber, J.; du Boulay, C.; Primrose, J.; Burn, J.; Fodde, R.

    1996-01-01

    Desmoid tumors are slowly growing fibrous tumors highly resistant to therapy and often fatal. Here, we report hereditary desmoid disease (HDD), a novel autosomal dominant trait with 100% penetrance affecting a three-generation kindred. Desmoid tumors are usually a complication of familial adenomatous polyposis, a predisposition to the early development of premalignant adenomatous polyps in the colorectum due to chain-terminating mutations of the APC gene. In general, one or more members in approximately 10% of the FAP families manifest desmoid tumors. Affected individuals from the HDD kindred are characterized by multifocal fibromatosis of the paraspinal muscles, breast, occiput, arms, lower ribs, abdominal wall, and mesentery. Osteomas, epidermal cysts, and other congenital features were also observed. We show that HDD segregates with an unusual germ-line chain-terminating mutation at the 3' end of the APC gene (codon 1924) with somatic loss of the wild-type allele leading to tumor development. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8940264

  8. Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8.

    PubMed

    Garbe, David; Doto, Jeffrey B; Sundaram, Meera V

    2004-06-01

    Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression.

  9. Caenorhabditis elegans lin-35/Rb, efl-1/E2F and other synthetic multivulva genes negatively regulate the anaphase-promoting complex gene mat-3/APC8.

    PubMed Central

    Garbe, David; Doto, Jeffrey B; Sundaram, Meera V

    2004-01-01

    Retinoblastoma (Rb)/E2F complexes repress expression of many genes important for G(1)-to-S transition, but also appear to regulate gene expression at other stages of the cell cycle. In C. elegans, lin-35/Rb and other synthetic Multivulva (SynMuv) group B genes function redundantly with other sets of genes to regulate G(1)/S progression, vulval and pharyngeal differentiation, and other unknown processes required for viability. Here we show that lin-35/Rb, efl-1/E2F, and other SynMuv B genes negatively regulate a component of the anaphase-promoting complex or cyclosome (APC/C). The APC/C is a multisubunit complex that promotes metaphase-to-anaphase progression and G(1) arrest by targeting different substrates for ubiquitination and proteasome-mediated destruction. The C. elegans APC/C gene mat-3/APC8 has been defined by temperature-sensitive embryonic lethal alleles that strongly affect germline meiosis and mitosis but only weakly affect somatic development. We describe severe nonconditional mat-3 alleles and a hypomorphic viable allele (ku233), all of which affect postembryonic cell divisions including those of the vulval lineage. The ku233 lesion is located outside of the mat-3 coding region and reduces mat-3 mRNA expression. Loss-of-function alleles of lin-35/Rb and other SynMuv B genes suppress mat-3(ku233) defects by restoring mat-3 mRNA to wild-type levels. Therefore, Rb/E2F complexes appear to repress mat-3 expression. PMID:15238519

  10. The Schizosaccharomyces pombe dim1+ Gene Interacts with the Anaphase-Promoting Complex or Cyclosome (APC/C) Component lid1+ and Is Required for APC/C Function

    PubMed Central

    Berry, Lynne D.; Feoktistova, Anna; Wright, Melanie D.; Gould, Kathleen L.

    1999-01-01

    The Schizosaccharomyces pombe dim1+ gene is required for entry into mitosis and for chromosome segregation during mitosis. To further understand dim1p function, we undertook a synthetic lethal screen with the temperature-sensitive dim1-35 mutant and isolated lid (for lethal in dim1-35) mutants. Here, we describe the temperature-sensitive lid1-6 mutant. At the restrictive temperature of 36°C, lid1-6 mutant cells arrest with a “cut” phenotype similar to that of cut4 and cut9 mutants. An epitope-tagged version of lid1p is a component of a multiprotein ∼20S complex; the presence of lid1p in this complex depends upon functional cut9+. lid1p-myc coimmunoprecipitates with several other proteins, including cut9p and nuc2p, and the presence of cut9p in a 20S complex depends upon the activity of lid1+. Further, lid1+ function is required for the multiubiquitination of cut2p, an anaphase-promoting complex or cyclosome (APC/C) target. Thus, lid1p is a component of the S. pombe APC/C. In dim1 mutants, the abundances of lid1p and the APC/C complex decline significantly, and the ubiquitination of an APC/C target is abolished. These data suggest that at least one role of dim1p is to maintain or establish the steady-state level of the APC/C. PMID:10082519

  11. ApcD, ApcF and ApcE are not required for the Orange Carotenoid Protein related phycobilisome fluorescence quenching in the cyanobacterium Synechocystis PCC 6803.

    PubMed

    Jallet, Denis; Gwizdala, Michal; Kirilovsky, Diana

    2012-08-01

    In cyanobacteria, strong blue-green light induces a photoprotective mechanism involving an increase of energy thermal dissipation at the level of phycobilisome (PB), the cyanobacterial antenna. This leads to a decrease of the energy arriving to the reaction centers. The photoactive Orange Carotenoid Protein (OCP) has an essential role in this mechanism. The binding of the red photoactivated OCP to the core of the PB triggers energy and PB fluorescence quenching. The core of PBs is constituted of allophycocyanin trimers emitting at 660 or 680nm. ApcD, ApcF and ApcE are the responsible of the 680nm emission. In this work, the role of these terminal emitters in the photoprotective mechanism was studied. Single and double Synechocystis PCC 6803 mutants, in which the apcD or/and apcF genes were absent, were constructed. The Cys190 of ApcE which binds the phycocyanobilin was replaced by a Ser. The mutated ApcE attached an unusual chromophore emitting at 710nm. The activated OCP was able to induce the photoprotective mechanism in all the mutants. Moreover, in vitro reconstitution experiments showed similar amplitude and rates of fluorescence quenching. Our results demonstrated that ApcD, ApcF and ApcE are not required for the OCP-related fluorescence quenching and they strongly suggested that the site of quenching is one of the APC trimers emitting at 660nm. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial. PMID:22172739

  12. Expression and promoter methylation status of hMLH1, MGMT, APC, and CDH1 genes in patients with colon adenocarcinoma

    PubMed Central

    Michailidi, Christina; Theocharis, Stamatios; Tsourouflis, Gerasimos; Pletsa, Vasiliki; Kouraklis, Gregorios; Patsouris, Efstratios; Papavassiliou, Athanasios G

    2015-01-01

    Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis. PMID:25908636

  13. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles.

    PubMed

    DeWitt, Marsha; Johnson, Robert L; Snyder, Paul; Fleet, James C

    2015-04-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions of mice with colon-specific deletion of both APC gene alleles (CAC; APC(Δ580/Δ580)). At four weeks of age, CAC; APC(Δ580/Δ580) and control mice were injected with vehicle or 1,25(OH)2D (1μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APC(Δ580/Δ580) mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APC(Δ580/Δ580) and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APC(Δ580/Δ580) or control mice. VDR mRNA and protein levels were lower (-65% and -90%) in the colon of CAC; APC(Δ580/Δ580) mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of transient receptor potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APC(Δ580/Δ580) mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. This article is part of a special issue entitled '17th Vitamin D Workshop'.

  14. Specific immunotherapy by genetically engineered APCs: the "guided missile" strategy.

    PubMed

    Wu, B; Wu, J M; Miagkov, A; Adams, R N; Levitsky, H I; Drachman, D B

    2001-04-01

    We tested the hypothesis that APCs genetically engineered to present an Ag and to express Fas ligand (FasL) simultaneously can target and eliminate Ag-specific T cells. Transgenic T cells specific for influenza hemagglutinin (HA) were used as targets. We prepared recombinant vaccinia virus vectors (VVV) to transfer the gene constructs individually or simultaneously into APCs. We prevented unwanted viral replication by attenuating the VVVs with psoralen-UV light treatment. For presentation of the HA Ag, APCs were transduced with cDNA for HA flanked by sequences of the lysosome-associated membrane protein that direct efficient processing and presentation of the Ag by APCs. As a "warhead" for the APCs, we transduced them with the gene for FasL, which induces apoptosis of Fas-expressing activated T cells. To protect the transduced APCs from self-destruction by FasL, we transferred cDNA for a truncated form of Fas-associated death domain, which inhibits Fas-mediated cell death. Our results show that the engineered APCs effectively expressed the genes of interest. APCs transduced with VVV carrying all three gene constructs specifically killed HA-transgenic T cells in culture. Coculture with T cells specific for an unrelated Ag (OVA) had no significant effect. Our in vitro findings show that APCs can be genetically engineered to target and kill Ag-specific T cells and represent a promising novel strategy for the specific treatment of autoimmune diseases. PMID:11254740

  15. Analysis of APC mutation in human ameloblastoma and clinical significance.

    PubMed

    Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

    2016-01-01

    As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype. PMID:27065015

  16. Type 1 serrated polyposis represents a predominantly female disease with a high prevalence of dysplastic serrated adenomas, without germline mutation in MUTYH, APC, and PTEN genes

    PubMed Central

    Petronio, Marco; Pinson, Stephane; Walter, Thomas; Joly, Marie-Odile; Hervieu, Valerie; Forestier, Julien

    2015-01-01

    Aim The aim of this article is to clarify the epidemiologic, clinical, endoscopic, biological and genetic characteristics of type 1 serrated polyposis patients. Patients and methods Consecutive patients responding to the WHO definition of type 1 serrated polyposis in one reference center for polyposis patients accepted genetic counseling. Detailed data on previous endoscopies, histology, and life habits were recorded, after informed consent, germline analysis of MUTYH, APC, and PTEN germline mutations. Molecular biology was tested on available fixed tissue from different lesion types. Results We included 29 patients (mean age 53.5 years, 21 women (72.4%)), four with a personal history of colorectal cancer (CRC), with a mean of 11.6 SSAs, with associated hyperplastic polyps in 93.1% and adenomas in 82.8%. SSAs showed no dysplasia in 46.9% of lesions (three of 29 patients), LGD in 51.9% (22/29 patients), and HGD in 1.2% (four of 29 patients). Dysplasia was more frequent in proximal SSAs and in women. Colectomy 15 patients (51.7%), upper digestive neoplasms: eight patients (27.5%); smokers: 24 patients (82.8%); family history of CRC: 16 patients (55.2%). Biology: MSI-H phenotype in one SSA, V600E BRAF mutation in 95% of SSAs; MGMT hypermethylation in three of 17 SSAs. No germline mutation was detected in MYH, APC or PTEN genes. Conclusion Type 1 serrated polyposis corresponds to a majority of women, with a high prevalence of smokers, a high prevalence of dysplastic serrated adenomas, particularly in females, without identified germline mutation in targeted predisposing genes. PMID:27087961

  17. Methylated APC and GSTP1 genes in serum DNA correlate with the presence of circulating blood tumor cells and are associated with a more aggressive and advanced breast cancer disease

    PubMed Central

    2010-01-01

    Background Tumor-related methylated DNA and circulating tumor cells (CTC) in the peripheral blood might be of prognostic importance in breast cancer. Thus, the aim of our study was to examine free methylated DNA and CTC in the blood from breast cancer patients and to correlate it with clinicopathological features known to influence prognosis. Materials and methods We prospectively obtained serum samples from 85 patients with breast cancer and 22 healthy volunteers. Sera were analysed by methylation specific PCR (MethyLight PCR) for five genes: adenomatous polyposis coli (APC), ras association domain family protein 1A (RASSF1A), estrogen receptor 1 (ESR1), CDKN2A (p16) and glutathione s-transferase pi 1 (GSTP1). Beta actin (ACTB) served as control. In parallel matched peripheral blood of 63 patients was used to assay for circulating tumor cells in the peripheral blood by a modified immunomagnetic AdnaTest BreastCancerSelect with PCR detection for EPCAM, MUC1, MGB1 and SPDEF. Results We found a hypermethylation in the APC gene in 29% (25/85), in RASSF1A in 26% (22/85), in GSTP1 in 18% (14/76) and in ESR1 in 38% (32/85) of all breast cancer patients. No hypermethylation of CDKN2A was found (0/25). Blood samples of patients were defined CTC positive by detecting the EPCAM 13% (8/63), MUC1 16% (10/63), MGB 9% (5/55), SPDEF 12% (7/58) and in 27% detecting one or more genes (15/55). A significant difference was seen in methylated APC DNA between cancer patients and healthy volunteers. Moreover, methylated APC, RASSF1 and CTC were significantly different in metastatic versus non-metastatic disease. In addition, the presence of methylated APC, RASSF1A and CTC correlated significantly with AJCC-staging (p = 0.001, p = 0.031 and 0.002, respectively). High incidences of methylations were found for the genes RASSF1 and ESR1 in healthy individuals (both 23% 5/22). Methylated GSTP1 was predominantly found in the serum of patients with large primaries (p = 0.023) and was highly

  18. A multigene mutation classification of 468 colorectal cancers reveals a prognostic role for APC

    PubMed Central

    Schell, Michael J.; Yang, Mingli; Teer, Jamie K.; Lo, Fang Yin; Madan, Anup; Coppola, Domenico; Monteiro, Alvaro N. A.; Nebozhyn, Michael V.; Yue, Binglin; Loboda, Andrey; Bien-Willner, Gabriel A.; Greenawalt, Danielle M.; Yeatman, Timothy J.

    2016-01-01

    Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC. PMID:27302369

  19. Differential RNA-seq analysis comparing APC-defective and APC-restored SW480 colorectal cancer cells

    PubMed Central

    King, Lauren E.; Love, Christopher G.; Sieber, Oliver M.; Faux, Maree C.; Burgess, Antony W.

    2016-01-01

    The adenomatous polyposis coli (APC) tumour suppressor gene is mutated in about 80% of colorectal cancers (CRC) Brannon et al. (2014) [1]. APC is a large multifunctional protein that regulates many biological functions including Wnt signalling (through the regulation of beta-catenin stability) Reya and Clevers (2005) [2], cell migration Kroboth et al. (2007), Sansom et al. (2004) [3], [4], mitosis Kaplan et al. (2001) [5], cell adhesion Faux et al. (2004), Carothers et al. (2001) [6], [7] and differentiation Sansom et al. (2004) [4]. Although the role of APC in CRC is often described as the deregulation of Wnt signalling, its other biological functions suggest that there are other factors at play that contribute to the onset of adenomas and the progression of CRC upon the truncation of APC. To identify genes and pathways that are dysregulated as a consequence of loss of function of APC, we compared the gene expression profiles of the APC mutated human CRC cell line SW480 following reintroduction of wild-type APC (SW480 + APC) or empty control vector (SW480 + vector control) Faux et al. (2004) . Here we describe the RNA-seq data derived for three biological replicates of parental SW480, SW480 + vector control and SW480 + APC cells, and present the bioinformatics pipeline used to test for differential gene expression and pathway enrichment analysis. A total of 1735 genes showed significant differential expression when APC was restored and were enriched for genes associated with cell polarity, Wnt signalling and the epithelial to mesenchymal transition. There was additional enrichment for genes involved in cell–cell adhesion, cell–matrix junctions, angiogenesis, axon morphogenesis and cell movement. The raw and analysed RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE76307. This dataset is useful for further investigations of the impact of APC mutation on the properties of colorectal cancer cells

  20. Extra nuchal-type fibroma associated with elastosis, traumatic neuroma, a rare APC gene missense mutation, and a very rare MUTYH gene polymorphism: a case report and review of the literature*.

    PubMed

    Linos, Konstantinos; Sedivcová, Monika; Cerna, Katerina; Sima, Radek; Kazakov, Dmitry V; Nazeer, Tipu; Glazyrin, Alexey; Valerian, Brian T; Carlson, J Andrew

    2011-11-01

    We report a case of an extra nuchal-type fibroma in a 51-year-old male suspected to have attenuated familial adenomatous polyposis (Gardner's syndrome), who presented with a longstanding buttock mass excised due to enlargement and pain. Histopathologically, lobules of haphazard, hypocellular, hyalinized collagen bundles replaced the dermis and subcutis and entrapped nerve bundles, mimicking Morton neuroma. Ramifying nerve twigs found around larger nerve fascicles showed the co-existence of traumatic neuroma. Elastic tissue stain revealed elastosis characterized by large, arborizing fibers lying between and within the hyalinized collagen bundles. Modified Masson's trichrome stain showed light blue staining of collagen bundles producing the hyalinized nodules with irregular, light red staining of collagen bundles at their periphery and within tumor collagen. Compression and/or degeneration of collagen and secondary elastosis with later entrapment by tumor collagen could explain this microscopic phenotype. By immunohistochemistry, tumor spindle cells expressed nuclear β-catenin and cyclin D1, mostly within regions of fibrosis implicating activation of the adenomatous polyposis coli (APC)-Wnt pathway. Genetic analysis showed a missense mutation in APC gene (c.7504G>A, p.G2502S in exon 15) and a functional homozygous polymorphism in the MUTYH gene (c.36+325G>C, (IVS1+5G/C)). Nuchal-type fibroma has been associated with Gardner's syndrome and trauma. In this patient, genetic predisposition coupled with repetitive, localized trauma and collagen degeneration may have provided the stimulus for the development of extra nuchal-type fibroma.

  1. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans.

    PubMed

    Wang, Julia; Jennings, Alexandra K; Kowalski, Jennifer R

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  2. The Anaphase-Promoting Complex (APC) ubiquitin ligase affects chemosensory behavior in C. elegans

    PubMed Central

    Wang, Julia; Jennings, Alexandra K.

    2016-01-01

    The regulation of fundamental aspects of neurobiological function has been linked to the ubiquitin signaling system (USS), which regulates the degradation and activity of proteins and is catalyzed by E1, E2, and E3 enzymes. The Anaphase-Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that controls diverse developmental and signaling processes in post-mitotic neurons; however, potential roles for the APC in sensory function have yet to be explored. In this study, we examined the effect of the APC ubiquitin ligase on chemosensation in Caenorhabditis elegans by testing chemotaxis to the volatile odorants, diacetyl, pyrazine, and isoamyl alcohol, to which wild-type worms are attracted. Animals with loss of function mutations in either of two alleles (g48 and ye143) of the gene encoding the APC subunit EMB-27 APC6 showed increased chemotaxis towards diacetyl and pyrazine, odorants sensed by AWA neurons, but exhibited normal chemotaxis to isoamyl alcohol, which is sensed by AWC neurons. The statistically significant increase in chemotaxis in the emb-27 APC6 mutants suggests that the APC inhibits AWA-mediated chemosensation in C. elegans. Increased chemotaxis to pyrazine was also seen with mutants lacking another essential APC subunit, MAT-2 APC1; however, mat-2 APC1 mutants exhibited wild type responses to diacetyl. The difference in responsiveness of these two APC subunit mutants may be due to differential strength of these hypomorphic alleles or may indicate the presence of functional sub-complexes of the APC at work in this process. These findings are the first evidence for APC-mediated regulation of chemosensation and lay the groundwork for further studies aimed at identifying the expression levels, function, and targets of the APC in specific sensory neurons. Because of the similarity between human and C. elegans nervous systems, the role of the APC in sensory neurons may also advance our understanding of human sensory function and disease. PMID

  3. Identification of APC mutations and evaluation of their expression level using a functional screening assay

    SciTech Connect

    Varesco, L.; Gismondi, V.; Bafico, A.

    1994-09-01

    A functional screen for chain-terminating mutations in the APC gene recently has been developed. It is based on the PCR and cloning of a segment of the gene in-frame with a colorimetric marker gene (lacz) followed by screening for the level of activity of the marker polypeptide (beta-galactosidase). This method scores colony number with different blue colors that are produced by bacteria containing normal and mutant APC segments. In the present work this method was used to screen the entire APC coding region by using eight primer pairs. DNA segments with known APC mutations at different positions in the gene were used as controls and were clearly identifiable with this assay. In addition, the entire APC coding region has been examined in 21 APC patients in whom PCR-SSCP did not identify an APC mutation. Novel mutations (n=14) were identified by the blue/white assay and were all confirmed by sequence analysis. This method also was used to quantitate the expression of paternal and maternal APC alleles taking advantage of an RsaI site polymorphism at position 1458 in a small number of informative individuals. Differential expression of some known mutant APC mRNAs was observed.

  4. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles

    PubMed Central

    DeWitt, Marsha; Johnson, Robert L.; Snyder, Paul; Fleet, James C.

    2015-01-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions ofmice with colon-specific deletion of both APC gene alleles (CAC; APCΔ580/Δ580). At four weeks of age, CAC; APCΔ580/Δ580 and control mice were injected with vehicle or 1,25(OH)2D (1 μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APCΔ580/Δ580 mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APCΔ580/Δ580 and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APCΔ580/Δ580 or control mice. VDR mRNA and protein levels were lower (−65% and −90%) in the colon of CAC; APCΔ580/Δ580 mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of Transient Receptor Potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APCΔ580/Δ580 mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. PMID:25597951

  5. APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

    PubMed Central

    Parisi, Alice; Lacour, Floriane; Giordani, Lorenzo; Colnot, Sabine; Maire, Pascal

    2015-01-01

    The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues. PMID:26304725

  6. APC is required for muscle stem cell proliferation and skeletal muscle tissue repair.

    PubMed

    Parisi, Alice; Lacour, Floriane; Giordani, Lorenzo; Colnot, Sabine; Maire, Pascal; Le Grand, Fabien

    2015-08-31

    The tumor suppressor adenomatous polyposis coli (APC) is a crucial regulator of many stem cell types. In constantly cycling stem cells of fast turnover tissues, APC loss results in the constitutive activation of a Wnt target gene program that massively increases proliferation and leads to malignant transformation. However, APC function in skeletal muscle, a tissue with a low turnover rate, has never been investigated. Here we show that conditional genetic disruption of APC in adult muscle stem cells results in the abrogation of adult muscle regenerative potential. We demonstrate that APC removal in adult muscle stem cells abolishes cell cycle entry and leads to cell death. By using double knockout strategies, we further prove that this phenotype is attributable to overactivation of β-catenin signaling. Our results demonstrate that in muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression and radically diverge from previous observations concerning stem cells in actively self-renewing tissues. PMID:26304725

  7. Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain

    PubMed Central

    Cronin, Nora B.; Yang, Jing; Zhang, Ziguo; Kulkarni, Kiran; Chang, Leifu; Yamano, Hiroyuki; Barford, David

    2015-01-01

    Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5N). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5N has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5N shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes. PMID:26343760

  8. Spontaneous Intestinal Tumorigenesis in Apc/Min+ Mice Requires Altered T Cell Development with IL-17A

    PubMed Central

    Chae, Wook-Jin; Bothwell, Alfred L. M.

    2015-01-01

    The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression. Here we address the inhibitory role of Tregs on intestinal tumorigenesis in the Apc/Min+ mouse model that resembles human familial adenomatous polyposis (FAP). Apc/Min+ mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria. To address the role of heterozygous Apc gene mutation in Tregs, we generated Foxp3-Cre, Apcflox/+ mice. Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transfer into Apc/Min+ mice, demonstrating that the heterozygous Apc gene mutation in Tregs does not induce the loss of control over tumor microenvironment. Adoptive transfer of in vitro generated Apc/Min+ iTregs (inducible Tregs) failed to inhibit intestinal tumorigenesis, suggesting that naïve CD4 T cells generated from Apc/Min+ mice thymus were impaired. We also showed that adoptively transferred IL-17A-deficient Apc/Min+ Tregs inhibited tumor growth, suggesting that IL-17A was critical to impair the tumor regression function of Apc/Min+ Tregs. Taken together, our results suggest that both T cell development in a functional thymus and IL-17A control the ability of Treg to inhibit intestinal tumorigenesis in Apc/Min+ mice. PMID:26146642

  9. Structure of an APC3-APC16 complex: Insights into assembly of the Anaphase Promoting Complex/Cyclosome

    PubMed Central

    Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; Weissmann, Florian; Miller, Darcie J.; VanderLinden, Ryan; Brown, Nicholas G.; Frye, Jeremiah J.; Peters, Jan-Michael; Schulman, Brenda A.

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-twofold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3’s TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7, and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3’s loop and IR-tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer, and together with biochemistry and prior data explain how APC16 recruits APC7 to APC3, show how APC3’s C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR-tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C. PMID:25490258

  10. APC15 mediates CDC20 auto-ubiquitylation by APC/CMCC and MCC disassembly

    PubMed Central

    Uzunova, Kristina; Dye, Billy T.; Schutz, Hannelore; Ladurner, Rene; Petzold, Georg; Toyoda, Yusuke; Jarvis, Marc A.; Brown, Nicholas G.; Poser, Ina; Novatchkova, Maria; Mechtler, Karl; Hyman, Anthony A.; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2012-01-01

    The anaphase-promoting complex/cyclosome bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) which inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which has been proposed to require CDC20 auto-ubiquitylation. Here we characterized APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C’s MCC binding site, is required for APC/CMCC-dependent CDC20 auto-ubiquitylation and degradation, and for timely anaphase initiation, but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the view that MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and that CDC20 auto-ubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators, and report the first generation of recombinant human APC/C. PMID:23007861

  11. Colorectal Tumors from APC*I1307K Carriers Principally Harbor Somatic APC Mutations outside the A8 Tract

    PubMed Central

    Zauber, Peter; Bishop, Timothy; Taylor, Claire; Sabbath-Solitare, Marlene; Marotta, Stephen; Tomlinson, Ian

    2014-01-01

    Purpose APC*I1307K (c.3920T>A) is an inherited variant associated with colorectal tumour risk found almost exclusively in those of Ashkenazi Jewish ancestry. A single nucleotide substitution creates an oligo-adenine tract (A8) that appears to be inherently prone to further mis-pairing and slippage. The reported multiple tumor phenotype of carriers is not easily reconciled with molecular and population genetics data. We postulated that some c.3920T>A carriers with multiple adenomas have other unidentified APC germ line or somatic mutations. Methods DNA from 82 colonic tumours and accompanying normal tissue collected from 29 carriers with multiple colorectal tumors was directly sequenced between codons 716 and 1604. We also assessed APC gene loss of heterozygosity. Results One patient (3.4%) was found to have an additional APC germ line mutation. Twenty-five of the tumours showed no significant somatic molecular change, 36 showed one change, 20 showed two, and one tumour showed more than 2 changes. Our data suggest a correlation between advancing histology and fewer beta-catenin binding sites remaining in the mutant proteins. Conclusions There were no other common germ line variants identified within the region of the APC gene examined, suggesting that any effect from this region on tumour production is attributable to the c.3920T>A allele. Our findings further suggest the only somatic genetic change clearly attributable to the c.3920T>A mutation is the c.3924_3925insA. PMID:24416237

  12. A knock-in mouse model reveals roles for nuclear Apc in cell proliferation, Wnt signal inhibition and tumor suppression

    PubMed Central

    Zeineldin, Maged; Cunningham, Jamie; McGuinness, William; Alltizer, Preston; Cowley, Brett; Blanchat, Bryan; Xu, Wenhao; Pinson, David; Neufeld, Kristi L.

    2011-01-01

    Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt signaling pathway by targeting proto-oncogene β-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, β-catenin can serve as a transcription co-factor for genes required for cell proliferation such as cyclin D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and cytoplasm, with nuclear APC implicated in inhibition of Wnt target gene expression. Taking a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals of Apc (ApcmNLS). ApcmNLS/mNLS mice are viable and fractionation of embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc compared to Apc+/+ MEFs. The levels of Apc and β-catenin protein were not significantly altered in small intestinal epithelia from ApcmNLS/mNLS mice. Compared to Apc+/+ mice, ApcmNLS/mNLS mice displayed increased proliferation in epithelial cells from the jejunum, ileum, and colon. These same tissues from ApcmNLS/mNLS mice displayed more mRNA from three genes up-regulated in response to canonical Wnt signal, c-Myc, Axin2, and Cyclin D1, and less mRNA from Hath 1 which is down-regulated in response to Wnt. These observations suggest a role for nuclear Apc in inhibition of canonical Wnt signaling and control of epithelial proliferation in intestinal tissue. Furthermore, we found ApcMin/+ mice, which harbor a mutation that truncates Apc, have increased polyp size and multiplicity if they also carry the ApcmNLS allele. Taken together, this analysis of the novel ApcmNLS mouse model supports a role for nuclear Apc in control of Wnt target genes, intestinal epithelial cell proliferation and polyp formation. PMID

  13. Dietary soy sphingolipids suppress tumorigenesis and gene expression in 1,2-dimethylhydrazine-treated CF1 mice and ApcMin/+ mice.

    PubMed

    Symolon, Holly; Schmelz, Eva M; Dillehay, Dirck L; Merrill, Alfred H

    2004-05-01

    Dietary supplementation with milk sphingolipids inhibits colon tumorigenesis in CF1 mice treated with a colon carcinogen [1,2-dimethylhydrazine (DMH)] and in multiple intestinal neoplasia (Min) mice, which develop intestinal tumors spontaneously. Plant sphingolipids differ structurally from those of mammals [soy glucosylceramide (GlcCer) consists predominantly of a 4,8-sphingadiene backbone and alpha-hydroxy-palmitic acid], which might affect their bioactivity. Soy GlcCer was added to the AIN-76A diet (which contains <0.005% sphingolipid) to investigate whether it would also suppress tumorigenesis in these mouse models. Soy GlcCer reduced colonic cell proliferation in the upper half of the crypts in mice treated with DMH by 50 and 56% (P < 0.05) at 0.025 and 0.1% of the diet (wt/wt), respectively, and reduced the number of aberrant colonic crypt foci (an early marker of colon carcinogenesis) by 38 and 52% (P < 0.05). Min mice fed diets containing 0.025 and 0.1% (wt/wt) soy GlcCer developed 22 and 37% fewer adenomas (P < 0.05), respectively. The effects of dietary sphingolipids on gene expression in the intestinal mucosal cells of Min mice were analyzed using Affymetrix GeneChip microarrays. Soy GlcCer affected the expression of 96 genes by > or = 2-fold in a dose-dependent manner, increasing 32 and decreasing 64. Decreases in the mRNA expression of two transcription factors associated with cancer, hypoxia-induced factor 1 alpha (HIF1 alpha) and transcription factor 4 (TCF4), were confirmed by quantitative RT-PCR. In conclusion, soy GlcCer suppressed colon tumorigenesis in two mouse models; hence, plant sphingolipids warrant further investigation as inhibitors of colon cancer. Because soy contains relatively high amounts of GlcCer, sphingolipids may partially account for the anticancer benefits attributed to soy-based foods.

  14. Inactivation and Disassembly of the Anaphase-Promoting Complex during Human Cytomegalovirus Infection Is Associated with Degradation of the APC5 and APC4 Subunits and Does Not Require UL97-Mediated Phosphorylation of Cdh1 ▿

    PubMed Central

    Tran, Karen; Kamil, Jeremy P.; Coen, Donald M.; Spector, Deborah H.

    2010-01-01

    Infection of quiescent cells by human cytomegalovirus (HCMV) elicits severe cell cycle deregulation, resulting in a G1/S arrest, which can be partly attributed to the inactivation of the anaphase-promoting complex (APC). As we previously reported, the premature phosphorylation of its coactivator Cdh1 and/or the dissociation of the core complex can account for the inactivation. We have expanded on these results and further delineated the key components required for disabling the APC during HCMV infection. The viral protein kinase UL97 was hypothesized to phosphorylate Cdh1, and consistent with this, phosphatase assays utilizing a virus with a UL97 deletion mutation (ΔUL97 virus) indicated that Cdh1 is hypophosphorylated at early times in the infection. Mass spectrometry analysis demonstrated that UL97 can phosphorylate Cdh1 in vitro, and the majority of the sites identified correlated with previously characterized cyclin-dependent kinase (Cdk) consensus sites. Analysis of the APC core complex during ΔUL97 virus infection showed APC dissociation occurring at the same time as during infection with wild-type virus, suggesting that the UL97-mediated phosphorylation of Cdh1 is not required for this to occur. Further investigation of the APC subunits showed a proteasome-dependent loss of the APC5 and APC4 subunits that was temporally associated with the disassembly of the APC. Immediate early viral gene expression was not sufficient for the degradation of APC4 and APC5, indicating that a viral early gene product(s), possibly in association with a de novo-synthesized cellular protein(s), is involved. PMID:20686030

  15. Black raspberries suppress colonic adenoma development in ApcMin/+ mice: relation to metabolite profiles.

    PubMed

    Pan, Pan; Skaer, Chad W; Wang, Hsin-Tzu; Stirdivant, Steven M; Young, Matthew R; Oshima, Kiyoko; Stoner, Gary D; Lechner, John F; Huang, Yi-Wen; Wang, Li-Shu

    2015-10-01

    Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients.

  16. Mechanism of APC/CCDC20 activation by mitotic phosphorylation

    PubMed Central

    Qiao, Renping; Weissmann, Florian; Yamaguchi, Masaya; Brown, Nicholas G.; VanderLinden, Ryan; Imre, Richard; Jarvis, Marc A.; Brunner, Michael R.; Davidson, Iain F.; Litos, Gabriele; Haselbach, David; Mechtler, Karl; Stark, Holger; Schulman, Brenda A.; Peters, Jan-Michael

    2016-01-01

    Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis. PMID:27114510

  17. Structure of an APC3–APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome

    SciTech Connect

    Yamaguchi, Masaya; Yu, Shanshan; Qiao, Renping; Weissmann, Florian; Miller, Darcie J.; VanderLinden, Ryan; Brown, Nicholas G.; Frye, Jeremiah J.; Peters, Jan-Michael; Schulman, Brenda A.

    2015-08-21

    The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the “Platform” centers around a cullin-RING-like E3 ligase catalytic core; the “Arc Lamp” is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3's TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a > 200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3's loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3's C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.

  18. Integrated metrology: an enabler for advanced process control (APC)

    NASA Astrophysics Data System (ADS)

    Schneider, Claus; Pfitzner, Lothar; Ryssel, Heiner

    2001-04-01

    Advanced process control (APC) techniques become more and more important as short innovation cycles in microelectronics and a highly competitive market requires cost-effective solutions in semiconductor manufacturing. APC marks a paradigm shift from statistically based techniques (SPC) using monitor wafers for sampling measurement data towards product wafer control. The APC functionalities including run-to-run control, fault detection, and fault analysis allow to detect process drifts and excursions at an early stage and to minimize the number of misprocessed wafers. APC is being established as part of factory control systems through the definition of an APC framework. A precondition for APC is the availability of sensors and measurement methods providing the necessary wafer data. This paper discusses integrated metrology as an enabler for APC and demonstrates practical implementations in semiconductor manufacturing.

  19. Studies on the Contribution of Human Cytomegalovirus UL21a and UL97 to Viral Growth and Inactivation of the Anaphase-Promoting Complex/Cyclosome (APC/C) E3 Ubiquitin Ligase Reveal a Unique Cellular Mechanism for Downmodulation of the APC/C Subunits APC1, APC4, and APC5

    PubMed Central

    Clark, Elizabeth

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) deregulates the cell cycle by several means, including inactivation of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Viral proteins UL97 and UL21a, respectively, affect the APC/C by phosphorylation of APC/C coactivator Cdh1 and by inducing the degradation of subunits APC4 and APC5, which along with APC1 form the APC/C platform subcomplex. The aim of this study was to further characterize the mechanism of APC/C inactivation and define the relative contributions of UL21a and UL97 to APC/C substrate accumulation and to viral growth. We show that in uninfected cells, UL21a but not UL97 can disrupt APC/C function, leading to the accumulation of substrates. We find that UL21a is necessary and sufficient to induce the degradation of APC1, in addition to the previously reported APC4 and APC5. We also demonstrate that there is a previously unreported cellular mechanism for a specific decrease in the levels of all three platform subunits, APC1, APC4, and APC5, upon the depletion of any one of these subunits or of subunit APC8. Finally, we show that at a low multiplicity of infection, either UL97 or UL21a can partially complement a growth-defective mutant virus lacking both UL21a and UL97, with significantly greater benefit afforded by the expression of both proteins. This double mutant also can be partially rescued by inactivation of the APC/C using small interfering RNAs against specific subunits. These results further our understanding of HCMV's interaction with the cell cycle machinery and reveal a new cellular pattern of APC/C subunit downmodulation. IMPORTANCE HCMV lytic infection subverts the host cell cycle machinery in multiple ways. A major effect is inactivation of the APC/C, which plays a central role in the control of cell cycle progression. This study provides further insight into the mechanism of inactivation. We discovered that the APC1 subunit, which along with APC4 and APC5 form the platform

  20. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    PubMed

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-04-27

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  1. Chlorinated Water Modulates the Development of Colorectal Tumors with Chromosomal Instability and Gut Microbiota in Apc-Deficient Mice.

    PubMed

    Sasada, Tatsunari; Hinoi, Takao; Saito, Yasufumi; Adachi, Tomohiro; Takakura, Yuji; Kawaguchi, Yasuo; Sotomaru, Yusuke; Sentani, Kazuhiro; Oue, Naohide; Yasui, Wataru; Ohdan, Hideki

    2015-01-01

    The gastrointestinal tract is continuously exposed to a variety of chemicals and commensal bacteria. Recent studies have shown that changes in gut microbial populations caused by chlorine or other chemicals in the drinking water influence the development of human colorectal cancer, although the mechanism of tumorigenesis in the gut epithelium is obfuscated by the diversity of microflora and complexity of the tumor microenvironment. In this regard, mouse models that recapitulate human colorectal cancer are an invaluable tool. In this study, we used two conditional adenomatous polyposis coli (Apc) knockout mouse models to investigate the effect of chlorinated water on tumorigenesis in the digestive tract. Mice with colon-specific carcinoma--caused by either chromosomal (CDX2P 9.5-NLS Cre;Apc(+/flox), abbreviated to CPC;Apc) or microsatellite (CDX2P9.5-G19Cre;Apc(flox/flox) and CDX2P9.5-G22Cre;Apc(flox/flox)) instability, respectively--were administered chlorinated (10.0 mg/L chlorine) or tap (0.7 mg/L chlorine) water and evaluated for colon polyp formation. In CPC;Apc mice given chlorinated drinking water, tumors tended to develop in the colon, whereas in those that drank tap water, tumors were mostly observed in the small intestine. There was no difference in the rate of tumor formation of CDX2P9.5-G19Cre;Apc(flox/flox) and CDX2P9.5-G22Cre;Apc(flox/flox) mice consuming chlorinated as compared to tap water, suggesting that microsatellite instability in the Apc gene does not significantly affect tumorigenesis. Chlorinated water altered the enteric environment by reducing the fecal populations of the obligatory anaerobes Clostridium perfringens and C. difficile, as well as species belonging to the Atopobium cluster, including Enterobacteriaceae and Staphylococcus sp., which was associated with colon tumorigenesis in CPC;Apc mice. These results suggest that differences in tumorigenesis among CPC;Apc mice consuming chlorinated versus tap water may be due to differences

  2. Regulation of APC Activity by Phosphorylation and Regulatory Factors

    PubMed Central

    Kotani, Shuji; Tanaka, Hirofumi; Yasuda, Hideyo; Todokoro, Kazuo

    1999-01-01

    Ubiquitin-dependent proteolysis of Cut2/Pds1 and Cyclin B is required for sister chromatid separation and exit from mitosis, respectively. Anaphase-promoting complex/cyclosome (APC) specifically ubiquitinates Cut2/Pds1 at metaphase–anaphase transition, and ubiquitinates Cyclin B in late mitosis and G1 phase. However, the exact regulatory mechanism of substrate-specific activation of mammalian APC with the right timing remains to be elucidated. We found that not only the binding of the activators Cdc20 and Cdh1 and the inhibitor Mad2 to APC, but also the phosphorylation of Cdc20 and Cdh1 by Cdc2-Cyclin B and that of APC by Polo-like kinase and cAMP-dependent protein kinase, regulate APC activity. The cooperation of the phosphorylation/dephosphorylation and the regulatory factors in regulation of APC activity may thus control the precise progression of mitosis. PMID:10459014

  3. The postsynaptic adenomatous polyposis coli (APC) multiprotein complex is required for localizing neuroligin and neurexin to neuronal nicotinic synapses in vivo.

    PubMed

    Rosenberg, Madelaine M; Yang, Fang; Mohn, Jesse L; Storer, Elizabeth K; Jacob, Michele H

    2010-08-18

    Synaptic efficacy requires that presynaptic and postsynaptic specializations align precisely and mature coordinately. The underlying mechanisms are poorly understood, however. We propose that adenomatous polyposis coli protein (APC) is a key coordinator of presynaptic and postsynaptic maturation. APC organizes a multiprotein complex that directs nicotinic acetylcholine receptor (nAChR) localization at postsynaptic sites in avian ciliary ganglion neurons in vivo. We hypothesize that the APC complex also provides retrograde signals that direct presynaptic active zones to develop in register with postsynaptic nAChR clusters. In our model, the APC complex provides retrograde signals via postsynaptic neuroligin that interacts extracellularly with presynaptic neurexin. S-SCAM (synaptic cell adhesion molecule) and PSD-93 (postsynaptic density-93) are scaffold proteins that bind to neuroligin. We identify S-SCAM as a novel component of neuronal nicotinic synapses. We show that S-SCAM, PSD-93, neuroligin and neurexin are enriched at alpha3*-nAChR synapses. PSD-93 and S-SCAM bind to APC and its binding partner beta-catenin, respectively. Blockade of selected APC and beta-catenin interactions, in vivo, leads to decreased postsynaptic accumulation of S-SCAM, but not PSD-93. Importantly, neuroligin synaptic clusters are also decreased. On the presynaptic side, there are decreases in neurexin and active zone proteins. Further, presynaptic terminals are less mature structurally and functionally. We define a novel neural role for APC by showing that the postsynaptic APC multiprotein complex is required for anchoring neuroligin and neurexin at neuronal synapses in vivo. APC human gene mutations correlate with autism spectrum disorders, providing strong support for the importance of the association, demonstrated here, between APC, neuroligin and neurexin.

  4. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer

    PubMed Central

    MATTHAIOS, DIMITRIOS; BALGKOURANIDOU, IOANNA; KARAYIANNAKIS, ANASTASIOS; BOLANAKI, HELEN; XENIDIS, NIKOLAOS; AMARANTIDIS, KYRIAKOS; CHELIS, LEONIDAS; ROMANIDIS, KONSTANTINOS; CHATZAKI, AIKATERINI; LIANIDOU, EVI; TRYPSIANIS, GRIGORIOS; KAKOLYRIS, STYLIANOS

    2016-01-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli (APC) and Ras association domain family 1 isoform A (RASSF1A) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, P<0.001) and advanced disease (37±7 vs. 15±3 months, P<0.001), compared with patients with methylated APC. Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, P<0.001) and advanced disease (28±4 vs. 16±3 months, P<0.001) than patients with

  5. Synergism between K-rasVal12 and mutant Apc accelerates murine large intestinal tumourigenesis.

    PubMed

    Luo, Feijun; Poulogiannis, George; Ye, Hongtao; Hamoudi, Rifat; Arends, Mark J

    2011-07-01

    K-ras (KRAS) is mutated in 40-50% of human colorectal adenomas and carcinomas and plays key roles in cell proliferation, apoptosis, motility and differentiation, but its functional contribution to intestinal tumourigenesis in vivo remains incompletely understood. We have previously crossed K-rasVal12 transgenic mice with Ah-Cre mice to produce K-rasVal12/Cre offspring that inducibly express K-rasVal12 4A and 4B in the intestines, but this alone showed no significant effect on intestinal adenoma formation. Here, we crossed these mice with Min mice to evaluate the effect of K-rasVal12 and Apc mutation on intestinal tumourigenesis in vivo. The double mutant K-rasVal12/Cre/ApcMin/+ mice showed a moderate (1.86-fold) increase in adenomas in the small intestines, but a striking acceleration (6-fold increase) of large intestinal adenoma formation (P<0.01) and significantly reduced survival (by ~5 weeks) compared with control ApcMin/+ mice (P<0.01). There was recombination of the mutant K-rasVal12 transgene in 80% of large intestinal adenomas with expression of both K-rasVal12 4A and 4B isoform transcripts and expression of K-RasVal12 protein. The large intestinal adenomas showed immunohistochemical evidence of activation of MapK, Akt and Wnt signaling pathways and this was confirmed by quantitative RT-PCR analysis of relative transcript expression levels of target genes using a panel of 23 selected genes evaluated in both adenomas and non-tumour-bearing intestines. Several genes including Tiam1, Gastrin, CD44, uPA, Igfbp4, VEGF and Cox-2 that are known to be transcriptionally regulated by activation of the Wnt signaling pathway were found to be expressed at higher levels in the large intestinal adenomas from K-rasVal12/Cre/ApcMin/+ mice compared with those from controls, although other Wnt signaling pathway target genes remained unchanged. These data show that intestinal expression of K-rasVal12 accelerates Apc-initiated intestinal adenomagenesis in vivo with

  6. WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.

    PubMed

    Li, Qiuhong; Chang, Leifu; Aibara, Shintaro; Yang, Jing; Zhang, Ziguo; Barford, David

    2016-09-20

    The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

  7. WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity.

    PubMed

    Li, Qiuhong; Chang, Leifu; Aibara, Shintaro; Yang, Jing; Zhang, Ziguo; Barford, David

    2016-09-20

    The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin-RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1(WD40)). To understand how Apc1(WD40) contributes to APC/C activity, a mutant form of the APC/C with Apc1(WD40) deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1(WD40) abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C-Cdh1 complex with Apc1(WD40) deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1(WD40) is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C. PMID:27601667

  8. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed

    Alman, B A; Li, C; Pajerski, M E; Diaz-Cano, S; Wolfe, H J

    1997-08-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  9. Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

    PubMed Central

    Alman, B. A.; Li, C.; Pajerski, M. E.; Diaz-Cano, S.; Wolfe, H. J.

    1997-01-01

    Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues

  10. A limited role for p53 in modulating the immediate phenotype of Apc loss in the intestine

    PubMed Central

    Reed, Karen R; Meniel, Valerie S; Marsh, Victoria; Cole, Alicia; Sansom, Owen J; Clarke, Alan R

    2008-01-01

    Background p53 is an important tumour suppressor with a known role in the later stages of colorectal cancer, but its relevance to the early stages of neoplastic initiation remains somewhat unclear. Although p53-dependent regulation of Wnt signalling activity is known to occur, the importance of these regulatory mechanisms during the early stages of intestinal neoplasia has not been demonstrated. Methods We have conditionally deleted the Adenomatous Polyposis coli gene (Apc) from the adult murine intestine in wild type and p53 deficient environments and subsequently compared the phenotype and transcriptome profiles in both genotypes. Results Expression of p53 was shown to be elevated following the conditional deletion of Apc in the adult small intestine. Furthermore, p53 status was shown to impact on the transcription profile observed following Apc loss. A number of key Wnt pathway components and targets were altered in the p53 deficient environment. However, the aberrant phenotype observed following loss of Apc (rapid nuclear localisation of β-catenin, increased levels of DNA damage, nuclear atypia, perturbed cell death, proliferation, differentiation and migration) was not significantly altered by the absence of p53. Conclusion p53 related feedback mechanisms regulating Wnt signalling activity are present in the intestine, and become activated following loss of Apc. However, the physiological Wnt pathway regulation by p53 appears to be overwhelmed by Apc loss and consequently the activity of these regulatory mechanisms is not sufficient to modulate the immediate phenotypes seen following Apc loss. Thus we are able to provide an explanation to the apparent contradiction that, despite having a Wnt regulatory capacity, p53 loss is not associated with early lesion development. PMID:18533991

  11. Measuring APC/C-Dependent Ubiquitylation In Vitro.

    PubMed

    Jarvis, Marc A; Brown, Nicholas G; Watson, Edmond R; VanderLinden, Ryan; Schulman, Brenda A; Peters, Jan-Michael

    2016-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.

  12. Panta rhei: The APC/C at steady state

    PubMed Central

    2013-01-01

    The anaphase-promoting complex or cyclosome (APC/C) is a conserved, multisubunit E3 ubiquitin (Ub) ligase that is active both in dividing and in postmitotic cells. Its contributions to life are especially well studied in the domain of cell division, in which the APC/C lies at the epicenter of a regulatory network that controls the directionality and timing of cell cycle events. Biochemical and structural work is shedding light on the overall organization of APC/C subunits and on the mechanism of substrate recognition and Ub chain initiation and extension as well as on the molecular mechanisms of a checkpoint that seizes control of APC/C activity during mitosis. Here, we review how these recent advancements are modifying our understanding of the APC/C. PMID:23589490

  13. Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner.

    PubMed

    Song, Min Sup; Carracedo, Arkaitz; Salmena, Leonardo; Song, Su Jung; Egia, Ainara; Malumbres, Marcos; Pandolfi, Pier Paolo

    2011-01-21

    PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K/AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor-suppressive activity; however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor-suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor-suppressive activity of catalytically inactive PTEN. Importantly, we demonstrate that PTEN mutant and PTEN null states are not synonymous as they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora kinases. This finding identifies a strategy for cancer patient stratification and, thus, optimization of targeted therapies. PAPERCLIP:

  14. Nuclear PTEN regulates the APC-CDH1 tumor suppressive complex in a phosphatase-independent manner

    PubMed Central

    Song, Min Sup; Carracedo, Arkaitz; Salmena, Leonardo; Song, Su Jung; Egia, Ainara; Malumbres, Marcos; Pandolfi, Pier Paolo

    2011-01-01

    SUMMARY PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K-AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor suppressive activity, however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor suppressive activity of catalytically-inactive PTEN. Importantly, we demonstrate that PTEN-mutant and PTEN-null states are not synonymous since they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora Kinases. This finding identifies a strategy for cancer patient stratification and thus, optimization of targeted therapies. PMID:21241890

  15. Correlation between the expression of DNMT1, and GSTP1 and APC, and the methylation status of GSTP1 and APC in association with their clinical significance in prostate cancer

    PubMed Central

    ZHANG, WEIJIE; JIAO, HONGLIANG; ZHANG, XUDONG; ZHAO, RUIHUA; WANG, FENG; HE, WEI; ZONG, HONG; FAN, QINGXIA; WANG, LIUXING

    2015-01-01

    The aim of the present study was to investigate the correlation between the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1), glutathione S-transferase-P1 (GSTP1) and adenomatous polyposis coli (APC), and the methylation status of GSTP1 and APC in prostate cancer (PCa) and benign prostatic hyperplasia (BPH), and to examine its clinical significance. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of DNMT1, GSTP1 and APC in 56 samples of PCa tissue and 10 samples of BPH tissue. Methylation-specific-PCR was used to detect the methylation status of the CpG island promoters of GSTP1 and APC. The positive rate of expression of DNMT1 in poorly-differentiated PCa, moderately-differentiated PCa, well-differentiated PCa and BPH was 86.7%, 70.6%, 55.6% and 30.0%, respectively (P<0.05); for GSTP1, the positive rate was 13.3%, 29.4%, 44.4% and 90.0%, respectively (P<0.05); and for APC, the positive rate was 23.3%, 47.6%, 55.6% and 70.0%, respectively (P<0.05). The correlation coefficient for the association between the expression of DNMT1 and GSTP1 was −0.891 (P<0.05). Between the expression of DNMT1 and APC, the correlation coefficient was −0.721 (P<0.05). GSTP1 and APC were hypermethylated in the majority of PCa tissue samples. The positive rate of methylation of these genes in poorly-differentiated PCa was 83.3% and 73.3%, respectively. By contrast, hypomethylation (or demethylation) was observed in BPH samples, in which the positive rate of methylation was 10.0% and 20.0%, respectively (P<0.05). The increased expression of DNMT1, and the reduced expression of GSTP1 and APC have an important role in the development of PCa. Due to the high expression of DNMT1 mRNA, it is likely that the hypermethylation of CpG islands contributed to the silencing of GSTP1 and APC in PCa tissues. PMID:25738352

  16. Los1p, involved in yeast pre-tRNA splicing, positively regulates members of the SOL gene family

    SciTech Connect

    Shen, W.C.; Stanford, D.R.; Hopper, A.K.

    1996-06-01

    To understand the role of Los1p in pre-tRNA splicing, we sought los1 multicopy suppressors. We found SOL1 that suppresses both point and null LOS1 mutations. Since, when fused to the Gal4p DNA-binding domain, Los1p activates transcription, we tested whether Los1p regulates SOL1. We found that los1 mutants have depleted levels of SOL1 mRNA and Sol1p. Thus, LOS1 appears to positively regulate SOL1. SOL1 belongs to a multigene family with at least two additional members, SOL2 and SOL3. Sol proteins have extensive similarity to an unusual group of glucose-6-phosphate dehydrogenases (G6PDs). As the similarities are restricted to areas separate from the catalytic domain, these G6PDs may have more than one function. The SOL gene disruptions negatively affect tRNA-mediated nonsense suppression and the severity increases with the number of mutant SOL genes. However, tRNA levels do not vary with either multicopy SOL genes or with SOL disruptions. Therefore, the Sol proteins affect tRNA expression/function at steps other than transcription or splicing. We propose that LOS1 regulates gene products involved in tRNA expression/function as well as pre-tRNA splicing. 64 refs., 6 figs., 6 tabs.

  17. Specific immunotherapy of experimental myasthenia by genetically engineered APCs: the "guided missile" strategy.

    PubMed

    Drachman, D B; Wu, J-M; Miagkov, A; Williams, M A; Adams, R N; Wu, B

    2003-09-01

    Although treatment of MG with general immunosuppressive agents is often effective, it has important drawbacks, including suppression of the immune system as a whole, with the risks of infection and neoplasia, and numerous other adverse side effects. Ideally, treatment of MG should eliminate the specific pathogenic autoimmune response to AChR, without otherwise suppressing the immune system or producing other adverse side effects. Although antibodies to AChR are directly responsible for the loss of AChRs at neuromuscular junctions in MG, the AChR antibody response is T cell-dependent, and immunotherapy directed at T cells can abrogate the autoantibody response, with resulting benefit. As in other autoimmune diseases, the T cell response in MG is highly heterogeneous. The design of specific immunotherapy must take this heterogeneity into account and target the entire repertoire of AChR-specific T cells. We describe our investigation of a novel strategy for specific immunotherapy of MG, involving gene transfer to convert antigen-presenting cells (APCs) to "guided missiles" that target AChR-specific T cells, and that induce apoptosis and elimination of those T cells. This strategy uses the ability of APCs from a given individual to present the entire spectrum of AChR epitopes unique for that individual, and thereby to target the entire repertoire of antigen-specific T cells of the same individual. Using viral vectors, we have genetically engineered the APCs to process and present the most important domain of the AChR molecule, and to express a "warhead" of Fas ligand (FasL) to eliminate the activated AChR-specific T cells with which they interact. Our results show that the APCs express the appropriate gene products, and effectively and specifically eliminate AChR-specific T cells by the Fas/FasL pathway, while sparing T cells of other specificities. PMID:14592923

  18. Olfactomedin 4 deletion induces colon adenocarcinoma in ApcMin/+ mice

    PubMed Central

    Liu, W; Li, H; Hong, S-H; Piszczek, G P; Chen, W; Rodgers, G P

    2016-01-01

    Colon carcinogenesis is a multiple-step process involving the accumulation of a series of genetic and epigenetic alterations. The most commonly initiating event of intestinal carcinogenesis is mutation of the adenomatous polyposis coli (APC) gene, which leads to activation of the Wnt/β-catenin pathway. Olfactomedin 4 (OLFM4) has emerged as an intestinal stem-cell marker, but its biological function in the intestine remains to be determined. Here we show that Olfm4 deletion induced colon adenocarcinoma in the distal colon of ApcMin/+ mice. Mechanistically, we found that OLFM4 is a target gene of the Wnt/β-catenin pathway and can downregulate β-catenin signaling by competing with Wnt ligands for binding to Frizzled receptors, as well as by inhibition of the Akt-GSK-3β (Akt-glycogen synthase kinase-3β) pathway. We have shown that both Wnt and nuclear factor-κB (NF-κB) signaling were boosted in tumor tissues of Apc Olfm4 double-mutant mice. These data establish OLFM4 as a critical negative regulator of the Wnt/β-catenin and NF-κB pathways that inhibits colon-cancer development initiated by APC mutation. In addition, Olfm4 deletion significantly enhanced intestinal-crypt proliferation and inflammation induced by azoxymethane/dextran sodium sulfate. Thus, OLFM4 has an important role in the regulation of intestinal inflammation and tumorigenesis, and could be a potential therapeutic target for intestinal malignant tumors. Unlike the human colonic epithelium, the mouse colonic epithelium does not express OLFM4, but nevertheless, systemic OLFM4 deletion promotes colon tumorigenesis and that loss from mucosal neutrophils may have a role to play. PMID:26973250

  19. Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

    PubMed

    Boekhout, Michiel; Yuan, Ruixue; Wondergem, Annelotte P; Segeren, Hendrika A; van Liere, Elsbeth A; Awol, Nesibu; Jansen, Imke; Wolthuis, Rob M F; de Bruin, Alain; Westendorp, Bart

    2016-03-01

    E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

  20. Zomepirac sodium vs APC with codeine for oral surgery pain.

    PubMed

    Mehlisch, D R; Joy, E D

    1981-06-01

    In this double-blind, repeat-dose study, 323 outpatients with moderate to severe pain after oral surgery assessed zomepirac sodium, a new oral, single-entity, nonnarcotic analgesic, and APC with codeine, 30 mg, a reference standard. Pain relief obtained with 100 mg of zomepirac sodium was significantly superior to that of APC with codeine, 30 mg; 50 mg of zomepirac sodium was as effective as the reference drug. The analgesic acceptability was highest for 100 mg of zomepirac sodium. Both doses of this new drug produced significantly fewer adverse reactions than APC with codeine, 30 mg.

  1. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    PubMed

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p < 0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p = 0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. PMID:26073472

  2. Antigen targeting to APC: from mice to veterinary species.

    PubMed

    Alvarez, B; Poderoso, T; Alonso, F; Ezquerra, A; Domínguez, J; Revilla, C

    2013-10-01

    Antigen delivery to receptors expressed on antigen presenting cells (APC) has shown to improve immunogenicity of vaccines in mice. An enhancement of cytotoxic T lymphocyte (CTL), helper T cell or humoral responses was obtained depending on the type of APC and the surface molecule targeted. Although this strategy is being also evaluated in livestock animals with promising results, some discrepancies have been found between species and pathogens. The genetic diversity of livestock animals, the different pattern of expression of some receptors among species, the use of different markers to characterize APC in large animals and sometimes the lack of reagents make difficult to compare results obtained in different species. In this review, we summarize the data available regarding antigen targeting to APC receptors in cattle, sheep and pig and discuss the results found in these animals in the context of what has been obtained in mice. PMID:23648645

  3. Antigen targeting to APC: from mice to veterinary species.

    PubMed

    Alvarez, B; Poderoso, T; Alonso, F; Ezquerra, A; Domínguez, J; Revilla, C

    2013-10-01

    Antigen delivery to receptors expressed on antigen presenting cells (APC) has shown to improve immunogenicity of vaccines in mice. An enhancement of cytotoxic T lymphocyte (CTL), helper T cell or humoral responses was obtained depending on the type of APC and the surface molecule targeted. Although this strategy is being also evaluated in livestock animals with promising results, some discrepancies have been found between species and pathogens. The genetic diversity of livestock animals, the different pattern of expression of some receptors among species, the use of different markers to characterize APC in large animals and sometimes the lack of reagents make difficult to compare results obtained in different species. In this review, we summarize the data available regarding antigen targeting to APC receptors in cattle, sheep and pig and discuss the results found in these animals in the context of what has been obtained in mice.

  4. Identification of Mom12 and Mom13, two novel modifier loci of Apc (Min) -mediated intestinal tumorigenesis.

    PubMed

    Crist, Richard C; Roth, Jacquelyn J; Lisanti, Michael P; Siracusa, Linda D; Buchberg, Arthur M

    2011-04-01

    Colorectal cancer is a heterogeneous disease resulting from a combination of genetic and environmental factors. The C57BL/6J (B6) Apc (Min/+) mouse develops polyps throughout the gastrointestinal tract and has been a valuable model for understanding the genetic basis of intestinal tumorigenesis. Apc (Min/+) mice have been used to study known oncogenes and tumor suppressor genes on a controlled genetic background. These studies often utilize congenic knockout alleles, which can carry an unknown amount of residual donor DNA. The Apc (Min) model has also been used to identify modifer loci, known as Modifier of Min (Mom) loci, which alter Apc (Min) -mediated intestinal tumorigenesis. B6 mice carrying a knockout allele generated in WW6 embryonic stem cells were crossed to B6 Apc (Min/+) mice to determine the effect on polyp multiplicity. The newly generated colony developed significantly more intestinal polyps than Apc (Min/+) controls. Polyp multiplicity did not correlate with inheritance of the knockout allele, suggesting the presence of one or more modifier loci segregating in the colony. Genotyping of simple sequence length polymorphism (SSLP) markers revealed residual 129X1/SvJ genomic DNA within the congenic region of the parental knockout line. An analysis of polyp multiplicity data and SSLP genotyping indicated the presence of two Mom loci in the colony: 1) Mom12, a dominant modifier linked to the congenic region on chromosome 6, and 2) Mom13, which is unlinked to the congenic region and whose effect is masked by Mom12. The identification of Mom12 and Mom13 demonstrates the potential problems resulting from residual heterozygosity present in congenic lines. PMID:21386660

  5. Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2

    PubMed Central

    Ruane, Peter T.; Gumy, Laura F.; Bola, Becky; Anderson, Beverley; Wozniak, Marcin J.; Hoogenraad, Casper C.; Allan, Victoria J.

    2016-01-01

    Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons. PMID:27272132

  6. LOS2, a genetic locus required for cold-responsive gene transcription encodes a bi-functional enolase.

    PubMed

    Lee, Hojoung; Guo, Yan; Ohta, Masaru; Xiong, Liming; Stevenson, Becky; Zhu, Jian-Kang

    2002-06-01

    The Arabidopsis mutation, los2, impairs cold-responsive gene transcription, acquired freezing tolerance and plant resistance to chilling under certain conditions. LOS2 was isolated through positional cloning and shown to encode an enolase in the glycolytic pathway. In animal cells, enolase has also been known to function as a transcription factor that represses the expression of c-myc by binding to the c-myc gene promoter. LOS2 fused to green fluorescent protein is targeted to the nucleus as well as to the cytoplasm. LOS2/enolase protein can bind to the cis-element of the human c-myc gene promoter and to the gene promoter of STZ/ZAT10, a zinc finger transcriptional repressor from Arabidopsis. STZ/ZAT10 expression is induced rapidly and transiently by cold in the wild type, and this induction is stronger and more sustained in the los2 mutant. Furthermore, the expression of a RD29A-LUC reporter gene is repressed significantly by STZ/ZAT10 in transient expression assays in Arabidopsis leaves. Our results demonstrate that cold-responsive gene transcription in plants is controlled by a bi-functional enolase.

  7. SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs

    PubMed Central

    van der Lelij, Petra; Stocsits, Roman R; Ladurner, Rene; Petzold, Georg; Kreidl, Emanuel; Koch, Birgit; Schmitz, Julia; Neumann, Beate; Ellenberg, Jan; Peters, Jan-Michael

    2014-01-01

    Although splicing is essential for the expression of most eukaryotic genes, inactivation of splicing factors causes specific defects in mitosis. The molecular cause of this defect is unknown. Here, we show that the spliceosome subunits SNW1 and PRPF8 are essential for sister chromatid cohesion in human cells. A transcriptome-wide analysis revealed that SNW1 or PRPF8 depletion affects the splicing of specific introns in a subset of pre-mRNAs, including pre-mRNAs encoding the cohesion protein sororin and the APC/C subunit APC2. SNW1 depletion causes cohesion defects predominantly by reducing sororin levels, which causes destabilisation of cohesin on DNA. SNW1 depletion also reduces APC/C activity and contributes to cohesion defects indirectly by delaying mitosis and causing “cohesion fatigue”. Simultaneous expression of sororin and APC2 from intron-less cDNAs restores cohesion in SNW1-depleted cells. These results indicate that the spliceosome is required for mitosis because it enables expression of genes essential for cohesion. Our transcriptome-wide identification of retained introns in SNW1- and PRPF8-depleted cells may help to understand the aetiology of diseases associated with splicing defects, such as retinosa pigmentosum and cancer. PMID:25257309

  8. Insights into anaphase promoting complex TPR subdomain assembly from a CDC26-APC6 structure

    SciTech Connect

    Wang, Jing; Dye, Billy T; Rajashankar, Kanagalaghatta R; Kurinov, Igor; Schulman, Brenda A

    2009-12-01

    The multisubunit anaphase promoting complex (APC) is an essential cell-cycle regulator. Although CDC26 is known to have a role in APC assembly, its molecular function has remained unclear. Biophysical, structural and genetic studies presented here reveal that CDC26 stabilizes the structure of APC6, a core TPR protein required for APC integrity. Notably, CDC26-APC6 association involves an intermolecular TPR mimic composed of one helix from each protein.

  9. The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators

    PubMed Central

    Hagting, Anja; Izawa, Daisuke; Mansfeld, Jörg; Gibson, Toby J.; Pines, Jonathon

    2016-01-01

    The APC/C is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the Spindle Assembly Checkpoint (SAC). How the APC/C recognises its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in Cyclin A, BUBR1, BUB1 and Acm1, and show that it binds to the APC/C co-activator CDC20. The ABBA motif in Cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC. PMID:25669885

  10. The UMD-APC database, a model of nation-wide knowledge base: update with data from 3,581 variations.

    PubMed

    Grandval, Philippe; Blayau, Martine; Buisine, Marie-Pierre; Coulet, Florence; Maugard, Christine; Pinson, Stéphane; Remenieras, Audrey; Tinat, Julie; Uhrhammer, Nancy; Béroud, Christophe; Olschwang, Sylviane

    2014-05-01

    Familial adenomatous polyposis (FAP) is a rare autosomal-inherited disease that highly predisposes to colorectal cancer, characterized by a diffuse duodenal and colorectal polyposis associated with various extradigestive tumors and linked to germline mutations within the APC gene. A French consortium of laboratories involved in APC mutation screening has progressively improved the description of the variation spectrum, inferred functional significance of nontruncating variations, and delineated phenotypic characteristics of the disease. The current version of the UMD-APC database is described here. The total number of variations has risen to 5,453 representing 1,473 distinct variations. The published records initially registered into the database were extended with 3,581 germline variations found through genetic testing performed by the eight licensed laboratories belonging to the French APC network. Sixty six of 149 variations of previously unknown significance have now been classified as (likely) causal or neutral. The database is available on the Internet (http://www.umd.be/APC/) and updated twice per year according to the consensus rules of the network. The UMD-APC database is thus expected to facilitate functional classification of rare synonymous, nonsynonymous, and intronic mutations and consequently improve genetic counseling and medical care in FAP families.

  11. Association of APC, GSTP1 and SOCS1 promoter methylation with the risk of hepatocellular carcinoma: a meta-analysis.

    PubMed

    Liu, Meng; Cui, Lian-Hua; Li, Cheng-Cheng; Zhang, Li

    2015-11-01

    Studies of the relationships of adenomatous polyposis coli (APC), glutathione-S-transferase P1 (GSTP1) and suppressor of the cytokine signalling 1 (SOCS1) promoter region methylation with the risk of hepatocellular carcinoma (HCC) have yielded inconsistent results. We carried out the current meta-analysis to comprehensively assess the associations between APC, GSTP1 and SOCS1 promoter methylation frequency and the risk of HCC. All relevant reports were identified by searching the PubMed, Embase, Web of Science, CNKI and the Chinese BioMedical Literature databases before 1 March 2014, with restriction to articles published in the Chinese and English languages. Pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to investigate the rates of APC, GSTP1 and SOCS1 promoter methylation and the risk of HCC. Our meta-analysis identified relationships of APC (12 studies with 592 HCC tumour tissues), GSTP1 (14 studies including 646 HCC tumour tissues) and SOCS1 (11 studies with 512 HCC tumour tissues) promoter methylation with the risk of HCC. Compared with paracancerous tissues, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 5.32 (95% CI=2.96-9.56), 5.65, (95% CI=3.41-9.35) and 2.73 (95% CI=1.37-5.44), respectively. Compared with normal liver tissues as controls, the pooled ORs of APC, GSTP1 and SOCS1 promoter region methylation in HCC cancer tissues were 20.43 (95% CI=5.56-75.08), 18.78 (95% CI=5.76-61.19) and 13.00 (95% CI=5.20-32.47), respectively. Subgroup analysis by ethnicity showed that APC, GSTP1 and SOCS1 promoter methylation was associated significantly with the risk of HCC in both Asian and White populations (all P<0.05). Our meta-analysis suggested strong associations between APC, GSTP1 and SOCS1 gene promoter methylation and the risk of HCC, suggesting these to be promising biomarkers for HCC. PMID:25853848

  12. Some fine-structural findings on the thyroid gland in Apc1638T/1638T mice that express a C-terminus lacking truncated Apc.

    PubMed

    Yokoyama, Atsushi; Nomura, Ryuji; Kurosumi, Masafumi; Shimomura, Atsushi; Onouchi, Takanori; Iizuka-Kogo, Akiko; Smits, Ron; Fodde, Riccardo; Itoh, Mditsuyasu; Senda, Takao

    2012-06-01

    Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc(1638T/1638T) mice that express a truncated Apc lacking the C-terminal domain. The Apc(1638T/1638T) mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc(1638T/1638T) mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc(1638T/1638T) and Apc(+/+) mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc(1638T/1638T) mice were significantly different whereas the Apc(+/+) mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc(1638T/1638T) follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc(+/+) follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc(1638T/1638T) mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc(+/+) mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc(1638T/1638T) mice and suggest suppressed secretory activities of these cells.

  13. Phosphorylation and dephosphorylation regulate APC/CCdh1 substrate degradation

    PubMed Central

    Simpson-Lavy, Kobi J; Zenvirth, Drora; Brandeis, Michael

    2015-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase activated by its G1 specific adaptor protein Cdh1 is a major regulator of the cell cycle. The APC/CCdh1 mediates degradation of dozens of proteins, however, the kinetics and requirements for their degradation are largely unknown. We demonstrate that overexpression of the constitutive active CDH1m11 mutant that is not inhibited by phosphorylation results in mitotic exit in the absence of the FEAR and MEN pathways, and DNA re-replication in the absence of Cdc7 activity. This mode of mitotic exit also reveals additional requirements for APC/CCdh1 substrate degradation, which for some substrates such as Pds1 or Clb5 is dephosphorylation, but for others such as Cdc5 is phosphorylation. PMID:26252546

  14. Dietary Methyl Donor Depletion Protects Against Intestinal Tumorigenesis in ApcMin/+ Mice

    PubMed Central

    Kadaveru, Krishna; Protiva, Petr; Greenspan, Emily J; Kim, Young-In; Rosenberg, Daniel W

    2012-01-01

    Despite recent population data, the influence of dietary folate supplementation on colon cancer risk remains controversial. This study examines the effects of folate deficiency, in combination with choline, methionine and vitamin B12 depletion, on intestinal tumorigenesis in ApcMin/+ mice. Methyl donor sufficient (MDS) and deficient (MDD) diets were started at 5 or 10 weeks of age and tumors evaluated at 16 weeks. MDD suppressed intestinal tumor formation in ApcMin/+ mice (~80%) when started at 5 weeks of age. The protective effect was lost when MDD was initiated at 10 weeks of age, indicating an important time-dependency on cancer suppression. Concomitant with cancer protection, MDD restricted body weight gain. Therefore, a second study was conducted in which MDS was given ad libitum or pair-fed with MDD. While small intestinal tumors were reduced 54% in pair-fed MDS mice, MDD caused a further reduction (96%). In colon, although MDD did not affect tumor numbers, tumor size was reduced. Gene expression profiling of normal-appearing colonic mucosa after 11 weeks on MDD identified a total of 493 significantly down-regulated genes relative to the MDS group. Pathway analysis placed many of these genes within general categories of inflammatory signaling and cell cycle regulation, consistent with recently published human data obtained during folate depletion (1). Further studies are warranted to investigate the complex interplay of methyl donor status and cancer protection in high-risk populations. PMID:22677908

  15. Cdh1 regulates craniofacial development via APC-dependent ubiquitination and activation of Goosecoid.

    PubMed

    Shao, Rui; Liu, Jia; Yan, Guang; Zhang, Jinfang; Han, Yujiao; Guo, Jianfeng; Xu, Zhan; Yuan, Zhu; Liu, Jiankang; Malumbres, Marcos; Wan, Lixin; Wei, Wenyi; Zou, Weiguo

    2016-06-01

    Craniofacial anomalies (CFAs) characterized by birth defects of skull and facial bones are the most frequent congenital disease. Genomic analysis has identified multiple genes responsible for CFAs; however, the underlying genetic mechanisms for the majority of CFAs remain largely unclear. Our previous study revealed that the Wwp2 E3 ubiquitin ligase facilitates craniofacial development in part through inducing monoubiquitination and activation of the paired-like homeobox transcription factor, Goosecoid (Gsc). Here we report that Gsc is also ubiquitinated and activated by the APC(Cdh1) E3 ubiquitin ligase, leading to transcriptional activation of various Gsc target genes crucial for craniofacial development. Consistenly, neural crest-specific Cdh1-knockout mice display similar bone malformation as Wwp2-deficient mice in the craniofacial region, characterized by a domed skull, a short snout and a twisted nasal bone. Mechanistically, like Wwp2-deficient mice, mice with Cdh1 deficiency in neural crest cells exhibit reduced Gsc/Sox6 transcriptional activities. Simultaneous deletion of Cdh1 and Wwp2 results in a more severe craniofacial defect compared with single gene deletion, suggesting a synergistic augmentation of Gsc activity by these two E3 ubiquitin ligases. Hence, our study reveals a novel role for Cdh1 in craniofacial development through promoting APC-dependent non-proteolytic ubiquitination and activation of Gsc.

  16. High performance APCS conceptual design and evaluation scoping study

    SciTech Connect

    Soelberg, N.; Liekhus, K.; Chambers, A.; Anderson, G.

    1998-02-01

    This Air Pollution Control System (APCS) Conceptual Design and Evaluation study was conducted to evaluate a high-performance (APC) system for minimizing air emissions from mixed waste thermal treatment systems. Seven variations of high-performance APCS designs were conceptualized using several design objectives. One of the system designs was selected for detailed process simulation using ASPEN PLUS to determine material and energy balances and evaluate performance. Installed system capital costs were also estimated. Sensitivity studies were conducted to evaluate the incremental cost and benefit of added carbon adsorber beds for mercury control, specific catalytic reduction for NO{sub x} control, and offgas retention tanks for holding the offgas until sample analysis is conducted to verify that the offgas meets emission limits. Results show that the high-performance dry-wet APCS can easily meet all expected emission limits except for possibly mercury. The capability to achieve high levels of mercury control (potentially necessary for thermally treating some DOE mixed streams) could not be validated using current performance data for mercury control technologies. The engineering approach and ASPEN PLUS modeling tool developed and used in this study identified APC equipment and system performance, size, cost, and other issues that are not yet resolved. These issues need to be addressed in feasibility studies and conceptual designs for new facilities or for determining how to modify existing facilities to meet expected emission limits. The ASPEN PLUS process simulation with current and refined input assumptions and calculations can be used to provide system performance information for decision-making, identifying best options, estimating costs, reducing the potential for emission violations, providing information needed for waste flow analysis, incorporating new APCS technologies in existing designs, or performing facility design and permitting activities.

  17. Characterization of Colorectal Cancer Development in Apc (min/+) Mice.

    PubMed

    Nalbantoglu, ILKe; Blanc, Valerie; Davidson, Nicholas O

    2016-01-01

    The Apc (min/+) mouse provides an excellent experimental model for studying genetic, environmental, and therapeutic aspects of intestinal neoplasia in humans. In this chapter, we will describe techniques for studying colon cancer development in Apc (min/+) mice on C57BL/6J (B6) background, focusing on the roles of environmental modifiers, including Dextran Sulfate Sodium (DSS), high fat diet, and bile acid supplementation in the context of experimental colorectal cancer. This chapter also includes protocols describing extraction and purification of DSS-contaminated RNA, as well as sampling, harvesting, and tissue processing. The common pathologic lesions encountered in these animals are described in detail. PMID:27246043

  18. Chemoprevention of Familial Adenomatous Polyposis by Bromo-noscapine (EM011) in the ApcMin/+ Mouse Model

    PubMed Central

    Li, Shiwang; Ghaleb, Amr M.; He, Jing; Bughani, Usha; Bialkowska, Agnieszka B.; Yang, Vincent W.; Joshi, Harish C.

    2011-01-01

    Germline mutation of the tumor suppressor gene, adenomatous polyposis coli (APC), is responsible for familial adenomatous polyposis (FAP) with nearly 100% risk for colon cancer at an early age. Although FAP is involved in only 1% of all colon cancer cases, over 80% of sporadic cancers harbor somatic mutations of APC. We show here that bromo-onoscapine (EM011), a rationally-designed synthetic derivative of a natural non-toxic tubulin-binding alkaloid-noscapine, that reduces the dynamics of microtubules, causes a reversible G2/M arrest in wild type mouse embryonic fibroblasts (MEFs), but an aberrant exit from a brief mitotic block, followed by apoptosis in MEFs after APC deletion with siRNA. Furthermore, both β-catenin levels and activity fell to half the original levels with a concomitant reduction of cell proliferation-inducing cyclin D1, c-Myc, and induction of cytostatic protein p21 prior to caspase-3 activation. Additionally, we show a statistically significant reduction in the number of newly emerging intestinal polyps (to 35% compared with untreated mice) as well as the mean size of polyps (to 42% compared with untreated mice) in EM011-treated ApcMin/+ mice as compared to their sham-treated control littermates. The remaining polyps in the EM011 treated group of ApcMin/+ mice showed evidence of elevated apoptosis as revealed by immunohistochemistry. We failed to detect any evidence of histopathological and hematological toxicities following EM011 treatment. Taken together, our data are persuasive that a clinical trial of EM011 is possible for the prevention/amelioration of polyposis in FAP patients. PMID:22052467

  19. Chlorinated Water Modulates the Development of Colorectal Tumors with Chromosomal Instability and Gut Microbiota in Apc-Deficient Mice

    PubMed Central

    Sasada, Tatsunari; Hinoi, Takao; Saito, Yasufumi; Adachi, Tomohiro; Takakura, Yuji; Kawaguchi, Yasuo; Sotomaru, Yusuke; Sentani, Kazuhiro; Oue, Naohide; Yasui, Wataru; Ohdan, Hideki

    2015-01-01

    The gastrointestinal tract is continuously exposed to a variety of chemicals and commensal bacteria. Recent studies have shown that changes in gut microbial populations caused by chlorine or other chemicals in the drinking water influence the development of human colorectal cancer, although the mechanism of tumorigenesis in the gut epithelium is obfuscated by the diversity of microflora and complexity of the tumor microenvironment. In this regard, mouse models that recapitulate human colorectal cancer are an invaluable tool. In this study, we used two conditional adenomatous polyposis coli (Apc) knockout mouse models to investigate the effect of chlorinated water on tumorigenesis in the digestive tract. Mice with colon-specific carcinoma—caused by either chromosomal (CDX2P 9.5-NLS Cre;Apc+/flox, abbreviated to CPC;Apc) or microsatellite (CDX2P9.5-G19Cre;Apcflox/flox and CDX2P9.5-G22Cre;Apcflox/flox) instability, respectively—were administered chlorinated (10.0 mg/L chlorine) or tap (0.7 mg/L chlorine) water and evaluated for colon polyp formation. In CPC;Apc mice given chlorinated drinking water, tumors tended to develop in the colon, whereas in those that drank tap water, tumors were mostly observed in the small intestine. There was no difference in the rate of tumor formation of CDX2P9.5-G19Cre;Apcflox/flox and CDX2P9.5-G22Cre;Apcflox/flox mice consuming chlorinated as compared to tap water, suggesting that microsatellite instability in the Apc gene does not significantly affect tumorigenesis. Chlorinated water altered the enteric environment by reducing the fecal populations of the obligatory anaerobes Clostridium perfringens and C. difficile, as well as species belonging to the Atopobium cluster, including Enterobacteriaceae and Staphylococcus sp., which was associated with colon tumorigenesis in CPC;Apc mice. These results suggest that differences in tumorigenesis among CPC;Apc mice consuming chlorinated versus tap water may be due to differences in

  20. The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

    PubMed

    Cao, Juxiang; Dai, Xiangpeng; Wan, Lixin; Wang, Hongshen; Zhang, Jinfang; Goff, Philip S; Sviderskaya, Elena V; Xuan, Zhenyu; Xu, Zhixiang; Xu, Xiaowei; Hinds, Philip; Flaherty, Keith T; Faller, Douglas V; Goding, Colin R; Wang, Yongjun; Wei, Wenyi; Cui, Rutao

    2015-09-01

    The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma. PMID:26329581

  1. Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes

    PubMed Central

    Zur, Amit; Brandeis, Michael

    2002-01-01

    The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. We studied how d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. We characterized the degradation of substrates with swapped d-boxes. Cyclin B1 with KEN was degraded by APC/Cfzr only. Fzy with RXXL could be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy- but not APC/Cfzr-specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/Cfzr is activated in early G1. These observations demonstrate how d-box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr. PMID:12198152

  2. APC/C-CCS52A complexes control meristem maintenance in the Arabidopsis root.

    PubMed

    Vanstraelen, Marleen; Baloban, Mikhail; Da Ines, Olivier; Cultrone, Antonietta; Lammens, Tim; Boudolf, Véronique; Brown, Spencer C; De Veylder, Lieven; Mergaert, Peter; Kondorosi, Eva

    2009-07-14

    Plant organs originate from meristems where stem cells are maintained to produce continuously daughter cells that are the source of different cell types. The cell cycle switch gene CCS52A, a substrate specific activator of the anaphase promoting complex/cyclosome (APC/C), controls the mitotic arrest and the transition of mitotic cycles to endoreduplication (ER) cycles as part of cell differentiation. Arabidopsis, unlike other organisms, contains 2 CCS52A isoforms. Here, we show that both of them are active and regulate meristem maintenance in the root tip, although through different mechanisms. The CCS52A1 activity in the elongation zone of the root stimulates ER and mitotic exit, and contributes to the border delineation between dividing and expanding cells. In contrast, CCS52A2 acts directly in the distal region of the root meristem to control identity of the quiescent center (QC) cells and stem cell maintenance. Cell proliferation assays in roots suggest that this control involves CCS52A2 mediated repression of mitotic activity in the QC cells. The data indicate that the CCS52A genes favor a low mitotic state in different cell types of the root tip that is required for meristem maintenance, and reveal a previously undescribed mechanism for APC/C mediated control in plant development.

  3. Dietary acrylamide intake and the risk of colorectal cancer with specific mutations in KRAS and APC.

    PubMed

    Hogervorst, Janneke G F; de Bruijn-Geraets, Daisy; Schouten, Leo J; van Engeland, Manon; de Kok, Theo M C M; Goldbohm, R Alexandra; van den Brandt, Piet A; Weijenberg, Matty P

    2014-05-01

    Acrylamide, a probable human carcinogen, is present in heat-treated carbohydrate-rich foods. Epidemiological studies have not shown a clear association between acrylamide intake and colorectal cancer (CRC) risk. This may be due to the molecular heterogeneity in colorectal tumors, which was not taken into consideration before. Since the acrylamide metabolite glycidamide induces specific DNA mutations in rodents, we investigated whether acrylamide is associated with CRC risk characterized by mutations in Kirsten-ras (KRAS) and adenomatous polyposis coli (APC); key genes in colorectal carcinogenesis. This case-cohort analysis, within the Netherlands Cohort Study on diet and cancer, was based on 7.3 years of follow-up. Acrylamide intake was assessed with a food frequency questionnaire. Mutation analysis of codons 1286-1520 in exon 15 in APC and codons 12 and 13 in exon 1 in KRAS was performed on tumor tissue of 733 cases. Hazard ratios (HR) were calculated using Cox proportional hazards analysis. Among men, acrylamide intake was statistically significantly associated with an increased risk of particularly tumors with an activating KRAS mutation {HR fourth versus first quartile: 2.12 [95% confidence interval (CI): 1.16-3.87], P trend: 0.01}. Among women, acrylamide intake was statistically significantly associated with a decreased risk of particularly tumors with a truncating APC mutation (fourth versus first quartile: 0.47 (95% CI: 0.23-0.94), P trend: 0.02), but only in the highest quartile of intake. This is the first study to show that acrylamide might be associated with CRC with specific somatic mutations, differentially in men and women. More research is needed to corroborate or refute these findings.

  4. Metabolomics of Apc Min/+ mice genetically susceptible to intestinal cancer

    PubMed Central

    2014-01-01

    Background To determine how diets high in saturated fat could increase polyp formation in the mouse model of intestinal neoplasia, Apc Min/+ , we conducted large-scale metabolome analysis and association study of colon and small intestine polyp formation from plasma and liver samples of Apc Min/+ vs. wild-type littermates, kept on low vs. high-fat diet. Label-free mass spectrometry was used to quantify untargeted plasma and acyl-CoA liver compounds, respectively. Differences in contrasts of interest were analyzed statistically by unsupervised and supervised modeling approaches, namely Principal Component Analysis and Linear Model of analysis of variance. Correlation between plasma metabolite concentrations and polyp numbers was analyzed with a zero-inflated Generalized Linear Model. Results Plasma metabolome in parallel to promotion of tumor development comprises a clearly distinct profile in Apc Min/+ mice vs. wild type littermates, which is further altered by high-fat diet. Further, functional metabolomics pathway and network analyses in Apc Min/+ mice on high-fat diet revealed associations between polyp formation and plasma metabolic compounds including those involved in amino-acids metabolism as well as nicotinamide and hippuric acid metabolic pathways. Finally, we also show changes in liver acyl-CoA profiles, which may result from a combination of Apc Min/+ -mediated tumor progression and high fat diet. The biological significance of these findings is discussed in the context of intestinal cancer progression. Conclusions These studies show that high-throughput metabolomics combined with appropriate statistical modeling and large scale functional approaches can be used to monitor and infer changes and interactions in the metabolome and genome of the host under controlled experimental conditions. Further these studies demonstrate the impact of diet on metabolic pathways and its relation to intestinal cancer progression. Based on our results, metabolic signatures

  5. Single-molecule spectroscopy and femtosecond transient absorption studies on the excitation energy transfer process in ApcE(1-240) dimers.

    PubMed

    Long, Saran; Zhou, Meng; Tang, Kun; Zeng, Xiao-Li; Niu, Yingli; Guo, Qianjin; Zhao, Kai-Hong; Xia, Andong

    2015-05-28

    ApcE(1-240) dimers with one intrinsic phycocyanobilin (PCB) chromophore in each monomer that is truncated from the core-membrane linker (ApcE) of phycobilisomes (PBS) in Nostoc sp. PCC 7120 show a sharp and significantly red-shifted absorption. Two explanations either conformation-dependent Förster resonance energy transfer (FRET) or the strong exciton coupling limit have been proposed for red-shifted absorption. This is a classic example of the special pair in the photosynthetic light harvesting proteins, but the mechanism of this interaction is still a matter of intense debate. We report the studies using single-molecule and transient absorption spectra on the interaction in the special pair of ApcE dimers. Our results demonstrate the presence of conformation-dependent FRET between the two PCB chromophores in ApcE dimers. The broad distributions of fluorescence intensities, lifetimes and polarization difference from single-molecule measurements reveal the heterogeneity of local protein-pigment environments in ApcE dimers, where the same molecular structures but different protein environments are the main reason for the two PCB chromophores with different spectral properties. The excitation energy transfer rate between the donor and the acceptor about (110 ps)(-1) is determined from transient absorption measurements. The red-shifted absorption in ApcE dimers could result from more extending conformation, which shows another type of absorption redshift that does not depend on strong exciton coupling. The results here stress the importance of conformation-controlled spectral properties of the chemically identical chromophores, which could be a general feature to control energy/electron transfer, widely existing in the light harvesting complexes. PMID:25925197

  6. Lovastatin, but not orlistat, reduces intestinal polyp volume in an ApcMin/+ mouse model.

    PubMed

    Notarnicola, Maria; Barone, Michele; Francavilla, Antonio; Tutino, Valeria; Bianco, Giusy; Tafaro, Angela; Minoia, Mario; Polimeno, Lorenzo; Napoli, Anna; Scavo, Maria Principia; Caruso, Maria Gabriella

    2016-08-01

    The statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and orlistat, an inhibitor of fatty acid synthase (FAS), inhibit tumor cell growth by restricting cholesterol and fatty acid synthesis, respectively. We previously demonstrated that an omega (ω)-3 polyunsaturated fatty acid (PUFA)- or olive oil-enriched diet reduced the polyp number and volume in ApcMin/+ mice. This phenomenon was associated with a significant inhibition of FAS and HMGCoAR, as well as an increase in the estrogen receptor (ER)β/α ratio. Herein, we evaluated the effect of lovastatin and orlistat on polyp development and ER expression in ApcMin/+ mice, in order to confirm previous data obtained with ω‑3-PUFAs and olive oil. As expected, the use of lovastatin and orlistat significantly reduced HMGCoAR and FAS enzymatic activities and gene expression in colonic tissues, but did not affect the number of intestinal polyps, while there was a statistically significant reduction in polyp volume only in the mouse group treated with lovastatin. In the mice receiving orlistat, we observed a significant increase in cell proliferation in the polyp tissue, as well as enhanced expression of ERα. Moreover, the overexpression of ERα was associated with a statistically significant increase in PES1, Shh and Gli1 protein levels, considered ERα-related molecular targets. PMID:27277576

  7. 75 FR 78246 - Medicare Program; Re-Chartering of the Advisory Panel on Ambulatory Payment Classification (APC...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-15

    ... Charter effective through November 21, 2012. FOR FURTHER INFORMATION CONTACT: Shirl Ackerman-Ross, (410... new technology APCs to clinical APCs). Evaluating APC group weights. Reviewing packaging the cost of... methodology for packaging and the impact of packaging on APC group structure and payment. Removing...

  8. Heterozygous deletion of ATG5 in Apc(Min/+) mice promotes intestinal adenoma growth and enhances the antitumor efficacy of interferon-gamma.

    PubMed

    Wang, Lu; Wang, Yan; Lu, Yuyin; Zhang, Qianyun; Qu, Xianjun

    2015-01-01

    Autophagy related gene 5 (ATG5) was lost in 23% of the patients with colorectal cancer (CRC) and the role of loss of ATG5 in the pathogenesis of CRC remains unclear. Knockdown of ATG5 in cancer cells enhances the antitumor efficacy of lots of chemotherapeutic agents. However, there is still no animal model to validate these in vitro observations in vivo. In this study, we found that heterozygous deletion of ATG5 in Apc(Min/+) mice increased the number and size of adenomas as compared with those in Apc(Min/+)ATG5(+/+) mice. To investigate whether ATG5 deficiency could sensitize tumors to chemotherapies, we compared the antitumor effects of Interferon-gamma (IFN-γ) between Apc(Min/+)ATG5(+/+) and Apc(Min/+)ATG5(+/-) mice, as IFN-γ is a potential tumor suppressor for CRC and has been used clinically as an efficient adjuvant to chemotherapy of cancer. We revealed that heterozygous deletion of ATG5 significantly enhanced the antitumor efficacy of IFN-γ. Early treatment of Apc(Min/+)ATG5(+/-) mice with IFN-γ decreased tumor incidence rate to 16.7% and reduced the number of adenomas by 95.5% and late treatment led to regression of tumor. Moreover, IFN-γ treatment did not cause any evident toxic reaction. Mechanistic analysis revealed that heterozygous deletion of ATG5 activated EGFR/ERK1/2 and Wnt/β-catenin pathways in adenomas of Apc(Min/+) mice and enhanced the effects of IFN-γ-dependent inhibition of these 2 pathways. Our results demonstrate that ATG5 plays important roles in intestinal tumor growth and combination of IFN-γ and ATG5 deficiency or ATG5-targeted inhibition is a promising strategy for prevention and treatment of CRC. PMID:25695667

  9. Structural insights into the EB1–APC interaction

    PubMed Central

    Honnappa, Srinivas; John, Corinne M; Kostrewa, Dirk; Winkler, Fritz K; Steinmetz, Michel O

    2005-01-01

    EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 μM affinity. The conserved APC Ile2805–Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789–Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC–EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms. PMID:15616574

  10. Involvement of MET/TWIST/APC Combination or the Potential Role of Ossification Factors in Pediatric High-Grade Osteosarcoma Oncogenesis1

    PubMed Central

    Entz-Werle, Natacha; Lavaux, Thomas; Metzger, Nadia; Stoetzel, Corinne; Lasthaus, Christelle; Marec, Perrine; Kalifa, Chantal; Brugieres, Laurence; Pacquement, Hélène; Schmitt, Claudine; Tabone, Marie-Dominique; Gentet, Jean-Claude; Lutz, Patrick; Babin, Annie; Oudet, Pierre; Gaub, Marie Pierre; Perrin-Schmitt, Fabienne

    2007-01-01

    Dysregulated cell growth or differentiation due to misexpression of developmental critical factors seems to be a decisive event in oncogenesis. As osteosarcomas are histologically defined by malignant osteoblasts producing an osteoid component, we prospected in pediatric osteosarcomas treated with OS94 protocol the genomic status of several genes implied in ossification processes. In 91 osteosarcoma cases, we focused on the analysis of the fibroblast growth factor receptors (FGFRs) TWIST, APC, and MET by allelotyping, real-time quantitative polymerase chain reaction, gene sequencing, and protein polymorphism study. Our study supports the frequent role of TWIST, APC, and MET as osteosarcoma markers (50%, 62%, and 50%, respectively). TWIST and MET were mainly found to be deleted, and no additional APC mutation was identified. Surprisingly, FGFRs are abnormal in only < 30%. Most of these factors and their abnormalities seem to be linked more or less to one clinical subgroup, but the most significant correlation is the link of MET, TWIST, and APC abnormalities to a worse outcome and their combination within abnormal tumors. A wider cohort is mandatory to define more robust molecular conclusions, but these results are to be considered as the beginning of a more accurate basis for diagnosis, in search of targeted therapies, and to further characterize prognostic markers. PMID:17786187

  11. Comparative characterization of the virulence gene clusters (lipooligosacharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species

    PubMed Central

    Richards, Vincent P.; Lefébure, Tristan; Pavinski Bitar, Paulina D.; Stanhope, Michael J.

    2013-01-01

    Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosacharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking. PMID:23279811

  12. Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

    PubMed

    Richards, Vincent P; Lefébure, Tristan; Pavinski Bitar, Paulina D; Stanhope, Michael J

    2013-03-01

    Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.

  13. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

    PubMed

    Luo, Feijun; Brooks, David G; Ye, Hongtao; Hamoudi, Rifat; Poulogiannis, George; Patek, Charles E; Winton, Douglas J; Arends, Mark J

    2009-10-01

    Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant

  14. Hematopoietic prostaglandin D synthase suppresses intestinal adenomas in ApcMin/+ mice.

    PubMed

    Park, Jae Man; Kanaoka, Yoshihide; Eguchi, Naomi; Aritake, Kosuke; Grujic, Sava; Materi, Alicia M; Buslon, Virgilio S; Tippin, Brigette L; Kwong, Alan M; Salido, Eduardo; French, Samuel W; Urade, Yoshihiro; Lin, Henry J

    2007-02-01

    Aspirin and other nonsteroidal anti-inflammatory drugs prevent some cases of colon cancer by inhibiting prostaglandin (PG) synthesis. PGE(2) promotes colon neoplasia, as shown by knockout mouse studies on enzymes and receptors in the PG cascade. A few experiments 20 to 30 years ago suggested that PGD(2) may suppress tumors, but a role for biosynthetic enzymes for PGD(2) in tumor development has not been studied. We report here that disruption of the gene for hematopoietic PGD synthase in Apc(Min/+) mice led to approximately 50% more intestinal adenomas compared with controls. Tumor size was not affected. By immunohistochemistry, we detected hematopoietic PGD synthase mainly in macrophages and monocytes of the gut mucosa. The mean number of tumors did not increase with knockout of the gene for the lipocalin type of the enzyme, which is not produced in the intestine. On the other hand, Apc(Min/+) mice with transgenic human hematopoietic PGD synthase tended to have 80% fewer intestinal adenomas. The transgene produced high mRNA levels (375-fold over endogenous). There was a suggestion of higher urinary excretion of 11beta-PGF(2alpha) and a lower excretion of a PGE(2) metabolite in transgenic mice, but differences (30-40%) were not statistically significant. The results support an interpretation that hematopoietic PGD synthase controls an inhibitory effect on intestinal tumors. Further studies will be needed to prove possible mechanisms, such as routing of PG production away from protumorigenic PGE(2) or inhibition of the nuclear factor-kappaB cascade by PGD(2) metabolites. PMID:17283118

  15. The Unique N-terminus of the UbcH10 E2 Enzyme Controls the Threshold for APC Activation and Enhances Checkpoint Regulation of the APC

    PubMed Central

    Summers, Matthew K.; Pan, Borlan; Mukhyala, Kiran; Jackson, Peter K.

    2008-01-01

    Summary In vitro, the Anaphase Promoting Complex (APC) E3 ligase functions with E2 ubiquitin conjugating enzymes of the E2–C and Ubc4/5 families to ubiquitinate substrates. However, only the use of the E2–C family, notably UbcH10, is genetically well validated. Here, we biochemically demonstrate preferential use of UbcH10 by the APC, specified by the E2 core domain. Importantly, an additional E2–E3 interaction mediated by the N-terminal extension of UbcH10 regulates APC activity. Mutating the highly conserved N-terminus increases substrate ubiquitination, the number of substrate lysines targeted, allows ubiquitination of APC substrates lacking their destruction-boxes, increases resistance to the APC inhibitors Emi1 and BubR1 in vitro, and bypasses the spindle checkpoint in vivo. Fusion of the UbcH10 N-terminus to UbcH5 restricts ubiquitination activity, but does not direct specific interactions with the APC. Thus, UbcH10 combines a specific E2–E3 interface and regulation via its N-terminal extension to limit APC activity for substrate selection and checkpoint control. PMID:18722180

  16. Synergistic inhibition of the APC/C by the removal of APC15 in HCT116 cells lacking UBE2C

    PubMed Central

    Garvanska, Dimitriya H.; Larsen, Marie Sofie Yoo

    2016-01-01

    ABSTRACT The spindle assembly checkpoint (SAC) inhibits the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores by generating a diffusible inhibitor termed the mitotic checkpoint complex (MCC). At metaphase, rapid activation of the APC/C requires removal of the MCC, a process that has been shown to depend on the APC/C E2 enzymes, UBE2C and UBE2S. Here we investigate the in vivo role of the APC/C E2 enzymes in SAC silencing using CRISPR/Cas9 genetically engineered HCT116 UBE2C or UBE2S null cell lines. Using live cell assays, we show that UBE2C and UBE2S make a minor contribution to SAC silencing in HCT116 cells. Strikingly, in cells specifically lacking UBE2C, we observe a strong synergistic inhibition of mitotic progression when we stabilize the MCC on the APC/C by depleting APC15, potentially reflecting increased competition between the MCC and the remaining initiating E2 enzyme UBE2D. In conclusion, we provide in vivo insight into the APC/C E2 module and its interplay with SAC silencing components. PMID:27591192

  17. Apc bridges Wnt/{beta}-catenin and BMP signaling during osteoblast differentiation of KS483 cells

    SciTech Connect

    Miclea, Razvan L.; Horst, Geertje van der; Robanus-Maandag, Els C.; Loewik, Clemens W.G.M.; Oostdijk, Wilma; Wit, Jan M.; Karperien, Marcel

    2011-06-10

    The canonical Wnt signaling pathway influences the differentiation of mesenchymal cell lineages in a quantitative and qualitative fashion depending on the dose of {beta}-catenin signaling. Adenomatous polyposis coli (Apc) is the critical intracellular regulator of {beta}-catenin turnover. To better understand the molecular mechanisms underlying the role of Apc in regulating the differentiation capacity of skeletal progenitor cells, we have knocked down Apc in the murine mesenchymal stem cell-like KS483 cells by stable expression of Apc-specific small interfering RNA. In routine culture, KSFrt-Apc{sub si} cells displayed a mesenchymal-like spindle shape morphology, exhibited markedly decreased proliferation and increased apoptosis. Apc knockdown resulted in upregulation of the Wnt/{beta}-catenin and the BMP/Smad signaling pathways, but osteogenic differentiation was completely inhibited. This effect could be rescued by adding high concentrations of BMP-7 to the differentiation medium. Furthermore, KSFrt-Apc{sub si} cells showed no potential to differentiate into chondrocytes or adipocytes. These results demonstrate that Apc is essential for the proliferation, survival and differentiation of KS483 cells. Apc knockdown blocks the osteogenic differentiation of skeletal progenitor cells, a process that can be overruled by high BMP signaling.

  18. Analytic Patch Configuration (APC) gateway version 1.0 user's guide

    NASA Technical Reports Server (NTRS)

    Bingel, Bradford D.

    1990-01-01

    The Analytic Patch Configuration (APC) is an interactive software tool which translates aircraft configuration geometry files from one format into another. This initial release of the APC Gateway accommodates six formats: the four accepted APC formats (89f, 89fd, 89u, and 89ud), the PATRAN 2.x phase 1 neutral file format, and the Integrated Aerodynamic Analysis System (IAAS) General Geometry (GG) format. Written in ANSI FORTRAN 77 and completely self-contained, the APC Gateway is very portable and was already installed on CDC/NOS, VAX/VMS, SUN, SGI/IRIS, CONVEX, and GRAY hosts.

  19. Controlling the response to DNA damage by the APC/C-Cdh1.

    PubMed

    de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

    2016-03-01

    Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

  20. APC: A New Code for Atmospheric Polarization Computations

    NASA Technical Reports Server (NTRS)

    Korkin, Sergey V.; Lyapustin, Alexei I.; Rozanov, Vladimir V.

    2014-01-01

    A new polarized radiative transfer code Atmospheric Polarization Computations (APC) is described. The code is based on separation of the diffuse light field into anisotropic and smooth (regular) parts. The anisotropic part is computed analytically. The smooth regular part is computed numerically using the discrete ordinates method. Vertical stratification of the atmosphere, common types of bidirectional surface reflection and scattering by spherical particles or spheroids are included. A particular consideration is given to computation of the bidirectional polarization distribution function (BPDF) of the waved ocean surface.

  1. Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset

    PubMed Central

    Listovsky, Tamar

    2013-01-01

    The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity. PMID:24100295

  2. An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination

    PubMed Central

    Zeng, Xing; King, Randall W.

    2012-01-01

    The Anaphase-Promoting Complex/Cyclosome (APC) is a ubiquitin ligase required for exit from mitosis. We previously showed that Tosyl Arginine Methyl Ester (TAME) inhibits APC-dependent proteolysis by competing with the C-terminal IR-tail of the APC activator Cdc20 for APC binding. Here we show that in the absence of APC substrates, TAME ejects Cdc20 from the APC by promoting Cdc20 auto-ubiquitination in its N-terminal region. Cyclin B1 antagonizes TAME's effect by promoting binding of free Cdc20 to the APC and suppressing Cdc20 auto-ubiquitination. Nevertheless, TAME stabilizes cyclin B1 in Xenopus extract by two mechanisms. First, it reduces the kcat of the APCCdc20/cyclin B1 complex without affecting the Km, slowing the initial ubiquitination of unmodified cyclin B1. Second, as cyclin B1 becomes ubiquitinated, it loses its ability to promote Cdc20 binding to the APC in the presence of TAME. As a result, cyclin B1 ubiquitination terminates before reaching the threshold necessary for proteolysis. PMID:22366722

  3. REMOVAL OF ARSENIC IN DRINKING WATER: ARS CFU-50 APC ELECTROFLOCCULATION AND FILTRATION WATER TREATMENT SYSTEM

    EPA Science Inventory

    ETV testing of the ARS CFU-50 APC Electroflocculation and Filtration Water Treatment System (ARS CFU-50 APC) for arsenic removal was conducted at the Town of Bernalillo Well #3 site from April 18 through May 2, 2006. The source water was chlorinated groundwater from two supply w...

  4. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    PubMed Central

    Barua, Dipak; Hlavacek, William S.

    2013-01-01

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases and , which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. We find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases and . Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by , we suggest that is a potential target for therapeutic intervention in colorectal cancer. Specific inhibition of is predicted to limit binding of β—catenin to truncated

  5. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    SciTech Connect

    Barua, Dipak; Hlavacek, William S.

    2013-09-26

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases CK1α and GSK–3β, which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. In this paper, we find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases CK1α and GSK–3β. Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1ϵ, we suggest that CK1ϵ is a potential target for therapeutic intervention in colorectal cancer. Finally, specific inhibition

  6. Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

    DOE PAGES

    Barua, Dipak; Hlavacek, William S.

    2013-09-26

    In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases CK1α and GSK–3β, which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of amore » rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. In this paper, we find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases CK1α and GSK–3β. Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1ϵ, we suggest that CK1ϵ is a potential target for therapeutic intervention in colorectal cancer. Finally, specific

  7. APC binds intermediate filaments and is required for their reorganization during cell migration.

    PubMed

    Sakamoto, Yasuhisa; Boëda, Batiste; Etienne-Manneville, Sandrine

    2013-02-01

    Intermediate filaments (IFs) are components of the cytoskeleton involved in most cellular functions, including cell migration. Primary astrocytes mainly express glial fibrillary acidic protein, vimentin, and nestin, which are essential for migration. In a wound-induced migration assay, IFs reorganized to form a polarized network that was coextensive with microtubules in cell protrusions. We found that the tumor suppressor adenomatous polyposis coli (APC) was required for microtubule interaction with IFs and for microtubule-dependent rearrangements of IFs during astrocyte migration. We also show that loss or truncation of APC correlated with the disorganization of the IF network in glioma and carcinoma cells. In migrating astrocytes, vimentin-associated APC colocalized with microtubules. APC directly bound polymerized vimentin via its armadillo repeats. This binding domain promoted vimentin polymerization in vitro and contributed to the elongation of IFs along microtubules. These results point to APC as a crucial regulator of IF organization and confirm its fundamental role in the coordinated regulation of cytoskeletons.

  8. Berberine potently attenuates intestinal polyps growth in ApcMin mice and familial adenomatous polyposis patients through inhibition of Wnt signalling

    PubMed Central

    Zhang, Junfang; Cao, Hailong; Zhang, Bing; Cao, Hanwei; Xu, Xiuqin; Ruan, Hang; Yi, Tingting; Tan, Li; Qu, Rui; Song, Gang; Wang, Bangmao; Hu, Tianhui

    2013-01-01

    As a traditional anti-inflammatory Chinese herbal medicine, Alkaloid berberine has been recently reported to exhibit anti-tumour effects against a wide spectrum of cancer. However, the mechanism was largely unknown. Gene chip array reveals that with berberine treatment, c-Myc, the target gene of Wnt pathway, was down-regulated 5.3-folds, indicating that berberine might inhibit Wnt signalling. TOPflash analysis revealed that Wnt activity was significantly reduced after berberine treatment, and the mechanism of which might be that berberine disrupted β-catenin transfer to nucleus through up-regulating the expression of adenomatous polyposis coli (APC) gene and stabilized APC-β-catenin complex. Berberine administration in ApcMin/+ mice exhibited fewer and smaller polyps in intestine, along with reduction in cyclin D1 and c-Myc expression. In clinical practice, oral administration of berberine also significantly reduced the familial adenomatous polyposis patients' polyp size along with the inhibition of cyclin D1 expression in polyp samples. These observations indicate that berberine inhibits colon tumour formation through inhibition of Wnt/β-catenin signalling and berberine might be a promising drug for the prevention of colon cancer. PMID:24015932

  9. Evaluation and analysis of SEASAT-A Scanning Multichannel Microwave Radiometer (SMMR) Antenna Pattern Correction (APC) algorithm

    NASA Technical Reports Server (NTRS)

    Kitzis, J. L.; Kitzis, S. N.

    1979-01-01

    An evaluation of the versions of the SEASAT-A SMMR antenna pattern correction (APC) algorithm is presented. Two efforts are focused upon in the APC evaluation: the intercomparison of the interim, box, cross, and nominal APC modes; and the development of software to facilitate the creation of matched spacecraft and surface truth data sets which are located together in time and space. The problems discovered in earlier versions of the APC, now corrected, are discussed.

  10. HIV-1 and HIV-2 differentially mature plasmacytoid dendritic cells into IFN-producing cells or APCs.

    PubMed

    Royle, Caroline M; Graham, David R; Sharma, Simone; Fuchs, Dietmar; Boasso, Adriano

    2014-10-01

    HIV-1 causes a progressive impairment of immune function. HIV-2 is a naturally attenuated form of HIV, and HIV-2 patients display a slow-progressing disease. The leading hypothesis for the difference in disease phenotype between HIV-1 and HIV-2 is that more efficient T cell-mediated immunity allows for immune-mediated control of HIV-2 infection, similar to that observed in the minority of HIV-1-infected long-term nonprogressors. Understanding how HIV-1 and HIV-2 differentially influence the immune function may highlight critical mechanisms determining disease outcome. We investigated the effects of exposing primary human peripheral blood cells to HIV-1 or HIV-2 in vitro. HIV-2 induced a gene expression profile distinct from HIV-1, characterized by reduced type I IFN, despite similar upregulation of IFN-stimulated genes and viral restriction factors. HIV-2 favored plasmacytoid dendritic cell (pDC) differentiation into cells with an APC phenotype rather than IFN-α-producing cells. HIV-2, but not HIV-1, inhibited IFN-α production in response to CpG-A. The balance between pDC maturation into IFN-α-producing cells or development of an APC phenotype differentiates the early response against HIV-1 and HIV-2. We propose that divergent paths of pDC differentiation driven by HIV-1 and HIV-2 cause the observed differences in pathogenicity between the two viruses.

  11. Evaluating the organizational effectiveness of APC implementation efforts.

    PubMed

    Patel, Rajan

    2002-06-01

    To optimize revenue under the Medicare outpatient prospective payment system's new coding-based ambulatory payment classifications (APCs), healthcare providers need to ensure several key steps are taken at the organizational level. Individuals who manage coding need to identify areas of overlap and adjust billing systems to reflect changes under the system. Billing managers should develop practices and protocols that provide detailed reviews of claims, implement a formal denial management program, track reasons for denials, and communicate denial information with their staffs. Proper evaluation of financial practices also is important. Financial managers need to develop formal ways to monitor financial performance consistently and on an ongoing basis and ensure the hospital is generating sufficient volume and keeping service costs in line with payments.

  12. Plant stanols induce intestinal tumor formation by up-regulating Wnt and EGFR signaling in Apc Min mice.

    PubMed

    Marttinen, Maija; Päivärinta, Essi; Storvik, Markus; Huikko, Laura; Luoma-Halkola, Heli; Piironen, Vieno; Pajari, Anne-Maria; Mutanen, Marja

    2013-01-01

    The rate of APC mutations in the intestine increases in middle-age. At the same period of life, plant sterol and stanol enriched functional foods are introduced to diet to lower blood cholesterol. This study examined the effect of plant stanol enriched diet on intestinal adenoma formation in the Apc(Min) mouse. Apc(Min) mice were fed 0.8% plant stanol diet or control diet for nine weeks. Cholesterol, plant sterols and plant stanols were analyzed from the caecum content and the intestinal mucosa. Levels of β-catenin, cyclin D1, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1/2 (ERK1/2) were measured from the intestinal mucosa by Western blotting. Gene expression was determined from the intestinal mucosa using Affymetrix and the data were analyzed for enriched categories and pathways. Plant stanols induced adenoma formation in the small intestine, however, the adenoma size was not affected. We saw increased levels of nuclear β-catenin, phosphorylated β-catenin (Ser675 and Ser552), nuclear cyclin D1, total and phosphorylated EGFR and phosphorylated ERK1/2 in the intestinal mucosa after plant stanol feeding. The Affymetrix data demonstrate that several enzymes of cholesterol synthesis pathway were up-regulated, although the cholesterol level in the intestinal mucosa was not altered. We show that plant stanols induce adenoma formation by activating Wnt and EGFR signaling. EGFR signaling seems to have promoted β-catenin phosphorylation and its translocation into the nucleus, where the expression of cyclin D1 was increased. Up-regulated cholesterol synthesis may partly explain the increased EGFR signaling in the plant stanol-fed mice.

  13. Copy number variants associated with 18p11.32, DCC and the promoter 1B region of APC in colorectal polyposis patients

    PubMed Central

    Masson, Amy L.; Talseth-Palmer, Bente A.; Evans, Tiffany-Jane; McElduff, Patrick; Spigelman, Allan D.; Hannan, Garry N.; Scott, Rodney J.

    2015-01-01

    Familial Adenomatous Polyposis (FAP) is the second most common inherited predisposition to colorectal cancer (CRC) associated with the development of hundreds to thousands of adenomas in the colon and rectum. Mutations in APC are found in ~ 80% polyposis patients with FAP. In the remaining 20% no genetic diagnosis can be provided suggesting other genes or mechanisms that render APC inactive may be responsible. Copy number variants (CNVs) remain to be investigated in FAP and may account for disease in a proportion of polyposis patients. A cohort of 56 polyposis patients and 40 controls were screened for CNVs using the 2.7M microarray (Affymetrix) with data analysed using ChAS (Affymetrix). A total of 142 CNVs were identified unique to the polyposis cohort suggesting their involvement in CRC risk. We specifically identified CNVs in four unrelated polyposis patients among CRC susceptibility genes APC, DCC, MLH1 and CTNNB1 which are likely to have contributed to disease development in these patients. A recurrent deletion was observed at position 18p11.32 in 9% of the patients screened that was of particular interest. Further investigation is necessary to fully understand the role of these variants in CRC risk given the high prevalence among the patients screened. PMID:26909336

  14. Stable MCC binding to the APC/C is required for a functional spindle assembly checkpoint

    PubMed Central

    Hein, Jamin B; Nilsson, Jakob

    2014-01-01

    The spindle assembly checkpoint (SAC) delays progression into anaphase until all chromosomes have aligned on the metaphase plate by inhibiting Cdc20, the mitotic co-activator of the APC/C. Mad2 and BubR1 bind and inhibit Cdc20, thereby forming the mitotic checkpoint complex (MCC), which can bind stably to the APC/C. Whether MCC formation per se is sufficient for a functional SAC or MCC association with the APC/C is required remains unclear. Here, we analyze the role of two conserved motifs in Cdc20, IR and C-Box, in binding of the MCC to the APC/C. Mutants in both motifs assemble the MCC normally, but IR motif integrity is particularly important for stable binding to the APC/C. Cells expressing Cdc20 with a mutated IR motif have a compromised SAC, as uninhibited Cdc20 can compete with the MCC for APC/C binding and activate it. We thus show that stable MCC association with the APC/C is critical for a functional SAC. PMID:24464857

  15. Oscillation of APC/C activity during cell cycle arrest promotes centrosome amplification

    PubMed Central

    Prosser, Suzanna L.; Samant, Mugdha D.; Baxter, Joanne E.; Morrison, Ciaran G.; Fry, Andrew M.

    2014-01-01

    Centrosome duplication is licensed by the disengagement, or ‘uncoupling’, of centrioles during late mitosis. However, arrest of cells in G2 can trigger premature centriole disengagement. Here, we show that premature disengagement results from untimely activation of the APC/C leading to securin degradation and release of active separase. APC/C activation during G2 arrest is dependent on Plk1-mediated degradation of the APC/C inhibitor, Emi1, but Plk1 also has a second APC/C-independent role in promoting disengagement. Importantly, APC/C and Plk1 activity also stimulate centriole disengagement in response to hydroxyurea or DNA damage-induced cell cycle arrest and this leads to centrosome amplification. However, the re-duplication of disengaged centrioles is dependent on Cdk2 activity and Cdk2 activation coincides with a subsequent inactivation of the APC/C and re-accumulation of cyclin A. Release from these arrests leads to mitotic entry but, due to the presence of disengaged and/or amplified centrosomes, formation of abnormal mitotic spindles that lead to chromosome missegregation. Thus, oscillation of APC/C activity during cell cycle arrest promotes both centrosome amplification and genome instability. PMID:22956538

  16. Diet- and Genetically-induced Obesity Produces Alterations in the Microbiome, Inflammation and Wnt Pathway in the Intestine of Apc+/1638N Mice: Comparisons and Contrasts

    PubMed Central

    Liu, Wei; Crott, Jimmy W.; Lyu, Lin; Pfalzer, Anna C.; Li, Jinchao; Choi, Sang-Woon; Yang, Yingke; Mason, Joel B.; Liu, Zhenhua

    2016-01-01

    Obesity is an established risk factor for colorectal cancer (CRC). Our previous study indicated that obesity increases activity of the pro-tumorigenic Wnt-signaling. Presently, we sought to further advance our understanding of the mechanisms by which obesity promotes CRC by examining associations between microbiome, inflammation and Wnt-signaling in Apc+/1638N mice whose obesity was induced by one of two modalities, diet- or genetically-induced obesity. Three groups were employed: Apc+/1638NLepr+/+ fed a low fat diet (10% fat), Apc+/1638NLepr+/+ fed a high fat diet (60% fat, diet-induced obesity), and Apc+/1638NLeprdb/db fed a low fat diet (genetically-induced obesity). All animals received diets for 16 weeks from 8 to 24 weeks of age. The abundance of 19 bowel cancer-associated bacterial taxa were examined by real-time PCR. The abundance of Turicibacter and Desulfovibrio decreased, but F. prausnitizii increased, in diet-induced obese mice (p < 0.05). In contrast, in genetically-induced obese mice, Bifidobacterium, A. muciniphila and E. rectale decreased, but Peptostrptococcus, and E. coli increased (p < 0.05). Both diet- and genetically-induced obesity altered the expression of genes involved in bacterial recognition (MyD88) and increased inflammation as indicated by elevated levels of cytokines (IFNγ and TNF-α for genetically-induced obesity, and IL-6 for diet-induced obesity). The elevated inflammation was associated with altered expression of genes that are integral components of the Wnt-signaling cascade in a fashion indicating its activation. These findings demonstrate that the composition of the small intestinal microbiome is affected differently in diet- and genetically-induced obesity, but both are associated with elevated intestinal inflammation and alterations of the Wnt pathway towards enhancing tumorigenesis.

  17. Diet- and Genetically-induced Obesity Produces Alterations in the Microbiome, Inflammation and Wnt Pathway in the Intestine of Apc+/1638N Mice: Comparisons and Contrasts

    PubMed Central

    Liu, Wei; Crott, Jimmy W.; Lyu, Lin; Pfalzer, Anna C.; Li, Jinchao; Choi, Sang-Woon; Yang, Yingke; Mason, Joel B.; Liu, Zhenhua

    2016-01-01

    Obesity is an established risk factor for colorectal cancer (CRC). Our previous study indicated that obesity increases activity of the pro-tumorigenic Wnt-signaling. Presently, we sought to further advance our understanding of the mechanisms by which obesity promotes CRC by examining associations between microbiome, inflammation and Wnt-signaling in Apc+/1638N mice whose obesity was induced by one of two modalities, diet- or genetically-induced obesity. Three groups were employed: Apc+/1638NLepr+/+ fed a low fat diet (10% fat), Apc+/1638NLepr+/+ fed a high fat diet (60% fat, diet-induced obesity), and Apc+/1638NLeprdb/db fed a low fat diet (genetically-induced obesity). All animals received diets for 16 weeks from 8 to 24 weeks of age. The abundance of 19 bowel cancer-associated bacterial taxa were examined by real-time PCR. The abundance of Turicibacter and Desulfovibrio decreased, but F. prausnitizii increased, in diet-induced obese mice (p < 0.05). In contrast, in genetically-induced obese mice, Bifidobacterium, A. muciniphila and E. rectale decreased, but Peptostrptococcus, and E. coli increased (p < 0.05). Both diet- and genetically-induced obesity altered the expression of genes involved in bacterial recognition (MyD88) and increased inflammation as indicated by elevated levels of cytokines (IFNγ and TNF-α for genetically-induced obesity, and IL-6 for diet-induced obesity). The elevated inflammation was associated with altered expression of genes that are integral components of the Wnt-signaling cascade in a fashion indicating its activation. These findings demonstrate that the composition of the small intestinal microbiome is affected differently in diet- and genetically-induced obesity, but both are associated with elevated intestinal inflammation and alterations of the Wnt pathway towards enhancing tumorigenesis. PMID:27698916

  18. β-Catenin destruction complex-independent regulation of Hippo–YAP signaling by APC in intestinal tumorigenesis

    PubMed Central

    Cai, Jing; Maitra, Anirban; Anders, Robert A.; Taketo, Makoto M.; Pan, Duojia

    2015-01-01

    Mutations in Adenomatous polyposis coli (APC) underlie familial adenomatous polyposis (FAP), an inherited cancer syndrome characterized by the widespread development of colorectal polyps. APC is best known as a scaffold protein in the β-catenin destruction complex, whose activity is antagonized by canonical Wnt signaling. Whether other effector pathways mediate APC's tumor suppressor function is less clear. Here we report that activation of YAP, the downstream effector of the Hippo signaling pathway, is a general hallmark of tubular adenomas from FAP patients. We show that APC functions as a scaffold protein that facilitates the Hippo kinase cascade by interacting with Sav1 and Lats1. Consistent with the molecular link between APC and the Hippo signaling pathway, genetic analysis reveals that YAP is absolutely required for the development of APC-deficient adenomas. These findings establish Hippo–YAP signaling as a critical effector pathway downstream from APC, independent from its involvement in the β-catenin destruction complex. PMID:26193883

  19. Influence of myeloperoxidase on colon tumor occurrence in inflamed versus non-inflamed colons of ApcMin/+ mice☆

    PubMed Central

    Al-Salihi, Mazin; Reichert, Ethan; Fitzpatrick, F.A.

    2015-01-01

    Control of colorectal cancer needs to be tailored to its etiology. Tumor promotion mechanisms in colitis-associated colon cancer differ somewhat from the mechanisms involved in hereditary and sporadic colorectal cancer. Unlike sporadic or inherited tumors, some experimental models show that colitis-associated colon tumors do not require cyclooxygenase (COX) expression for progression, and non-steroidal anti-inflammatory drugs (NSAIDs) which prevent sporadic or inherited colon cancer do not prevent colitis-associated colon cancer. We report that myeloperoxidase (MPO), an ancestor of the COX isoenzymes, is a determinant of colitis-associated colon tumors in ApcMin/+ mice. During experimentally induced colitis, inhibition of MPO by resorcinol dampened colon tumor development. Conversely, in the bowels of ApcMin/+ mice without colitis, resorcinol administration or ‘knockout’ of MPO gene coincided with a slight, but discernible increase in colon tumor incidence. Acrolein, a by-product of MPO catalysis, formed a covalent adduct with the phosphatase tensin homolog (PTEN) tumor suppressor and enhanced the activity of the Akt kinase proto-oncogene in vitro and in vivo. Thus, MPO may be an important determinant of diet and inflammation on colon cancer risk via its effect on endogenous exposure to oxidants and acrolein. We propose a hypothetical model to explain an apparent dichotomy between colon tumor occurrence and MPO inhibition in inflamed versus non-inflamed colons. PMID:26262998

  20. Grape antioxidant dietary fiber inhibits intestinal polyposis in ApcMin/+ mice: relation to cell cycle and immune response.

    PubMed

    Sánchez-Tena, Susana; Lizárraga, Daneida; Miranda, Anibal; Vinardell, Maria P; García-García, Francisco; Dopazo, Joaquín; Torres, Josep L; Saura-Calixto, Fulgencio; Capellà, Gabriel; Cascante, Marta

    2013-08-01

    Epidemiological and experimental studies suggest that fiber and phenolic compounds might have a protective effect on the development of colon cancer in humans. Accordingly, we assessed the chemopreventive efficacy and associated mechanisms of action of a lyophilized red grape pomace containing proanthocyanidin (PA)-rich dietary fiber [grape antioxidant dietary fiber (GADF)] on spontaneous intestinal tumorigenesis in the Apc(Min/+) mouse model. Mice were fed a standard diet (control group) or a 1% (w/w) GADF-supplemented diet (GADF group) for 6 weeks. GADF supplementation greatly reduced intestinal tumorigenesis, significantly decreasing the total number of polyps by 76%. Moreover, size distribution analysis showed a considerable reduction in all polyp size categories [diameter <1mm (65%), 1-2mm (67%) and >2mm (87%)]. In terms of polyp formation in the proximal, middle and distal portions of the small intestine, a decrease of 76, 81 and 73% was observed, respectively. Putative molecular mechanisms underlying the inhibition of intestinal tumorigenesis were investigated by comparison of microarray expression profiles of GADF-treated and non-treated mice. We observed that the effects of GADF are mainly associated with the induction of a G1 cell cycle arrest and the downregulation of genes related to the immune response and inflammation. Our findings show for the first time the efficacy and associated mechanisms of action of GADF against intestinal tumorigenesis in Apc(Min/+) mice, suggesting its potential for the prevention of colorectal cancer.

  1. A CDC20-APC/SOX2 Signaling Axis Regulates Human Glioblastoma Stem-like Cells.

    PubMed

    Mao, Diane D; Gujar, Amit D; Mahlokozera, Tatenda; Chen, Ishita; Pan, Yanchun; Luo, Jingqin; Brost, Taylor; Thompson, Elizabeth A; Turski, Alice; Leuthardt, Eric C; Dunn, Gavin P; Chicoine, Michael R; Rich, Keith M; Dowling, Joshua L; Zipfel, Gregory J; Dacey, Ralph G; Achilefu, Samuel; Tran, David D; Yano, Hiroko; Kim, Albert H

    2015-06-23

    Glioblastoma harbors a dynamic subpopulation of glioblastoma stem-like cells (GSCs) that can propagate tumors in vivo and is resistant to standard chemoradiation. Identification of the cell-intrinsic mechanisms governing this clinically important cell state may lead to the discovery of therapeutic strategies for this challenging malignancy. Here, we demonstrate that the mitotic E3 ubiquitin ligase CDC20-anaphase-promoting complex (CDC20-APC) drives invasiveness and self-renewal in patient tumor-derived GSCs. Moreover, CDC20 knockdown inhibited and CDC20 overexpression increased the ability of human GSCs to generate brain tumors in an orthotopic xenograft model in vivo. CDC20-APC control of GSC invasion and self-renewal operates through pluripotency-related transcription factor SOX2. Our results identify a CDC20-APC/SOX2 signaling axis that controls key biological properties of GSCs, with implications for CDC20-APC-targeted strategies in the treatment of glioblastoma.

  2. Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice.

    PubMed

    Velázquez, Kandy T; Enos, Reilly T; Narsale, Aditi A; Puppa, Melissa J; Davis, J Mark; Murphy, E Angela; Carson, James A

    2014-06-01

    Although there are currently no approved treatments for cancer cachexia, there is an intensified interest in developing therapies because of the high mortality index associated with muscle wasting diseases. Successful treatment of the cachectic patient focuses on improving or maintaining body weight and musculoskeletal function. Nutraceutical compounds, including the natural phytochemical quercetin, are being examined as potential treatments because of their anti-inflammatory, antioxidant, and anticarcinogenic properties. The purpose of this study was to determine the effect of quercetin supplementation on the progression of cachexia in the adenomatous polyposis coli (Apc)(Min/+) mouse model of colorectal cancer. At 15 wk of age, C57BL/6 and male Apc(Min/+) mice were supplemented with 25 mg/kg of quercetin or vehicle solution mix of Tang juice and water (V) daily for 3 wk. Body weight, strength, neuromuscular performance, and fatigue were assessed before and after quercetin or V interventions. Indicators of metabolic dysfunction and inflammatory signaling were also assessed. During the treatment period, the relative decrease in body weight in the Apc(Min/+) mice gavaged with V (Apc(Min/+)V; -14% ± 2.3) was higher than in control mice gavaged with V (+0.6% ± 1.0), control mice gavaged with quercetin (-2% ± 1.0), and Apc(Min/+) mice gavaged with quercetin (Apc(Min/+)Q; -9% ± 1.3). At 18 wk of age, the loss of grip strength and muscle mass shown in Apc(Min/+)V mice was significantly attenuated (P < 0.05) in Apc(Min/+)Q mice. Furthermore, Apc(Min/+)V mice had an induction of plasma interleukin-6 and muscle signal transducer and activator of transcription 3 phosphorylation, which were significantly (P < 0.05) mitigated in Apc(Min/+)Q mice, despite having a similar tumor burden. Quercetin treatment did not improve treadmill run-time-to-fatigue, hyperglycemia, or hyperlipidemia in cachectic Apc(Min/+) mice. Overall, quercetin supplementation positively affected

  3. A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

    PubMed Central

    Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

    2013-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

  4. Synergistic Blockade of Mitotic Exit by Two Chemical Inhibitors of the APC/C

    PubMed Central

    Sackton, Katharine L.; Dimova, Nevena; Zeng, Xing; Tian, Wei; Zhang, Mengmeng; Sackton, Timothy B.; Meaders, Johnathan; Pfaff, Kathleen L.; Sigoillot, Frederic; Yu, Hongtao; Luo, Xuelian; King, Randall W.

    2014-01-01

    Summary Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the Anaphase-Promoting Complex/Cyclosome (APC/C), a thirteen-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumor cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc205, which forms a co-receptor with the APC/C to recognize substrates containing a Destruction box (D-box)6-14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identified a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-L-arginine methyl ester (TAME), a small molecule that blocks the APC/C-Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine. PMID:25156254

  5. Evaluation and Analysis of Seasat a Scanning Multichannel Microwave Radiometer (SMMR) Antenna Pattern Correction (APC) Algorithm

    NASA Technical Reports Server (NTRS)

    Kitzis, S. N.; Kitzis, J. L.

    1979-01-01

    The accuracy of the SEASAT-A SMMR antenna pattern correction (APC) algorithm was assessed. Interim APC brightness temperature measurements for the SMMR 6.6 GHz channels are compared with surface truth derived sea surface temperatures. Plots and associated statistics are presented for SEASAT-A SMMR data acquired for the Gulf of Alaska experiment. The cross-track gradients observed in the 6.6 GHz brightness temperature data are discussed.

  6. Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apc-minus mice.

    PubMed

    Myzak, Melinda C; Dashwood, W Mohaiza; Orner, Gayle A; Ho, Emily; Dashwood, Roderick H

    2006-03-01

    Sulforaphane (SFN) is an isothiocyanate from broccoli that induces phase 2 detoxification enzymes. We recently reported that SFN acts as a histone deacetylase (HDAC) inhibitor in human colon cancer cells in vitro, and the present study sought to extend these findings in vivo. In mice treated with a single oral dose of 10 mumol SFN, there was significant inhibition of HDAC activity in the colonic mucosa after 6 h, and immunoblots revealed a concomitant increase in acetylated histones H3 and H4, which returned to control levels by 48 h. Longer-term treatment with SFN in the diet resulted in levels of acetylated histones and p21(WAF1) in the ileum, colon, prostate, and peripheral blood mononuclear cells that were elevated compared with controls. Consistent with these findings, SFN suppressed tumor development in Apc(min) mice, and there was an increase in acetylated histones in the polyps, including acetylated histones specifically associated with the promoter region of the P21 and bax genes. These results provide the first evidence for HDAC inhibition by SFN in vivo and imply that such a mechanism might contribute to the cancer chemoprotective and therapeutic effects of SFN, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

  7. Atomic structure of the APC/C and its mechanism of protein ubiquitination

    PubMed Central

    Yang, Jing; McLaughlin, Stephen H.; Barford, David

    2015-01-01

    The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

  8. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

    PubMed

    Li, Jun; Woods, Susan L; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R M; Spurdle, Amanda B; Simpson, Peter T; da Silva, Leonard; Lakhani, Sunil R; Clouston, Andrew D; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F; Wen, Xiaogang; Martin, Hilary C; Neklason, Deborah W; Davis, Sean R; Walker, Robert L; Calzone, Kathleen A; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N; Hulick, Peter J; Weissman, Scott M; Newlin, Anna; Rubinstein, Wendy S; Sampson, Jone E; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K; Huntsman, David G; Foulkes, William D; Carneiro, Fatima; Lindor, Noralane M; Edwards, Stacey L; French, Juliet D; Waddell, Nicola; Meltzer, Paul S; Worthley, Daniel L; Schrader, Kasmintan A; Chenevix-Trench, Georgia

    2016-05-01

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

  9. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

    PubMed

    Li, Jun; Woods, Susan L; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R M; Spurdle, Amanda B; Simpson, Peter T; da Silva, Leonard; Lakhani, Sunil R; Clouston, Andrew D; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F; Wen, Xiaogang; Martin, Hilary C; Neklason, Deborah W; Davis, Sean R; Walker, Robert L; Calzone, Kathleen A; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N; Hulick, Peter J; Weissman, Scott M; Newlin, Anna; Rubinstein, Wendy S; Sampson, Jone E; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K; Huntsman, David G; Foulkes, William D; Carneiro, Fatima; Lindor, Noralane M; Edwards, Stacey L; French, Juliet D; Waddell, Nicola; Meltzer, Paul S; Worthley, Daniel L; Schrader, Kasmintan A; Chenevix-Trench, Georgia

    2016-05-01

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present. PMID:27087319

  10. APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

    PubMed Central

    Mills, Kate M.; Brocardo, Mariana G.; Henderson, Beric R.

    2016-01-01

    Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations. PMID:26658612

  11. Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo.

    PubMed

    Cedeño, Cesyen; La Monaca, Esther; Esposito, Mara; Gutierrez, Gustavo J

    2016-01-01

    The anaphase-promoting complex or cyclosome (APC/C) is one of the major orchestrators of the cell division cycle in mammalian cells. The APC/C acts as a ubiquitin ligase that triggers sequential ubiquitylation of a significant number of substrates which will be eventually degraded by proteasomes during major transitions of the cell cycle. In this chapter, we present accessible methodologies to assess both in in vitro conditions and in cellular systems ubiquitylation reactions mediated by the APC/C. In addition, we also describe techniques to evidence the changes in protein stability provoked by modulation of the activity of the APC/C. Finally, specific methods to analyze interactors or posttranslational modifications of particular APC/C subunits are also discussed. Given the crucial role played by the APC/C in the regulation of the cell cycle, this review only focuses on its action and effects in actively proliferating cells. PMID:27613041

  12. APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

    PubMed Central

    Ginesta, Mireia M.; Diaz-Riascos, Zamira Vanessa; Busquets, Juli; Pelaez, Núria; Serrano, Teresa; Peinado, Miquel Àngel; Jorba, Rosa; García-Borobia, Francisco Javier; Capella, Gabriel; Fabregat, Joan

    2016-01-01

    Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71–80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms.

  13. APC promoter is frequently methylated in pancreatic juice of patients with pancreatic carcinomas or periampullary tumors

    PubMed Central

    Ginesta, Mireia M.; Diaz-Riascos, Zamira Vanessa; Busquets, Juli; Pelaez, Núria; Serrano, Teresa; Peinado, Miquel Àngel; Jorba, Rosa; García-Borobia, Francisco Javier; Capella, Gabriel; Fabregat, Joan

    2016-01-01

    Early detection of pancreatic and periampullary neoplasms is critical to improve their clinical outcome. The present authors previously demonstrated that DNA hypermethylation of adenomatous polyposis coli (APC), histamine receptor H2 (HRH2), cadherin 13 (CDH13), secreted protein acidic and cysteine rich (SPARC) and engrailed-1 (EN-1) promoters is frequently detected in pancreatic tumor cells. The aim of the present study was to assess their prevalence in pancreatic juice of carcinomas of the pancreas and periampullary area. A total of 135 pancreatic juices obtained from 85 pancreatic cancer (PC), 26 ampullary carcinoma (AC), 10 intraductal papillary mucinous neoplasm (IPMN) and 14 chronic pancreatitis (CP) patients were analyzed. The methylation status of the APC, HRH2, CDH13, SPARC and EN-1 promoters was analyzed using methylation specific-melting curve analysis (MS-MCA). Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were also tested with allele-specific quantitative polymerase chain reaction amplification. Out of the 5 promoters analyzed, APC (71%) and HRH2 (65%) were the most frequently methylated in PC juice. APC methylation was also detected at a high frequency in AC (76%) and IPMN (80%), but only occasionally observed in CP (7%). APC methylation had a high sensitivity (71–80%) for all types of cancer analyzed. The panel (where a sample scored as positive when ≥2 markers were methylated) did not outperform APC as a single marker. Finally, KRAS detection in pancreatic juice offered a lower sensitivity (50%) and specificity (71%) for detection of any cancer. APC hypermethylation in pancreatic juice, as assessed by MS-MCA, is a frequent event of potential clinical usefulness in the diagnosis of pancreatic and periampullary neoplasms. PMID:27602165

  14. Paternal B Vitamin Intake Is a Determinant of Growth, Hepatic Lipid Metabolism and Intestinal Tumor Volume in Female Apc1638N Mouse Offspring

    PubMed Central

    Sabet, Julia A.; Park, Lara K.; Iyer, Lakshmanan K.; Tai, Albert K.; Koh, Gar Yee; Pfalzer, Anna C.; Parnell, Laurence D.; Mason, Joel B.; Liu, Zhenhua; Byun, Alexander J.; Crott, Jimmy W.

    2016-01-01

    Background The importance of maternal nutrition to offspring health and risk of disease is well established. Emerging evidence suggests paternal diet may affect offspring health as well. Objective In the current study we sought to determine whether modulating pre-conception paternal B vitamin intake alters intestinal tumor formation in offspring. Additionally, we sought to identify potential mechanisms for the observed weight differential among offspring by profiling hepatic gene expression and lipid content. Methods Male Apc1638N mice (prone to intestinal tumor formation) were fed diets containing replete (control, CTRL), mildly deficient (DEF), or supplemental (SUPP) quantities of vitamins B2, B6, B12, and folate for 8 weeks before mating with control-fed wild type females. Wild type offspring were euthanized at weaning and hepatic gene expression profiled. Apc1638N offspring were fed a replete diet and euthanized at 28 weeks of age to assess tumor burden. Results No differences in intestinal tumor incidence or burden were found between male Apc1638N offspring of different paternal diet groups. Although in female Apc1638N offspring there were no differences in tumor incidence or multiplicity, a stepwise increase in tumor volume with increasing paternal B vitamin intake was observed. Interestingly, female offspring of SUPP and DEF fathers had a significantly lower body weight than those of CTRL fed fathers. Moreover, hepatic trigylcerides and cholesterol were elevated 3-fold in adult female offspring of SUPP fathers. Weanling offspring of the same fathers displayed altered expression of several key lipid-metabolism genes. Hundreds of differentially methylated regions were identified in the paternal sperm in response to DEF and SUPP diets. Aside from a few genes including Igf2, there was a striking lack of overlap between these genes differentially methylated in sperm and differentially expressed in offspring. Conclusions In this animal model, modulation of

  15. Geopolymers prepared from DC plasma treated air pollution control (APC) residues glass: properties and characterisation of the binder phase.

    PubMed

    Kourti, Ioanna; Devaraj, Amutha Rani; Bustos, Ana Guerrero; Deegan, David; Boccaccini, Aldo R; Cheeseman, Christopher R

    2011-11-30

    Air pollution control (APC) residues have been blended with glass-forming additives and treated using DC plasma technology to produce a high calcium aluminosilicate glass (APC glass). This has been used to form geopolymer-glass composites that exhibit high strength and density, low porosity, low water absorption, low leaching and high acid resistance. The composites have a microstructure consisting of un-reacted residual APC glass particles imbedded in a complex geopolymer and C-S-H gel binder phase, and behave as particle reinforced composites. The work demonstrates that materials prepared from DC plasma treated APC residues have potential to be used to form high quality pre-cast products.

  16. The Spindle Assembly Checkpoint Is Not Essential for Viability of Human Cells with Genetically Lowered APC/C Activity

    PubMed Central

    Wild, Thomas; Larsen, Marie Sofie Yoo; Narita, Takeo; Schou, Julie; Nilsson, Jakob; Choudhary, Chunaram

    2016-01-01

    Summary The anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes—UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C. PMID:26904940

  17. Polarizing T and B Cell Responses by APC-Targeted Subunit Vaccines

    PubMed Central

    Grødeland, Gunnveig; Fossum, Even; Bogen, Bjarne

    2015-01-01

    Current influenza vaccines mostly aim at the induction of specific neutralizing antibodies. While antibodies are important for protection against a particular virus strain, T cells can recognize epitopes that will offer broader protection against influenza. We have previously developed a DNA vaccine format by which protein antigens can be targeted specifically to receptors on antigen presenting cells (APCs). The DNA-encoded vaccine proteins are homodimers, each chain consisting of a targeting unit, a dimerization unit, and an antigen. The strategy of targeting antigen to APCs greatly enhances immune responses as compared to non-targeted controls. Furthermore, targeting of antigen to different receptors on APCs can polarize the immune response to different arms of immunity. Here, we discuss how targeting of hemagglutinin to MHC class II molecules increases Th2 and IgG1 antibody responses, whereas targeting to chemokine receptors XCR1 or CCR1/3/5 increases Th1 and IgG2a responses, in addition to CD8+ T cell responses. We also discuss these results in relation to work published by others on APC-targeting. Differential targeting of APC surface molecules may allow the induction of tailor-made phenotypes of adaptive immune responses that are optimal for protection against various infectious agents, including influenza virus. PMID:26257735

  18. APC-PC Combined Scheme in Gilbert Two State Model: Proposal and Study

    NASA Astrophysics Data System (ADS)

    Bulo, Yaka; Saring, Yang; Bhunia, Chandan Tilak

    2016-08-01

    In an automatic repeat request (ARQ) scheme, a packet is retransmitted if it gets corrupted due to transmission errors caused by the channel. However, an erroneous packet may contain both erroneous bits and correct bits and hence it may still contain useful information. The receiver may be able to combine this information from multiple erroneous copies to recover the correct packet. Packet combining (PC) is a simple and elegant scheme of error correction in transmitted packet, in which two received copies are XORed to obtain the bit location of erroneous bits. Thereafter, the packet is corrected by bit inversion of bit located as erroneous. Aggressive packet combining (APC) is a logic extension of PC primarily designed for wireless communication with objective of correcting error with low latency. PC offers higher throughput than APC, but PC does not correct double bit errors if occur in same bit location of erroneous copies of the packet. A hybrid technique is proposed to utilize the advantages of both APC and PC while attempting to remove the limitation of both. In the proposed technique, applications of APC-PC on Gilbert two state model has been studied. The simulation results show that the proposed technique offers better throughput than the conventional APC and lesser packet error rate than PC scheme.

  19. Production of geopolymers using glass produced from DC plasma treatment of air pollution control (APC) residues.

    PubMed

    Kourti, Ioanna; Rani, D Amutha; Deegan, D; Boccaccini, A R; Cheeseman, C R

    2010-04-15

    Air pollution control (APC) residues are the hazardous waste produced from cleaning gaseous emissions at energy-from-waste (EfW) facilities processing municipal solid waste (MSW). APC residues have been blended with glass-forming additives and treated using DC plasma technology to produce a high calcium alumino-silicate glass. This research has investigated the optimisation and properties of geopolymers prepared from this glass. Work has shown that high strength geopolymers can be formed and that the NaOH concentration of the activating solution significantly affects the properties. The broad particle size distribution of the APC residue glass used in these experiments results in a microstructure that contains unreacted glass particles included within a geopolymer binder phase. The high calcium content of APC residues may cause the formation of some amorphous calcium silicate hydrate (C-S-H) gel. A mix prepared with S/L=3.4, Si/Al=2.6 and [NaOH]=6M in the activating solution, produced high strength geopolymers with compressive strengths of approximately 130 MPa. This material had high density (2070 kg/m(3)) and low porosity. The research demonstrates for the first time that glass derived from DC plasma treatment of APC residues can be used to form high strength geopolymer-glass composites that have potential for use in a range of applications.

  20. Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function.

    PubMed

    de Lange, Job; Faramarz, Atiq; Oostra, Anneke B; de Menezes, Renee X; van der Meulen, Ida H; Rooimans, Martin A; Rockx, Davy A; Brakenhoff, Ruud H; van Beusechem, Victor W; King, Randall W; de Winter, Johan P; Wolthuis, Rob M F

    2015-01-01

    Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31(comet). A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers. PMID:26423134

  1. Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function

    PubMed Central

    de Lange, Job; Faramarz, Atiq; Oostra, Anneke B.; de Menezes, Renee X.; van der Meulen, Ida H.; Rooimans, Martin A.; Rockx, Davy A.; Brakenhoff, Ruud H.; van Beusechem, Victor W.; King, Randall W.; de Winter, Johan P.; Wolthuis, Rob M. F.

    2015-01-01

    Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers. PMID:26423134

  2. Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates

    PubMed Central

    Min, Mingwei; Mayor, Ugo; Lindon, Catherine

    2013-01-01

    Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting. PMID:24004664

  3. Co-regulation proteomics reveals substrates and mechanisms of APC/C-dependent degradation.

    PubMed

    Singh, Sasha A; Winter, Dominic; Kirchner, Marc; Chauhan, Ruchi; Ahmed, Saima; Ozlu, Nurhan; Tzur, Amit; Steen, Judith A; Steen, Hanno

    2014-02-18

    Using multiplexed quantitative proteomics, we analyzed cell cycle-dependent changes of the human proteome. We identified >4,400 proteins, each with a six-point abundance profile across the cell cycle. Hypothesizing that proteins with similar abundance profiles are co-regulated, we clustered the proteins with abundance profiles most similar to known Anaphase-Promoting Complex/Cyclosome (APC/C) substrates to identify additional putative APC/C substrates. This protein profile similarity screening (PPSS) analysis resulted in a shortlist enriched in kinases and kinesins. Biochemical studies on the kinesins confirmed KIFC1, KIF18A, KIF2C, and KIF4A as APC/C substrates. Furthermore, we showed that the APC/C(CDH1)-dependent degradation of KIFC1 regulates the bipolar spindle formation and proper cell division. A targeted quantitative proteomics experiment showed that KIFC1 degradation is modulated by a stabilizing CDK1-dependent phosphorylation site within the degradation motif of KIFC1. The regulation of KIFC1 (de-)phosphorylation and degradation provides insights into the fidelity and proper ordering of substrate degradation by the APC/C during mitosis. PMID:24510915

  4. Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer

    PubMed Central

    Simon, Janelle; Tschaharganeh, Darjus F; van Es, Johan H; Clevers, Hans; Lowe, Scott W

    2015-01-01

    The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals, and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC. PMID:26091037

  5. Effect of drying on leaching testing of treated municipal solid waste incineration APC-residues.

    PubMed

    Hu, Yuyan; Hyks, Jiri; Astrup, Thomas; Christensen, Thomas H

    2008-08-01

    Air-pollution-control (APC) residues from waste incinerators are hazardous waste according to European legislation and must be treated prior to landfilling. Batch and column leaching data determine which type of landfill can receive the treated APC-residues. CEN standards are prescribed for the batch and column leaching test; however, these standards do not specify whether or not the residue samples should be dried prior to the leaching testing. Laboratory tests were performed in parallel (dried/non-dried) on treated APC-residue samples and evaluated with respect to Cr, Cd, Cu, Pb and Zn leaching. The effect of drying of the wet APC-residue samples was particularly dramatic regarding the leaching of Cr. Drying resulted in 10-100 times more Cr leaching in both batch and columns test. Drying also affected the leaching of Cd, Cu and Pb. Initial Cd leaching was up to 100 times higher in column tests with dried APC-residue than in tests with wet residues. The effect of drying appeared to be a combination of decreasing the reduction capacity of the sample (Cr), decreasing pH (Cd, Cu) and in column tests also a wash-out of salts (probably affecting Cd and Pb). If the leaching tests are intended to mimic landfill conditions, the results of this paper suggest that the tests should be done on wet, non-dried residue samples, although this may be less practical than testing dried samples.

  6. Obesity promotes PhIP-induced small intestinal carcinogenesis in hCYP1A-db/db mice: involvement of mutations and DNA hypermethylation of Apc.

    PubMed

    Wang, Hong; Liu, Anna; Kuo, Yingyi; Chi, Eric; Yang, Xu; Zhang, Lanjing; Yang, Chung S

    2016-07-01

    Obesity is associated with an increased risk of cancer. To study the promotion of dietary carcinogen-induced gastrointestinal cancer by obesity, we employed 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to induce intestinal tumorigenesis in CYP1A-humanized (hCYP1A) mice, in which mouse Cyp1a1/1a2 was replaced with human CYP1A1/1A2 Obesity was introduced in hCYP1A mice by breeding with Lepr(db/+) mice to establish the genetically induced obese hCYP1A-Lepr(db/db) mice or by feeding hCYP1A mice a high-fat diet. PhIP induced the formation of small intestinal tumors at the ages of weeks 28-40 in obese hCYP1A mice, but not in lean hCYP1A mice. No tumors were found in colon and other gastrointestinal organs in the lean or obese mice. Using immunohistochemistry (IHC), we found strong positive staining of NF-κB p65, pSTAT3 and COX2 as well as elevated levels of nuclear β-catenin (Ctnnb1) in small intestinal tumors, but not in normal tissues. By sequencing Apc and Ctnnb1 genes, we found that most PhIP-induced small intestinal tumors in obese mice carried only a single heterozygous mutation in Apc By bisulfite-sequencing of CpG islands of Apc, we found DNA hypermethylation in a CpG cluster located in its transcription initiation site, which most likely caused the inactivation of the wild-type Apc allele. Our findings demonstrate that PhIP-induced small intestinal carcinogenesis in hCYP1A-db/db mice is promoted by obesity and involves Apc mutation and inactivation by DNA hypermethylation. This experimental result is consistent with the association of obesity and the increased incidence of small intestinal cancer in humans in recent decades. PMID:27207656

  7. AGILE integration into APC for high mix logic fab

    NASA Astrophysics Data System (ADS)

    Gatefait, M.; Lam, A.; Le Gratiet, B.; Mikolajczak, M.; Morin, V.; Chojnowski, N.; Kocsis, Z.; Smith, I.; Decaunes, J.; Ostrovsky, A.; Monget, C.

    2015-09-01

    mix logic Fab) in term of product and technology portfolio AGILE corrects for up to 120nm of product topography error on process layer with less than 50nm depth of focus Based on tool functionalities delivered by ASML and on high volume manufacturing requirement, AGILE integration is a real challenge. Regarding ST requirements "Automatic AGILE" functionality developed by ASML was not a turnkey solution and a dedicated functionality was needed. A "ST homemade AGILE integration" has been fully developed and implemented within ASML and ST constraints. This paper describes this integration in our Advanced Process Control platform (APC).

  8. Characterization of the male ApcMin/+ mouse as a hypogonadism model related to cancer cachexia

    PubMed Central

    White, James P.; Puppa, Melissa J.; Narsale, Aditi; Carson, James A.

    2013-01-01

    Summary Cancer cachexia, the unintentional loss of lean body mass, is associated with decreased quality of life and poor patient survival. Hypogonadism, involving a reduction in circulating testosterone, is associated with the cachectic condition. At this time there is a very limited understanding of the role of hypogonadism in cancer cachexia progression. This gap in our knowledge is related to a lack of functional hypogonadal models associated with cancer cachexia. The ApcMin/+ mouse is an established colorectal cancer model that develops an IL-6 dependent cachexia which is physiologically related to human disease due to the gradual progression of tumor development and cachexia. The purpose of this study was to assess the utility of the ApcMin/+ mouse for the examination of hypogonadism during cancer cachexia and to investigate if IL-6 has a role in this process. We report that ApcMin/+ mice that are weight stable have comparable testosterone levels and gonad size compared to wild type mice. Cachectic ApcMin/+ mice exhibit a reduction in circulating testosterone and gonad size, which has a significant association with the degree of muscle mass and functional strength loss. Circulating testosterone levels were also significantly associated with the suppression of myofibrillar protein synthesis. Skeletal muscle and testes androgen receptor expression were decreased with severe cachexia. Although testes STAT3 phosphorylation increased with severe cachexia, systemic IL-6 over-expression for 2 weeks was not sufficient to reduce either testes weight or circulating testosterone. Inhibition of systemic IL-6 signaling by an IL-6 receptor antibody to ApcMin/+ mice that had already initiated weight loss was sufficient to attenuate a reduction in testes size and circulating testosterone. In summary, the ApcMin/+ mouse becomes hypogonadal with the progression of cachexia severity and elevated circulating IL-6 levels may have a role in the development of hypogonadism during

  9. Multiple mucin depleted foci, high proliferation and low apoptotic response in the onset of colon carcinogenesis of the PIRC rat, mutated in Apc.

    PubMed

    Femia, Angelo Pietro; Luceri, Cristina; Soares, Paulo Victoria; Lodovici, Maura; Caderni, Giovanna

    2015-03-15

    PIRC rats (F344/NTac-Apc (am1137) ) mutated in the Apc gene spontaneously develop colon tumors thus mimicking familial adenomatous polyposis (FAP) and sporadic colorectal cancer (CRC) more closely than Apc-based rodent models developing tumors mostly in the small intestine. To understand whether microscopic dysplastic lesions precede the development of macroscopic tumors, PIRC rat colon was examined for the presence of mucin depleted foci (MDF), microadenomas of the rodent and human colon. Few MDF (about 4/animal) were already present in 1-month-old rats and their number rapidly increases to about 250 in 8-month-old rats. These lesions showed Wnt signaling activation (nuclear β-catenin accumulation) and were dramatically decreased by sulindac (320 ppm), a drug with chemopreventive activity (MDF/rat at 4 months: 156 ± 8 and 38 ± 6 in controls and sulindac-treated rats, respectively, means ± SE, p < 0.001). Since altered proliferation and apoptosis could underlie the early phases of carcinogenesis, we studied these processes in the apparently normal colon mucosa (NM) of 1-month-old PIRC and wt rats. Colon proliferation (PCNA expression) was significantly higher in PIRC rats. Notably, PIRC rat NM showed resistance to apoptosis since it sustained proliferation and had lower apoptosis after a cytotoxic insult with 1,2 dimethylhydrazine. Gene expression of Myc, p21, Birc5, Ogg1, Apex1 and Sod2 were significantly up-regulated in the NM of PIRC rat. The overall results put forward PIRC rat as useful model of colon carcinogenesis, either to study the process itself or to test in vivo chemopreventive agents in both short- and long-term studies.

  10. Asymmetric 1-alkyl-2-acyl phosphatidylcholine: a helper lipid for enhanced non-viral gene delivery.

    PubMed

    Huang, Zhaohua; Li, Weijun; Szoka, Francis C

    2012-05-01

    Rationally designed asymmetrical alkylacyl phosphatidylcholines (APC) have been synthesized and evaluated as helper lipids for non-viral gene delivery. A long aliphatic chain (C22-C24) was introduced at the 1-position of glycerol backbone, a branched lipid chain (C18) at the 2-position, and a phosphocholine head group at the 3-position. The fusogenicity of APC depends on the length and degree of saturation of the alkyl chain. Cationic lipids were formulated with APC as either lipoplexes or nanolipoparticles, and evaluated for their stability, transfection efficiency, and cytotoxicity. APC mediated high in vitro transfection efficiency, and had low cytotoxicity. Small nanolipoparticles (less than 100 nm) can be obtained with APC by applying as low as 0.1% PEG-lipid. Our study extends the type of helper lipids that are suitable for gene transfer and points the way to improve non-viral nucleic acid delivery system other than the traditional cationic lipids optimization.

  11. Characterization of elastic-plastic properties of AS4/APC-2 thermoplastic composite

    NASA Technical Reports Server (NTRS)

    Sun, C. T.; Yoon, K. J.

    1988-01-01

    Elastic and inelastic properties of AS4/APC-2 composites were characterized with respect to temperature variation by using a one-parameter orthotropic plasticity model and a one parameter failure criterion. Simple uniaxial off-axis tension tests were performed on coupon specimens of unidirectional AS4/APC-2 thermoplastic composite at various temperatures. To avoid the complication caused by the extension-shear coupling effect in off-axis testing, new tabs were designed and used on the test specimens. The experimental results showed that the nonlinear behavior of constitutive relations and the failure strengths can be characterized quite well using the one parameter plasticity model and the failure criterion, respectively.

  12. The CDC20-APC/SOX2 signaling axis: An achilles' heel for glioblastoma

    PubMed Central

    Gujar, Amit D.; Yano, Hiroko; Kim, Albert H.

    2016-01-01

    ABSTRACT Glioblastoma stem-like cells (GSCs) play a critical role in glioblastoma progression and recurrence. We discuss recent results on the role of the mitotic ubiquitin ligase cell division cycle 20–anaphase-promoting complex (CDC20-APC) in the governance of cardinal GSC functions through a mechanism involving the transcription factor sex-determining region Y-box 2 (SOX2). These findings expand the non-mitotic roles of CDC20-APC with implications for stem cell biology. PMID:27314081

  13. How holistic process control translates into high mix logic fab APC?

    NASA Astrophysics Data System (ADS)

    Le-Gratiet, B.; Gatefait, M.; Ducotè, J.; Decaunes, J.; Lam, A.; Beraud, B.; Mikolajczak, M.; Pelletier, A.; Orlando, B.; Sundermann, F.; Ostrovsky, A.; Lapeyre, C.

    2014-10-01

    Advanced CMOS nodes require more and more information to get the wafer process well setup. Process tool intrinsic capabilities are not sufficient to secure specifications. APC systems (Advanced Process Control) are being developed in waferfab to manage process context information to automatically adjust and tune wafer processing. The APC manages today Run to Run component from and between various process steps plus a sub-recipes/profiles corrections management. This paper will outline the architecture of an integrated/holistic process control system for a high mix advanced logic waferfoundry.

  14. An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition

    PubMed Central

    Ondracka, Andrej; Robbins, Jonathan A.; Cross, Frederick R.

    2016-01-01

    B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition. PMID:27410035

  15. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

    PubMed Central

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-01-01

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  16. An APC/C-Cdh1 Biosensor Reveals the Dynamics of Cdh1 Inactivation at the G1/S Transition.

    PubMed

    Ondracka, Andrej; Robbins, Jonathan A; Cross, Frederick R

    2016-01-01

    B-type cyclin-dependent kinase activity must be turned off for mitotic exit and G1 stabilization. B-type cyclin degradation is mediated by the anaphase-promoting complex/cyclosome (APC/C); during and after mitotic exit, APC/C is dependent on Cdh1. Cdh1 is in turn phosphorylated and inactivated by cyclin-CDK at the Start transition of the new cell cycle. We developed a biosensor to assess the cell cycle dynamics of APC/C-Cdh1. Nuclear exit of the G1 transcriptional repressor Whi5 is a known marker of Start; APC/C-Cdh1 is inactivated 12 min after Whi5 nuclear exit with little measurable cell-to-cell timing variability. Multiple phosphorylation sites on Cdh1 act in a redundant manner to repress its activity. Reducing the number of phosphorylation sites on Cdh1 can to some extent be tolerated for cell viability, but it increases variability in timing of APC/C-Cdh1 inactivation. Mutants with minimal subsets of phosphorylation sites required for viability exhibit striking stochasticity in multiple responses including budding, nuclear division, and APC/C-Cdh1 activity itself. Multiple cyclin-CDK complexes, as well as the stoichiometric inhibitor Acm1, contribute to APC/C-Cdh1 inactivation; this redundant control is likely to promote rapid and reliable APC/C-Cdh1 inactivation immediately following the Start transition.

  17. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  18. Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate

    PubMed Central

    Matsusaka, Takahiro; Enquist-Newman, Maria; Morgan, David O.; Pines, Jonathon

    2014-01-01

    ABSTRACT The Anaphase Promoting Complex or Cyclosome (APC/C) is critical to the control of mitosis. The APC/C is an ubiquitin ligase that targets specific mitotic regulators for proteolysis at distinct times in mitosis, but how this is achieved is not well understood. We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis. Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1. Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1. PMID:25217616

  19. APC/C-Cdh1 coordinates neurogenesis and cortical size during development

    NASA Astrophysics Data System (ADS)

    Delgado-Esteban, Maria; García-Higuera, Irene; Maestre, Carolina; Moreno, Sergio; Almeida, Angeles

    2013-12-01

    The morphology of the adult brain is the result of a delicate balance between neural progenitor proliferation and the initiation of neurogenesis in the embryonic period. Here we assessed whether the anaphase-promoting complex/cyclosome (APC/C) cofactor, Cdh1—which regulates mitosis exit and G1-phase length in dividing cells—regulates neurogenesis in vivo. We use an embryo-restricted Cdh1 knockout mouse model and show that functional APC/C-Cdh1 ubiquitin ligase activity is required for both terminal differentiation of cortical neurons in vitro and neurogenesis in vivo. Further, genetic ablation of Cdh1 impairs the ability of APC/C to promote neurogenesis by delaying the exit of the progenitor cells from the cell cycle. This causes replicative stress and p53-mediated apoptotic death resulting in decreased number of cortical neurons and cortex size. These results demonstrate that APC/C-Cdh1 coordinates cortical neurogenesis and size, thus posing Cdh1 in the molecular pathogenesis of congenital neurodevelopmental disorders, such as microcephaly.

  20. Cdh1/Hct1-APC is essential for the survival of postmitotic neurons.

    PubMed

    Almeida, Angeles; Bolaños, Juan P; Moreno, Sergio

    2005-09-01

    Cell division at the end of mitosis and G1 is controlled by Cdh1/Hct1, an activator of the E3-ubiquitin ligase anaphase-promoting complex (APC) that promotes the ubiquitylation and degradation of mitotic cyclins and other substrates. Cdh1-APC is active in postmitotic neurons, where it regulates axonal growth and patterning in the developing brain. However, it remains unknown whether Cdh1-APC is involved in preventing cell-cycle progression in terminally differentiated neurons. To address this issue, we used the small hairpin RNA strategy to deplete Cdh1 in postmitotic neurons. We observed that Cdh1 silencing rapidly triggered apoptotic neuronal death. To investigate the underlying mechanism, we focused on cyclin B1, a major Cdh1-APC substrate. Our results demonstrate that Cdh1 is required to prevent the accumulation of cyclin B1 in terminally differentiated neurons. Moreover, by keeping cyclin B1 low, Cdh1 prevented these neurons from entering an aberrant S phase that led to apoptotic cell death. These results provide an explanation for the mechanism of cyclin B1 reactivation that occurs in the brain of patients suffering from neurodegenerative diseases, such as Alzheimer's disease.

  1. A process for treatment of APC residues from municipal solid waste incinerators: Preliminary results

    SciTech Connect

    Hjelmar, O.; Birch, H.

    1997-12-01

    The problem of environmentally safe management of the residues from air pollution control (APC) systems at municipal solid waste (MSW) incinerators, particularly the residues from the semidry/dry acid gas cleaning processes (dry scrubber residues), has not yet been solved in a satisfactory and sustainable manner. These residues are in many cases simply stored indefinitely in big bags or they are landfilled under conditions that in the long term may not be able to prevent potentially harmful constituents from leaching and leaking into the environment. The APC residues, including fly ash, are in many countries classified as hazardous or special waste due to their high contents of soluble salts (particularly calcium chloride) and trace elements/heavy metals. The semidry/dry APC residues are strongly alkaline due to a content of excess lime, and the high pH favours the leaching of several contaminants, particularly lead. This paper presents preliminary results of a study of a process for treatment of semidry/dry APC residues and fly ash from MSW incinerators. In the process the contaminants are partly removed, partly immobilized thus improving the above mentioned situation and allowing for subsequent safe management (i.e. utilization or landfilling) of the treated residues.

  2. Control of APC/C-dependent ubiquitin chain elongation by reversible phosphorylation

    PubMed Central

    Craney, Allison; Kelly, Aileen; Jia, Luying; Fedrigo, Indro; Yu, Hongtao; Rape, Michael

    2016-01-01

    Most metazoan E3 ligases contain a signature RING domain that promotes the transfer of ubiquitin from the active site of E2 conjugating enzymes to lysine residues in substrates. Although these RING-E3s depend on E2 enzymes for catalysis, how they turn on their E2s at the right time and place remains poorly understood. Here we report a phosphorylation-dependent mechanism that ensures timely activation of the E2 Ube2S by its RING-E3, the anaphase-promoting complex (APC/C); while phosphorylation of a specific serine residue in the APC/C coactivator Cdc20 prevents delivery of Ube2S to the APC/C, removal of this mark by PP2AB56 allows Ube2S to bind the APC/C and catalyze ubiquitin chain elongation. PP2AB56 also stabilizes kinetochore–microtubule attachments to shut off the spindle checkpoint, suggesting that cells regulate the E2–E3 interplay to coordinate ubiquitination with critical events during cell division. PMID:26811472

  3. Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

    PubMed Central

    Lu, Dan; Hsiao, Jennifer Y.; Davey, Norman E.; Van Voorhis, Vanessa A.; Foster, Scott A.

    2014-01-01

    The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/CCdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/CCdc20 substrate destruction. PMID:25287299

  4. 42 CFR 419.31 - Ambulatory payment classification (APC) system and payment weights.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... services within a group are not comparable with respect to the use of resources if the highest median cost for an item or service within the group is more than 2 times greater than the lowest median cost for... the Federal Food, Drug and Cosmetic Act. (3) The payment rate determined for an APC group...

  5. Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in ApcMin/+ mice.

    PubMed

    Chen, Lei; Brar, Manreetpal S; Leung, Frederick C C; Hsiao, W L Wendy

    2016-05-24

    Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing. Study showed that GpS treatment significantly reduced the number of intestinal polyps in a preventive mode. More importantly, GpS feeding strikingly reduced the sulfate-reducing bacteria lineage, which are known to produce hydrogen sulfide and contribute to damage the intestinal epithelium or even promote cancer progression. Meanwhile, GpS also boosted the beneficial microbes. In the gut barrier of the ApcMin/+ mice, GpS treatment increased Paneth and goblet cells, up-regulated E-cadherin and down-regulated N-cadherin. In addition, GpS decreased the pro-oncogenic β-catenin, p-Src and the p-STAT3. Furthermore, GpS might also improve the inflamed gut epithelium of the ApcMin/+ mice by upregulating the anti-inflammatory cytokine IL-4, while downregulating pro-inflammatory cytokines TNF-α, IL-1β and IL-18. Intriguingly, GpS markedly stimulated M2 and suppressed M1 macrophage markers, indicating that GpS altered mucosal cytokine profile in favor of the M1 to M2 macrophages switching, facilitating intestinal tissue repair. In conclusion, GpS might reverse the host's inflammatory phenotype by increasing beneficial bacteria, decreasing sulfate-reducing bacteria, and alleviating intestinal inflammatory gut environment, which might contribute to its cancer preventive effects.

  6. Triterpenoid herbal saponins enhance beneficial bacteria, decrease sulfate-reducing bacteria, modulate inflammatory intestinal microenvironment and exert cancer preventive effects in ApcMin/+ mice

    PubMed Central

    Chen, Lei; Brar, Manreetpal S.; Leung, Frederick C. C.; Hsiao, W. L. Wendy

    2016-01-01

    Saponins derived from medicinal plants have raised considerable interest for their preventive roles in various diseases. Here, we investigated the impacts of triterpenoid saponins isolated from Gynostemma pentaphyllum (GpS) on gut microbiome, mucosal environment, and the preventive effect on tumor growth. Six-week old ApcMin/+ mice and their wild-type littermates were fed either with vehicle or GpS daily for the duration of 8 weeks. The fecal microbiome was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and 16S rRNA gene pyrosequencing. Study showed that GpS treatment significantly reduced the number of intestinal polyps in a preventive mode. More importantly, GpS feeding strikingly reduced the sulfate-reducing bacteria lineage, which are known to produce hydrogen sulfide and contribute to damage the intestinal epithelium or even promote cancer progression. Meanwhile, GpS also boosted the beneficial microbes. In the gut barrier of the ApcMin/+ mice, GpS treatment increased Paneth and goblet cells, up-regulated E-cadherin and down-regulated N-cadherin. In addition, GpS decreased the pro-oncogenic β-catenin, p-Src and the p-STAT3. Furthermore, GpS might also improve the inflamed gut epithelium of the ApcMin/+ mice by upregulating the anti-inflammatory cytokine IL-4, while downregulating pro-inflammatory cytokines TNF-β, IL-1β and IL-18. Intriguingly, GpS markedly stimulated M2 and suppressed M1 macrophage markers, indicating that GpS altered mucosal cytokine profile in favor of the M1 to M2 macrophages switching, facilitating intestinal tissue repair. In conclusion, GpS might reverse the host's inflammatory phenotype by increasing beneficial bacteria, decreasing sulfate-reducing bacteria, and alleviating intestinal inflammatory gut environment, which might contribute to its cancer preventive effects. PMID:27121311

  7. Curcumin suppresses intestinal polyps in APC Min mice fed a high fat diet.

    PubMed

    Pettan-Brewer, Christina; Morton, John; Mangalindan, Ruby; Ladiges, Warren

    2011-01-01

    Colorectal cancer (CRC) is a leading cause of cancer deaths in the United States. Various risk factors have been associated with CRC including increasing age and diet. Epidemiological and experimental studies have implicated a diet high in fat as an important risk factor for colon cancer. High fat diets can promote obesity resulting in insulin resistance and inflammation and the development of oxidative stress, increased cell proliferation, and suppression of apoptosis. Because of the high consumption of dietary fats, especially saturated fats, by Western countries, it is of interest to see if non-nutrient food factors might be effective in preventing or delaying CRC in the presence of high saturated fat intake. Curcumin (Curcuma longa), the main yellow pigment in turmeric, was selected to test because of its reported anti-tumor activity. APC Min mice, which develop intestinal polyps and have many molecular features of CRC, were fed a diet containing 35% pork fat, 33% sucrose, and a protein and vitamin mineral mixture (HFD) with or without 0.5% curcumin. These cohorts were compared to APC Min mice receiving standard rodent chow (RC) with 8% fat. APC Min mice fed the HFD for 3 months had a 23% increase in total number of polyps compared to APC Min mice on RC. Curcumin was able to significantly reverse the accelerated polyp development associated with the HFD suggesting it may be effective clinically in helping prevent colon cancer even when ingesting high amounts of fatty foods. The anti-tumor effect of curcumin was shown to be associated with enhanced apoptosis and increased efficiency of DNA repair. Since curcumin prevented the gain in body weight seen in APC Min mice ingesting the HFD, modulation of energy metabolism may also be a factor.

  8. Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit

    PubMed Central

    Meghini, Francesco; Martins, Torcato; Tait, Xavier; Fujimitsu, Kazuyuki; Yamano, Hiroyuki; Glover, David M.; Kimata, Yuu

    2016-01-01

    A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/CFzr substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity. PMID:27558644

  9. Targeting of Fzr/Cdh1 for timely activation of the APC/C at the centrosome during mitotic exit.

    PubMed

    Meghini, Francesco; Martins, Torcato; Tait, Xavier; Fujimitsu, Kazuyuki; Yamano, Hiroyuki; Glover, David M; Kimata, Yuu

    2016-08-25

    A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/C(Fzr) substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity.

  10. Dietary fat overcomes the protective activity of thrombospondin-1 signaling in the Apc(Min/+) model of colon cancer.

    PubMed

    Soto-Pantoja, D R; Sipes, J M; Martin-Manso, G; Westwood, B; Morris, N L; Ghosh, A; Emenaker, N J; Roberts, D D

    2016-01-01

    Thrombospondin 1 is a glycoprotein that regulates cellular phenotype through interactions with its cellular receptors and extracellular matrix-binding partners. Thrombospondin 1 locally regulates angiogenesis and inflammatory responses that contribute to colorectal carcinogenesis in Apc(Min/+) mice. The ability of thrombospondin 1 to regulate responses of cells and tissues to a variety of stresses suggested that loss of thrombospondin 1 may also have broader systemic effects on metabolism to modulate carcinogenesis. Apc(Min/+):Thbs1(-/-) mice exhibited decreased survival and higher tumor multiplicities in the small and large intestine relative to Apc(Min/+) mice when fed a low (5%) fat western diet. However, the protective effect of endogenous thrombospondin 1 was lost when the mice were fed a western diet containing 21% fat. Biochemical profiles of liver tissue identified systemic metabolic changes accompanying the effects of thrombospondin 1 and dietary lipid intake on tumorigenesis. A high-fat western diet differentially regulated elements of amino acid, energy and lipid metabolism in Apc(Min/+):Thbs1(-/-) mice relative to Apc(Min/+):Thbs1(+/+)mice. Metabolic changes in ketone body and tricarboxylic acid cycle intermediates indicate functional interactions between Apc and thrombospondin 1 signaling that control mitochondrial function. The cumulative diet-dependent differential changes observed in Apc(Min/+):Thbs1(-/-) versus Apc(Min/+) mice include altered amino acid and lipid metabolism, mitochondrial dysfunction, eicosanoids and ketone body formation. This metabolic profile suggests that the protective role of thrombospondin 1 to decrease adenoma formation in Apc(Min/+) mice results in part from improved mitochondrial function. PMID:27239962

  11. Assay for Detecting the I1307K Susceptibility Allele within the Adenomatous Polyposis ColiGene.

    PubMed

    Gruber, S B

    2001-01-01

    Most germline mutations of the adenomatous polyposis coli (APC) tumor suppressor gene result in a classic inherited cancer syndrome called familial adenomatous polyposis (FAP). FAP is characterized by thousands of colonic polyps, well-defined extracolonic manifestations that may include pigmented lesions of the ocular fundus, supernumerary teeth, osteomas, odontomas, desmoid tumors and epidermoid cysts, and a 100% lifetime risk of developing colorectal cancer. Shortly after the APC gene was cloned in 1991 (1,2) the molecular basis of an attenuated form of FAP was recognized to be related to germline mutations within APC that were most likely to be found in the 5' and 3' ends of the gene (3,4). The truncating mutations leading to classic FAP and attenuated FAP are quite rare, but recently a polymorphism of the APC gene was found among 6 to 7% of Ashkenazi Jews that approximately doubles the risk of colorectal cancer (5). PMID:21370146

  12. Self-pMHCII complexes are variably expressed in the thymus and periphery independent of mRNA expression but dependent on the activation state of the APCs

    PubMed Central

    Rodriguez, Stephanie N.; Jiang, Meizi; Bujo, Hideaki; Allen, Paul M.

    2014-01-01

    Self-peptide MHCII ligands are critical for selection of CD4+ T cells in the thymus, and maintenance in the periphery. To date, no investigation as to the exact thymic and peripheral expression of a naturally occurring positive selecting self-peptide MHCII (self-pMHCII) complex has taken place. We have generated a sensitive T cell hybridoma to functionally detect the endogenous presentation of a confirmed positive selecting self-pMHCII complex for a CD4+ transgenic T cell. Using this tool to survey and quantify the expression selecting self-pMHCII, we have shown unequivocal proof that a known CD4+ selecting ligand can be presented on both positive and negative selecting thymic APCs. We also show that peripheral presentation of this same selecting ligand is affected by activation state of the APCs. Furthermore, discrepancies between the gene expression and self-pMHCII complex presentation of this bona fide selecting ligand suggest that functional detection self-ligand complexes will be required to establish a complete view of the naturally presented endogenous self-pMHC landscape. PMID:25451972

  13. Human KIAA1018/FAN1 nuclease is a new mitotic substrate of APC/CCdh1

    PubMed Central

    Lai, Fenju; Hu, Kaishun; Wu, Yuanzhong; Tang, Jianjun; Sang, Yi; Cao, Jingying; Kang, Tiebang

    2012-01-01

    A recently identified protein, FAN1 (FANCD2-associated nuclease 1, previously known as KIAA1018), is a novel nuclease associated with monoubiquitinated FANCD2 that is required for cellular resistance against DNA interstrand crosslinking (ICL) agents. The mechanisms of FAN1 regulation have not yet been explored. Here, we provide evidence that FAN1 is degraded during mitotic exit, suggesting that FAN1 may be a mitotic substrate of the anaphase-promoting cyclosome complex (APC/C). Indeed, Cdh1, but not Cdc20, was capable of regulating the protein level of FAN1 through the KEN box and the D-box. Moreover, the up- and down-regulation of FAN1 affected the progression to mitotic exit. Collectively, these data suggest that FAN1 may be a new mitotic substrate of APC/CCdh1 that plays a key role during mitotic exit. PMID:22854063

  14. Cubism and the cell cycle: the many faces of the APC/C.

    PubMed

    Pines, Jonathon

    2011-07-01

    One does not often look to analytic cubism for insights into the control of the cell cycle, but Pablo Picasso beautifully encapsulated the fundamentals when he said that "every act of creation is, first of all, an act of destruction". The rapid destruction of specific cell cycle regulators at just the right moment in the cell cycle ensures that daughter cells receive an equal and identical set of chromosomes from their mother and that DNA replication always follows mitosis. Remarkably, one protein complex is responsible for this surgical precision, the APC/C (anaphase-promoting complex, also known as the cyclosome). The APC/C is tightly regulated by its co-activators and by the spindle assembly checkpoint. PMID:21633387

  15. Investigations of the mechanical behavior of graphite/PEEK (APC-2) composites

    SciTech Connect

    Lee, K.

    1991-01-01

    The purpose was to investigate the effects of two phenomena specific to thermoplastic resin composites. These phenomena are: (a) significant nonlinear creep, that occurs especially at high temperatures, which affects the residual thermal stresses in a geometrically constrained structure; and (b) spatially nonuniform transverse crystallinity that develops in some fiber-reinforced thermoplastic composites, which introduces spatial variability and nonlinearity in the stiffness of the composite. The first part is related to (a). It presents a computational scheme for the optimal temperature path for APC-2 composite laminate, modeled as nonlinear, thermorheologically complex, viscoelastic material. The laminate is assumed to be sufficiently thin to obviate accounting for temperature nonuniformities across the thickness. The second part concerns (b) and presents analytical/computational results for the in-plane stresses in a moderately thick, cross-ply, graphite/PEEK (APC-2) laminate accounting for effects of nonuniform PEEK crystallinity across the plate's thickness.

  16. Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation.

    PubMed

    Cannon, J L; Labno, C M; Bosco, G; Seth, A; McGavin, M H; Siminovitch, K A; Rosen, M K; Burkhardt, J K

    2001-08-01

    Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site. PMID:11520460

  17. Cubism and the cell cycle: the many faces of the APC/C.

    PubMed

    Pines, Jonathon

    2011-06-02

    One does not often look to analytic cubism for insights into the control of the cell cycle, but Pablo Picasso beautifully encapsulated the fundamentals when he said that "every act of creation is, first of all, an act of destruction". The rapid destruction of specific cell cycle regulators at just the right moment in the cell cycle ensures that daughter cells receive an equal and identical set of chromosomes from their mother and that DNA replication always follows mitosis. Remarkably, one protein complex is responsible for this surgical precision, the APC/C (anaphase-promoting complex, also known as the cyclosome). The APC/C is tightly regulated by its co-activators and by the spindle assembly checkpoint.

  18. APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

    PubMed Central

    Marrocco, Katia; Criqui, Marie-Claire; Zervudacki, Jérôme; Schott, Gregory; Eisler, Herfried; Parnet, Aude; Dunoyer, Patrice; Genschik, Pascal

    2012-01-01

    Background Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed. Conclusions/Significance Our work identified a first plant substrate of the APC/C, which is not a regulator of the cell cycle. Though we cannot exclude that APC/C-dependent degradation of DRB4 has some regulatory roles under specific growth conditions, our work rather points to a housekeeping function of APC/C in maintaining precise cellular

  19. Increased IκBα expression is essential for the tolerogenic property of TGF-β-exposed APCs

    PubMed Central

    Ghafoori, Paiman; Yoshimura, Takeru; Turpie, Bruce; Masli, Sharmila

    2009-01-01

    IκBα is an inhibitor of the transcriptional factor NF-κB, and it is an essential component of the signaling pathways that lead to expression of inflammatory molecules. These include cytokines and costimulatory molecules associated with antigen presentation in an inflammatory immune response. In this study, we report that antigen-presenting cells exposed to TGF-β induce peripheral tolerance by increasing IκBα expression. Exposure of antigen presenting cells (APCs) to TGF-β is known to impair their ability to secrete IL-12, and such impairment correlated with reduced NF-κB activity as indicated by significantly reduced nuclear levels of p50, an essential subunit of NF-κB for IL-12 transcription. Blockade of increased nuclear IκBα in APCs by expression of small interfering RNA molecules (siRNAs) targeting IκBα transcripts prevented IL-12 impairment and the decline in nuclear p50 levels. Furthermore, such IκBα blockade also interfered with the tolerogenic property of TGF-β- exposed APCs. However, increased expression of IκBα in APCs, independent of TGF-β exposure, reduced nuclear p50 levels and permitted tolerance induction by APCs. Thus, our findings attribute a direct and significant role to IκBα in the tolerogenic potential of APCs. Increased IκBα expression in APCs may therefore offer a therapeutic approach to achieve antigen-specific immunomodulation.—Ghafoori, P., Yoshimura, T., Turpie, B., Masli, S. Increased IκBα expression is essential for the tolerogenic property of TGF-β-exposed APCs. PMID:19237504

  20. Cohesion Fatigue Explains Why Pharmacological Inhibition of the APC/C Induces a Spindle Checkpoint-Dependent Mitotic Arrest

    PubMed Central

    Lara-Gonzalez, Pablo; Taylor, Stephen S.

    2012-01-01

    The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores by inhibiting the activity of the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. Once all the chromosomes have bioriented, SAC signalling is somehow silenced, which allows progression through mitosis. Recent studies suggest that the APC/C itself participates in SAC silencing by targeting an unknown factor for proteolytic degradation. Key evidence in favour of this model comes from the use of proTAME, a small molecule inhibitor of the APC/C. In cells, proTAME causes a mitotic arrest that is SAC-dependent. Even though this observation comes at odds with the current view that the APC/C acts downstream of the SAC, it was nonetheless argued that these results revealed a role for APC/C activity in SAC silencing. However, we show here that the mitotic arrest induced by proTAME is due to the induction of cohesion fatigue, a phenotype that is caused by the loss of sister chromatid cohesion following a prolonged metaphase. Under these conditions, the SAC is re-activated and APC/C inhibition is maintained independently of proTAME. Therefore, these results provide a simpler explanation for why the proTAME-induced mitotic arrest is also dependent on the SAC. While these observations question the notion that the APC/C is required for SAC silencing, we nevertheless show that APC/C activity does partially contribute to its own release from inhibitory complexes, and importantly, this does not depend on proteasome-mediated degradation. PMID:23145059

  1. Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

    PubMed

    Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R; Weissmann, Florian; Ordureau, Alban; Wu, Kuen-Phon; Zhang, Wei; Yu, Shanshan; Mercredi, Peter Y; Harrison, Joseph S; Davidson, Iain F; Qiao, Renping; Lu, Ying; Dube, Prakash; Brunner, Michael R; Grace, Christy R R; Miller, Darcie J; Haselbach, David; Jarvis, Marc A; Yamaguchi, Masaya; Yanishevski, David; Petzold, Georg; Sidhu, Sachdev S; Kuhlman, Brian; Kirschner, Marc W; Harper, J Wade; Peters, Jan-Michael; Stark, Holger; Schulman, Brenda A

    2016-06-01

    Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

  2. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

    SciTech Connect

    Aulia, Selina; Tang, Bor Luen . E-mail: bchtbl@nus.edu.sg

    2006-01-06

    The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons-a function that could become impaired in Alzheimer's and other neurodegenerative diseases.

  3. The Specificity of Targeted Vaccines for APC Surface Molecules Influences the Immune Response Phenotype

    PubMed Central

    Grødeland, Gunnveig; Mjaaland, Siri; Tunheim, Gro; Fredriksen, Agnete B.; Bogen, Bjarne

    2013-01-01

    Different diseases require different immune responses for efficient protection. Thus, prophylactic vaccines should prime the immune system for the particular type of response needed for protection against a given infectious agent. We have here tested fusion DNA vaccines which encode proteins that bivalently target influenza hemagglutinins (HA) to different surface molecules on antigen presenting cells (APC). We demonstrate that targeting to MHC class II molecules predominantly induced an antibody/Th2 response, whereas targeting to CCR1/3/5 predominantly induced a CD8+/Th1 T cell response. With respect to antibodies, the polarizing effect was even more pronounced upon intramuscular (i.m) delivery as compared to intradermal (i.d.) vaccination. Despite these differences in induced immune responses, both vaccines protected against a viral challenge with influenza H1N1. Substitution of HA with ovalbumin (OVA) demonstrated that polarization of immune responses, as a consequence of APC targeting specificity, could be extended to other antigens. Taken together, the results demonstrate that vaccination can be tailor-made to induce a particular phenotype of adaptive immune responses by specifically targeting different surface molecules on APCs. PMID:24244595

  4. The APC tumor suppressor binds to C-terminal binding protein to divert nuclear beta-catenin from TCF.

    PubMed

    Hamada, Fumihiko; Bienz, Mariann

    2004-11-01

    Adenomatous polyposis coli (APC) is an important tumor suppressor in the colon. APC antagonizes the transcriptional activity of the Wnt effector beta-catenin by promoting its nuclear export and its proteasomal destruction in the cytoplasm. Here, we show that a third function of APC in antagonizing beta-catenin involves C-terminal binding protein (CtBP). APC is associated with CtBP in vivo and binds to CtBP in vitro through its conserved 15 amino acid repeats. Failure of this association results in elevated levels of beta-catenin/TCF complexes and of TCF-mediated transcription. Notably, CtBP is neither associated with TCF in vivo nor does mutation of the CtBP binding motifs in TCF-4 alter its transcriptional activity. This questions the idea that CtBP is a direct corepressor of TCF. Our evidence indicates that APC is an adaptor between beta-catenin and CtBP and that CtBP lowers the availability of free nuclear beta-catenin for binding to TCF by sequestering APC/beta-catenin complexes. PMID:15525529

  5. Association between aberrant APC promoter methylation and breast cancer pathogenesis: a meta-analysis of 35 observational studies

    PubMed Central

    Zhou, Dan; Tang, Weiwei; Wang, Wenyi; Pan, Xiaoyan

    2016-01-01

    Background. Adenomatous polyposis coli (APC) is widely known as an antagonist of the Wnt signaling pathway via the inactivation of β-catenin. An increasing number of studies have reported that APC methylation contributes to the predisposition to breast cancer (BC). However, recent studies have yielded conflicting results. Methods. Herein, we systematically carried out a meta-analysis to assess the correlation between APC methylation and BC risk. Based on searches of the Cochrane Library, PubMed, Web of Science and Embase databases, the odds ratio (OR) with 95% confidence interval (CI) values were pooled and summarized. Results. A total of 31 articles involving 35 observational studies with 2,483 cases and 1,218 controls met the inclusion criteria. The results demonstrated that the frequency of APC methylation was significantly higher in BC cases than controls under a random effect model (OR = 8.92, 95% CI [5.12–15.52]). Subgroup analysis further confirmed the reliable results, regardless of the sample types detected, methylation detection methods applied and different regions included. Interestingly, our results also showed that the frequency of APC methylation was significantly lower in early-stage BC patients than late-stage ones (OR = 0.62, 95% CI [0.42–0.93]). Conclusion. APC methylation might play an indispensable role in the pathogenesis of BC and could be regarded as a potential biomarker for the diagnosis of BC. PMID:27478702

  6. Targeting the anaphase-promoting complex/cyclosome (APC/C)- bromodomain containing 7 (BRD7) pathway for human osteosarcoma

    PubMed Central

    Han, An-Jia; Shi, Hui-Juan; Wang, Fen; Wang, Xin; Zhong, Li; Duan, Tingmei; Wu, Yuanzhong; Cao, Jingying; Tang, Jianjun; Sang, Yi; Wang, Li; Lv, Xiaobin; Xu, Shuangbing; Zhang, Ru-Hua; Deng, Wu-Guo; Li, Sheng-Ping; Zeng, Yi-Xin; Kang, Tiebang

    2014-01-01

    Osteosarcoma is the most common primary malignant bone tumor in childhood and adolescence and has a propensity for local invasion and early lung metastasis. However, the current therapies often result in chemoresistance, and a therapeutic target is not available in the clinic for osteosarcoma. Here, we report that BRD7 forms a complex with the anaphase-promoting complex/cyclosome (APC/C) and is degraded by APC/Ccdh1 and APC/Ccdc20 during the cell cycle. Moreover, BRD7 is a tumor suppressor in osteosarcoma, and the BRD7 mutant resistant to degradation by APC/C is more efficient than the wild-type protein at suppressing proliferation, colony formation, and tumor growth of osteosarcoma in vitro and in vivo. The combination of proTAME, an inhibitor of APC/C, with chemotherapeutic drugs efficiently targets osteosarcoma in vitro. Furthermore, there is a strong inverse correlation of protein levels between BRD7 and Cdh1 or Cdc20, and lower BRD7 expression is an indicator for poor prognosis in patients with osteosarcoma. Collectively, our results indicate that targeting the APC/C-BRD7 pathway may be a novel strategy for treating osteosarcoma. PMID:24840027

  7. A novel approach to assess the spontaneous gastrointestinal bleeding risk of antithrombotic agents using Apc(min/+) mice.

    PubMed

    Wei, Huijun; Shang, Jin; Keohane, CarolAnn; Wang, Min; Li, Qiu; Ni, Weihua; O'Neill, Kim; Chintala, Madhu

    2014-06-01

    Assessment of the bleeding risk of antithrombotic agents is usually performed in healthy animals with some form of vascular injury to peripheral organs to induce bleeding. However, bleeding observed in patients with currently marketed antithrombotic drugs is typically spontaneous in nature such as intracranial haemorrhage (ICH) and gastrointestinal (GI) bleeding, which happens most frequently on top of preexisting pathologies such as GI ulcerations and polyps. Apc(min/+) mice are reported to develop multiple adenomas through the entire intestinal tract and display progressive anaemia.In this study, we evaluated the potential utility of Apc(min/+) mice as a model for assessing spontaneous GI bleeding with antithrombotic agents. Apc(min/+) mice exhibited progressive blood loss starting at the age of nine weeks. Despite the increase in bleeding, Apc(min/+) mice were in a hypercoagulable state and displayed an age-dependent increase in thrombin generation and circulating fibrinogen as well as a significant decrease in clotting times. We evaluated the effect of warfarin, dabigatran etexilate, apixaban and clopidogrel in this model by administering them in diet or in the drinking water to mice for 1-4 weeks. All of these marketed drugs significantly increased GI bleeding in Apc(min/+) mice, but not in wild-type mice. Although different exposure profiles of these antithrombotic agents make it challenging to compare the bleeding risk of compounds, our results indicate that the Apc(min/+) mouse may be a sensitive preclinical model for assessing the spontaneous GI bleeding risk of novel antithrombotic agents.

  8. Association between aberrant APC promoter methylation and breast cancer pathogenesis: a meta-analysis of 35 observational studies.

    PubMed

    Zhou, Dan; Tang, Weiwei; Wang, Wenyi; Pan, Xiaoyan; An, Han-Xiang; Zhang, Yun

    2016-01-01

    Background. Adenomatous polyposis coli (APC) is widely known as an antagonist of the Wnt signaling pathway via the inactivation of β-catenin. An increasing number of studies have reported that APC methylation contributes to the predisposition to breast cancer (BC). However, recent studies have yielded conflicting results. Methods. Herein, we systematically carried out a meta-analysis to assess the correlation between APC methylation and BC risk. Based on searches of the Cochrane Library, PubMed, Web of Science and Embase databases, the odds ratio (OR) with 95% confidence interval (CI) values were pooled and summarized. Results. A total of 31 articles involving 35 observational studies with 2,483 cases and 1,218 controls met the inclusion criteria. The results demonstrated that the frequency of APC methylation was significantly higher in BC cases than controls under a random effect model (OR = 8.92, 95% CI [5.12-15.52]). Subgroup analysis further confirmed the reliable results, regardless of the sample types detected, methylation detection methods applied and different regions included. Interestingly, our results also showed that the frequency of APC methylation was significantly lower in early-stage BC patients than late-stage ones (OR = 0.62, 95% CI [0.42-0.93]). Conclusion. APC methylation might play an indispensable role in the pathogenesis of BC and could be regarded as a potential biomarker for the diagnosis of BC. PMID:27478702

  9. Interphase APC/C–Cdc20 inhibition by cyclin A2–Cdk2 ensures efficient mitotic entry

    PubMed Central

    Hein, Jamin B.; Nilsson, Jakob

    2016-01-01

    Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or Cdc20. APC/C and Cdc20 are already present during interphase but APC/C–Cdc20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2–Cdk2 binds and phosphorylates Cdc20 in interphase and this inhibits APC/C–Cdc20 activity. Preventing Cdc20 phosphorylation results in pre-mature activation of the APC/C–Cdc20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2–Cdk2 inhibition of interphase APC/C–Cdc20 to allow further cyclin A2 accumulation and mitotic entry. PMID:26960431

  10. A relay mechanism between EB1 and APC facilitate STIM1 puncta assembly at endoplasmic reticulum-plasma membrane junctions.

    PubMed

    Asanov, Alexander; Sherry, Ryan; Sampieri, Alicia; Vaca, Luis

    2013-09-01

    The assembly of STIM1 protein puncta near endoplasmic reticulum-plasma membrane (ER-PM) junctions is required for optimal activation of store-operated channels (SOC). The mechanisms controlling the translocation of STIM1 puncta to ER-PM junctions remain largely unknown. In the present study, we have explored the role of the microtubule binding protein adenomatous polyposis coli (APC), on STIM1 puncta and store-operated calcium entry (SOCE). APC-depleted cells showed reduced STIM1 puncta near ER-PM junctions, instead puncta is found at the ER surrounding the cell nucleus. Reduced STIM1 puncta near ER-PM junctions in APC-depleted cells correlates with a strong inhibition of SOCE and diminished Orai whole-cell currents. Immunoprecipitation and confocal microscopy co-localization studies indicate that, upon depletion of the ER, STIM1 dissociates from EB1 and associates to APC. Deletion analysis identified an APC-binding domain in the carboxyl terminus of STIM1 (STIM1 650-685). These results together position APC as an important element in facilitating the translocation of STIM1 puncta near ER-PM junctions, which in turn is required for efficient SOCE and Orai activation upon depletion of the ER.

  11. Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly*

    PubMed Central

    Jaiswal, Richa; Stepanik, Vince; Rankova, Aneliya; Molinar, Olivia; Goode, Bruce L.; McCartney, Brooke M.

    2013-01-01

    Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla. PMID:23558679

  12. The APC I1307K allele conveys a significant increased risk for cancer.

    PubMed

    Leshno, Ari; Shapira, Shiran; Liberman, Eliezer; Kraus, Sarah; Sror, Miri; Harlap-Gat, Amira; Avivi, Doran; Galazan, Lior; David, Maayan; Maharshak, Nitsan; Moanis, Serhan; Arber, Nadir; Moshkowitz, Menachem

    2016-03-15

    This study is the first attempt to evaluate the association between the APC I1307K variant and overall cancer risk. It is unique in both its large sample size and in the reliability of data in the control group. The findings described in this article have major implications in terms of identifying asymptomatic individuals who are at increased risk to harbor cancer and therefore targeted to be enrolled in specific early detection and prevention programs. The prevalence of the APC I1307K missense mutation among Ashkenazi Jews is ∼ 6%. Carriers are at an increased risk for colorectal neoplasia. In this study, we examined the association of this variant with non-colorectal cancers. Consecutive 13,013 healthy subjects who underwent screening at the Integrated Cancer Prevention Center between 2006 and 2014 were enrolled. This population was supplemented with 1,611 cancer patients from the same institution. Demographics, medical history, and pathological data were recorded. Mortality data were obtained from the Ministry of Health's registry. The prevalence of APC I1307K in cancer patients and healthy subjects was compared. The APC I1307K variant was detected in 189 (11.8%) cancer patients compared to 614 (4.7%) healthy subjects, reflecting an adjusted age and sex odds ratio (OR) of 2.53 (p < 0.0001). History of two or more cancer types was associated with a positive carrier prevalence (OR = 4.38 p < 0.0001). Males had significantly increased carrier prevalence in lung, urologic, pancreatic, and skin cancers. The carrier prevalence among females was significantly higher only in breast and skin cancers. Female carriers developed cancer at a significantly older age compared to non-carriers (average 62.7 years vs. 57.8, respectively, p = 0.027), had better survival rates (HR = 0.58, p = 0.022) and overall increased longevity (average age of death 78.8 vs. 70.4 years, respectively, p = 0.003). In conclusion, the APC I1307K variant is a reliable marker for overall cancer risk

  13. Galactose-functionalized cationic polycarbonate diblock copolymer for targeted gene delivery to hepatocytes.

    PubMed

    Ong, Zhan Yuin; Yang, Chuan; Gao, Shu Jun; Ke, Xi-Yu; Hedrick, James L; Yan Yang, Yi

    2013-11-01

    To mediate selective gene delivery to hepatocytes via the asialoglycoprotein receptors (ASGP-Rs), we designed and synthesized well-defined and narrowly dispersed galactose- and glucose-functionalized cationic polycarbonate diblock copolymers (designated as Gal-APC and Glu-APC, respectively) using organocatalytic ring-opening polymerization of functionalized carbonate monomers, with a subsequent quaternization step using bis-tertiary amines to confer quaternary and tertiary amines for DNA binding and endosomal buffering, respectively. The sugar-functionalized diblock copolymers effectively bound and condensed DNA to form positively charged nanoparticles (<100 nm in diameter and ≈30 mV zeta-potential) that were stable under high physiological salt conditions. In comparison to the control Glu-APC/DNA complexes, Gal-APC/DNA complexes mediated significantly higher gene expression in ASGP-R positive HepG2 cells with no significant difference observed in ASGP-R negative HeLa cells. The co-incubation of Gal-APC/DNA complexes with a natural ASGP-R ligand effectively led to a decrease in gene expression, hence providing evidence for the ASGP-R mediated endocytosis of the polyplexes. Importantly, the Gal-APC/DNA complexes induced minimal cytotoxicities in HepG2 cells at the N/P ratios tested. Taken together, the galactose-functionalized cationic polycarbonate diblock copolymer has potential for use as a non-viral gene vector for the targeted delivery of therapeutic genes to hepatocytes in the treatment of liver diseases.

  14. Cdk8 deletion in the Apc(Min) murine tumour model represses EZH2 activity and accelerates tumourigenesis.

    PubMed

    McCleland, Mark L; Soukup, Tim M; Liu, Scot D; Esensten, Jonathan H; de Sousa e Melo, Felipe; Yaylaoglu, Murat; Warming, Soren; Roose-Girma, Merone; Firestein, Ron

    2015-12-01

    CDK8 is a dissociable kinase module of the Mediator complex and has been shown to play an important role in transcriptional regulation in organisms as diverse as yeast and humans. Recent studies suggest that CDK8 functions as an oncoprotein in melanoma and colon cancer. Importantly, these studies were conducted using in vitro cell line models and the role of CDK8 in tumourigenesis in vivo has not been explored. We have generated a mouse with a Cdk8 conditional knockout allele and examined the consequences of Cdk8 loss on normal tissue homeostasis and tumour development in vivo. Cdk8 deletion in the young adult mouse did not induce any gross or histopathological abnormalities, implying that Cdk8 is largely dispensable for somatic cellular homeostasis. In contrast, Cdk8 deletion in the Apc(Min) intestinal tumour model shortened the animals' survival and increased tumour burden. Although Cdk8 deletion did not affect tumour initiation, intestinal tumour size and growth rate were significantly increased in Cdk8-null animals. Transcriptome analysis performed on Cdk8-null intestinal cells revealed up-regulation of genes that are governed by the Polycomb group (PcG) complex. In support of these findings, Cdk8-null intestinal cells and tumours displayed a reduction in histone H3K27 trimethylation, both globally and at the promoters of a number of PcG-regulated genes involved in oncogenic signalling. Together, our findings uncover a tumour suppressor function for CDK8 in vivo and suggest that the role of CDK8 activity in driving oncogenesis is context-specific. Sequencing data were deposited at GEO (Accession No. GSE71385).

  15. Cdk8 deletion in the Apc(Min) murine tumour model represses EZH2 activity and accelerates tumourigenesis.

    PubMed

    McCleland, Mark L; Soukup, Tim M; Liu, Scot D; Esensten, Jonathan H; de Sousa e Melo, Felipe; Yaylaoglu, Murat; Warming, Soren; Roose-Girma, Merone; Firestein, Ron

    2015-12-01

    CDK8 is a dissociable kinase module of the Mediator complex and has been shown to play an important role in transcriptional regulation in organisms as diverse as yeast and humans. Recent studies suggest that CDK8 functions as an oncoprotein in melanoma and colon cancer. Importantly, these studies were conducted using in vitro cell line models and the role of CDK8 in tumourigenesis in vivo has not been explored. We have generated a mouse with a Cdk8 conditional knockout allele and examined the consequences of Cdk8 loss on normal tissue homeostasis and tumour development in vivo. Cdk8 deletion in the young adult mouse did not induce any gross or histopathological abnormalities, implying that Cdk8 is largely dispensable for somatic cellular homeostasis. In contrast, Cdk8 deletion in the Apc(Min) intestinal tumour model shortened the animals' survival and increased tumour burden. Although Cdk8 deletion did not affect tumour initiation, intestinal tumour size and growth rate were significantly increased in Cdk8-null animals. Transcriptome analysis performed on Cdk8-null intestinal cells revealed up-regulation of genes that are governed by the Polycomb group (PcG) complex. In support of these findings, Cdk8-null intestinal cells and tumours displayed a reduction in histone H3K27 trimethylation, both globally and at the promoters of a number of PcG-regulated genes involved in oncogenic signalling. Together, our findings uncover a tumour suppressor function for CDK8 in vivo and suggest that the role of CDK8 activity in driving oncogenesis is context-specific. Sequencing data were deposited at GEO (Accession No. GSE71385). PMID:26235356

  16. The four canonical tpr subunits of human APC/C form related homo-dimeric structures and stack in parallel to form a TPR suprahelix.

    PubMed

    Zhang, Ziguo; Chang, Leifu; Yang, Jing; Conin, Nora; Kulkarni, Kiran; Barford, David

    2013-11-15

    The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for >80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23(Nterm)). Cdc23(Nterm) is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23(Nterm) structure, we generated a model of full-length Cdc23. The resultant "V"-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes.

  17. The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix☆

    PubMed Central

    Zhang, Ziguo; Chang, Leifu; Yang, Jing; Conin, Nora; Kulkarni, Kiran; Barford, David

    2013-01-01

    The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for > 80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23Nterm). Cdc23Nterm is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23Nterm structure, we generated a model of full-length Cdc23. The resultant “V”-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes. PMID:23583778

  18. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

    PubMed

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-06-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity. PMID:26939887

  19. Insights into Degron Recognition by APC/C Coactivators from the Structure of an Acm1-Cdh1 Complex

    PubMed Central

    He, Jun; Chao, William C.H.; Zhang, Ziguo; Yang, Jing; Cronin, Nora; Barford, David

    2013-01-01

    Summary The anaphase-promoting complex/cyclosome (APC/C) regulates sister chromatid segregation and the exit from mitosis. Selection of most APC/C substrates is controlled by coactivator subunits (either Cdc20 or Cdh1) that interact with substrate destruction motifs—predominantly the destruction (D) box and KEN box degrons. How coactivators recognize D box degrons and how this is inhibited by APC/C regulatory proteins is not defined at the atomic level. Here, from the crystal structure of S. cerevisiae Cdh1 in complex with its specific inhibitor Acm1, which incorporates D and KEN box pseudosubstrate motifs, we describe the molecular basis for D box recognition. Additional interactions between Acm1 and Cdh1 identify a third protein-binding site on Cdh1 that is likely to confer coactivator-specific protein functions including substrate association. We provide a structural rationalization for D box and KEN box recognition by coactivators and demonstrate that many noncanonical APC/C degrons bind APC/C coactivators at the D box coreceptor. PMID:23707760

  20. APC/C and retinoblastoma interaction: cross-talk of retinoblastoma protein with the ubiquitin proteasome pathway.

    PubMed

    Ramanujan, Ajeena; Tiwari, Swati

    2016-10-01

    The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs.

  1. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn’s disease

    PubMed Central

    Robertson, Jack; Haas, Carolin T.; Pele, Laetitia C.; Monie, Tom P.; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E.; Powell, Jonathan J.

    2016-01-01

    Crohn’s disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn’s disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn’s disease show selective failure in PD-L1 expression. Therefore, in Crohn’s disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease. PMID:27226337

  2. Irreversible APC(Cdh1) Inactivation Underlies the Point of No Return for Cell-Cycle Entry.

    PubMed

    Cappell, Steven D; Chung, Mingyu; Jaimovich, Ariel; Spencer, Sabrina L; Meyer, Tobias

    2016-06-30

    Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle. PMID:27368103

  3. Aggregate material formulated with MSWI bottom ash and APC fly ash for use as secondary building material

    SciTech Connect

    Valle-Zermeño, R. del; Formosa, J.; Chimenos, J.M.; Martínez, M.; Fernández, A.I.

    2013-03-15

    Highlights: ► A concrete formulation was optimized using Bottom Ash and APC ash. ► 10% of APC ash achieves good compromise between economic and performance aspects. ► The crushed concrete was evaluated as secondary building granular material. ► The environmental behavior allows its use as secondary material. ► The abrasion resistance is not good enough for its use as a road sub-base material. - Abstract: The main goal of this paper is to obtain a granular material formulated with Municipal Solid Waste Incineration (MSWI) bottom ash (BA) and air pollution control (APC) fly ash to be used as secondary building material. Previously, an optimum concrete mixture using both MSWI residues as aggregates was formulated. A compromise between the environmental behavior whilst maximizing the reuse of APC fly ash was considered and assessed. Unconfined compressive strength and abrasion resistance values were measured in order to evaluate the mechanical properties. From these results, the granular mixture was not suited for certain applications owing to the high BA/APC fly ash content and low cement percentages used to reduce the costs of the final product. Nevertheless, the leaching test performed showed that the concentrations of all heavy metals were below the limits established by the current Catalan legislation for their reutilization. Therefore, the material studied might be mainly used in embankments, where high mechanical properties are not needed and environmental safety is assured.

  4. The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch

    PubMed Central

    Yang, Qiong; Ferrell, James E.

    2013-01-01

    Despite the complexity and variety of biological oscillators, their core design invariably includes an essential negative feedback loop. In the Xenopus laevis embryonic cell cycle oscillator, this loop consists of the kinase cyclin B-Cdk1 and the ubiquitin ligase APC/CCdc20; active Cdk1 activates APC/CCdc20, which then brings about cyclin B degradation and inactivates Cdk1. Here we ask how this negative feedback loop functions quantitatively, with the aim of understanding what mechanisms keep the Cdk1-APC/CCdc20 system from settling into a stable steady state with intermediate levels of Cdk1 and APC/CCdc20 activity. We found that the system operates as a time-delayed, digital switch, with a time lag of ~15 min between Cdk1 and APC/CCdc20 activation and a tremendously high degree of ultrasensitivity (nH ≈ 17). Computational modeling shows how these attributes contribute to the generation of robust, clock-like oscillations. Principles uncovered here may also apply to other activator-repressor oscillators and help in designing robust synthetic clocks. PMID:23624406

  5. APC/C and retinoblastoma interaction: cross-talk of retinoblastoma protein with the ubiquitin proteasome pathway

    PubMed Central

    Ramanujan, Ajeena; Tiwari, Swati

    2016-01-01

    The ubiquitin (Ub) ligase anaphase promoting complex/cyclosome (APC/C) and the tumour suppressor retinoblastoma protein (pRB) play key roles in cell cycle regulation. APC/C is a critical regulator of mitosis and G1-phase of the cell cycle whereas pRB keeps a check on proliferation by inhibiting transition to the S-phase. APC/C and pRB interact with each other via the co-activator of APC/C, FZR1, providing an alternative pathway of regulation of G1 to S transition by pRB using a post-translational mechanism. Both pRB and FZR1 have complex roles and are implicated not only in regulation of cell proliferation but also in differentiation, quiescence, apoptosis, maintenance of chromosomal integrity and metabolism. Both are also targeted by transforming viruses. We discuss recent advances in our understanding of the involvement of APC/C and pRB in cell cycle based decisions and how these insights will be useful for development of anti-cancer and anti-viral drugs. PMID:27402801

  6. Intestinal APCs of the endogenous nanomineral pathway fail to express PD-L1 in Crohn's disease.

    PubMed

    Robertson, Jack; Haas, Carolin T; Pele, Laetitia C; Monie, Tom P; Charalambos, Charles; Parkes, Miles; Hewitt, Rachel E; Powell, Jonathan J

    2016-05-26

    Crohn's disease is a chronic inflammatory condition most commonly affecting the ileum and colon. The aetiology of Crohn's disease is complex and may include defects in peptidoglycan recognition, and/or failures in the establishment of intestinal tolerance. We have recently described a novel constitutive endogenous delivery system for the translocation of nanomineral-antigen-peptidoglycan (NAP) conjugates to antigen presenting cells (APCs) in intestinal lymphoid patches. In mice NAP conjugate delivery to APCs results in high surface expression of the immuno-modulatory molecule programmed death receptor ligand 1 (PD-L1). Here we report that NAP conjugate positive APCs in human ileal tissues from individuals with ulcerative colitis and intestinal carcinomas, also have high expression of PD-L1. However, NAP-conjugate positive APCs in intestinal tissue from patients with Crohn's disease show selective failure in PD-L1 expression. Therefore, in Crohn's disease intestinal antigen taken up by lymphoid patch APCs will be presented without PD-L1 induced tolerogenic signalling, perhaps initiating disease.

  7. APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

    PubMed

    Choudhury, Rajarshi; Bonacci, Thomas; Arceci, Anthony; Lahiri, Debojyoti; Mills, Christine A; Kernan, Jennifer L; Branigan, Timothy B; DeCaprio, James A; Burke, Daniel J; Emanuele, Michael J

    2016-09-20

    The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems.

  8. Irreversible APC(Cdh1) Inactivation Underlies the Point of No Return for Cell-Cycle Entry.

    PubMed

    Cappell, Steven D; Chung, Mingyu; Jaimovich, Ariel; Spencer, Sabrina L; Meyer, Tobias

    2016-06-30

    Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.

  9. New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

    PubMed

    Ercilla, Amaia; Llopis, Alba; Feu, Sonia; Aranda, Sergi; Ernfors, Patrik; Freire, Raimundo; Agell, Neus

    2016-06-01

    Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.

  10. Substrate Recognition by the Cdh1 Destruction Box Receptor Is a General Requirement for APC/CCdh1-mediated Proteolysis.

    PubMed

    Qin, Liang; Guimarães, Dimitrius Santiago P S F; Melesse, Michael; Hall, Mark C

    2016-07-22

    The anaphase-promoting complex, or cyclosome (APC/C), is a ubiquitin ligase that selectively targets proteins for degradation in mitosis and the G1 phase and is an important component of the eukaryotic cell cycle control system. How the APC/C specifically recognizes its substrates is not fully understood. Although well characterized degron motifs such as the destruction box (D-box) and KEN-box are commonly found in APC/C substrates, many substrates apparently lack these motifs. A variety of alternative APC/C degrons have been reported, suggesting either that multiple modes of substrate recognition are possible or that our definitions of degron structure are incomplete. We used an in vivo yeast assay to compare the G1 degradation rate of 15 known substrates of the APC/C co-activator Cdh1 under normal conditions and conditions that impair binding of D-box, KEN-box, and the recently identified ABBA motif degrons to Cdh1. The D-box receptor was required for efficient proteolysis of all Cdh1 substrates, despite the absence of canonical D-boxes in many. In contrast, the KEN-box receptor was only required for normal proteolysis of a subset of substrates and the ABBA motif receptor for a single substrate in our system. Our results suggest that binding to the D-box receptor may be a shared requirement for recognition and processing of all Cdh1 substrates.

  11. APC/C and SCF(cyclin F) Constitute a Reciprocal Feedback Circuit Controlling S-Phase Entry.

    PubMed

    Choudhury, Rajarshi; Bonacci, Thomas; Arceci, Anthony; Lahiri, Debojyoti; Mills, Christine A; Kernan, Jennifer L; Branigan, Timothy B; DeCaprio, James A; Burke, Daniel J; Emanuele, Michael J

    2016-09-20

    The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase and core component of the cell-cycle oscillator. During G1 phase, APC/C binds to its substrate receptor Cdh1 and APC/C(Cdh1) plays an important role in restricting S-phase entry and maintaining genome integrity. We describe a reciprocal feedback circuit between APC/C and a second ubiquitin ligase, the SCF (Skp1-Cul1-F box). We show that cyclin F, a cell-cycle-regulated substrate receptor (F-box protein) for the SCF, is targeted for degradation by APC/C. Furthermore, we establish that Cdh1 is itself a substrate of SCF(cyclin F). Cyclin F loss impairs Cdh1 degradation and delays S-phase entry, and this delay is reversed by simultaneous removal of Cdh1. These data indicate that the coordinated, temporal ordering of cyclin F and Cdh1 degradation, organized in a double-negative feedback loop, represents a fundamental aspect of cell-cycle control. This mutual antagonism could be a feature of other oscillating systems. PMID:27653696

  12. Liver inflammation and metabolic signaling in ApcMin/+ mice: the role of cachexia progression.

    PubMed

    Narsale, Aditi A; Enos, Reilly T; Puppa, Melissa J; Chatterjee, Saurabh; Murphy, E Angela; Fayad, Raja; Pena, Majorette O'; Durstine, J Larry; Carson, James A

    2015-01-01

    The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer

  13. The ubiquitin ligase APC/CCdh1 puts the brakes on DNA-end resection

    PubMed Central

    Lafranchi, Lorenzo; Sartori, Alessandro A

    2015-01-01

    DNA double-strand breaks (DSBs) are highly deleterious lesions and their misrepair can promote genomic instability, a hallmark of cancer. DNA-end resection is a cell cycle-regulated mechanism that is required for the faithful repair of DSBs. We recently discovered that the anaphase-promoting complex/cyclosome-Cdh1 (APC/CCdh1) ubiquitin ligase is responsible for the timely degradation of CtBP-interacting protein (CtIP), a key DNA-end resection factor, providing a new layer of regulation of DSB repair in human cells. PMID:27308488

  14. Ink-jetting AJL8/APC for D-fiber electric field sensors.

    PubMed

    Kvavle, Joshua; Schultz, Stephen; Selfridge, Richard

    2009-10-01

    Spin casting electro-optic polymers for in-fiber device fabrication is problematic due to the flexibility and high-contrast topography of optical fibers. An ink-jetting method is developed for the deposition of AJL8/APC using a commercially available printer. The method results in more consistent control of film thickness and uses 1000 times less material than the spin-coating method. A D-fiber electric field sensor is fabricated using this deposition method and exhibits a sensitivity of 157 V/(m square root(Hz)) at a modulation frequency of 6 GHz.

  15. The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

    PubMed

    Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

    2016-06-10

    Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. PMID:27068743

  16. A functional network of the tumor suppressors APC, hDlg, and PTEN, that relies on recognition of specific PDZ-domains.

    PubMed

    Sotelo, Natalia S; Valiente, Miguel; Gil, Anabel; Pulido, Rafael

    2012-08-01

    APC and PTEN are tumor suppressor proteins that bind through their C-termini to the PDZ domain containing-hDlg scaffolding protein. We have found that co-expression of PTEN and hDlg enhanced the negative regulation of the PI3K/Akt pathway by PTEN, indicating the physiologic importance of these interactions. APC and PTEN share other PDZ domain containing-interacting partners, including the MAGI scaffolding proteins and the MAST family of protein kinases. Mutational analysis revealed that the C-terminal PDZ-binding motifs from APC and PTEN were differentially recognized by distinct PDZ domains. APC bound to the three PDZ domains from hDlg, whereas PTEN mainly bound to PDZ-2/hDlg. This indicates the existence of overlapping, but distinct PDZ-domain recognition patterns by APC and PTEN. Furthermore, a ternary complex formed by APC, PTEN, and hDlg was detected, suggesting that hDlg may serve as a platform to bring in proximity APC and PTEN tumor suppressor activities. In line with this, tumor-related mutations targeting the PDZ-2/hDlg domain diminished its interaction with APC and PTEN. Our results expand the PDZ-domain counterparts for the tumor suppressor APC, show that APC and PTEN share PDZ-domain partners but have individual molecular determinants for specific recognition of PDZ domains, and suggest the participation of the tumor suppressors APC, PTEN, and hDlg in PDZ-domain interaction networks which may be relevant in oncogenesis.

  17. Testing Models of the APC Tumor Suppressor/β-Catenin Interaction Reshapes Our View of the Destruction Complex in Wnt Signaling

    PubMed Central

    Yamulla, Robert J.; Kane, Eric G.; Moody, Alexandra E.; Politi, Kristin A.; Lock, Nicole E.; Foley, Andrew V. A.; Roberts, David M.

    2014-01-01

    The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling inactivated in >80% of colorectal cancers. APC participates in a multiprotein “destruction complex” that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high-affinity β-catenin-binding sites [20-amino-acid repeats (20Rs)] on APC. Here we test these models by generating a Drosophila APC2 mutant lacking all β-catenin-binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. Our results are inconsistent with current models, as we find that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, we generate an APC2 mutant lacking all β-catenin-binding sites (including the 15Rs) and find that a direct β-catenin/APC interaction is also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, our findings support a model whereby β-catenin-binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes we observe a separation of β-catenin destruction and Wg/Wnt signaling outputs and suggest that cytoplasmic retention of β-catenin likely accounts for this difference. PMID:24931405

  18. Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

    PubMed Central

    Fatehullah, A.; Sharma, S.; Newton, I. P.; Langlands, A. J.; Lay, H.; Nelson, S. A.; McMahon, R. K.; McIlvenny, N.; Appleton, P. L.; Cochran, S.; Näthke, I. S.

    2016-01-01

    Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic. PMID:27406832

  19. Polo-Like Kinase-1 Controls Aurora A Destruction by Activating APC/C-Cdh1

    PubMed Central

    van Leuken, Renske; Lim, Dan; Yao, XueBiao; Wolthuis, Rob M. F.; Yaffe, Michael B.; Medema, René H.; van Vugt, Marcel A. T. M.

    2009-01-01

    Polo-like kinase-1 (Plk1) is activated before mitosis by Aurora A and its cofactor Bora. In mitosis, Bora is degraded in a manner dependent on Plk1 kinase activity and the E3 ubiquitin ligase SCF-βTrCP. Here, we show that Plk1 is also required for the timely destruction of its activator Aurora A in late anaphase. It has been shown that Aurora A destruction is controlled by the auxiliary subunit Cdh1 of the Anaphase-Promoting Complex/Cyclosome (APC/C). Remarkably, we found that Plk1-depletion prevented the efficient dephosphorylation of Cdh1 during mitotic exit. Plk1 mediated its effect on Cdh1, at least in part, through direct phosphorylation of the human phosphatase Cdc14A, controlling the phosphorylation state of Cdh1. We conclude that Plk1 facilitates efficient Aurora A degradation through APC/C-Cdh1 activation after mitosis, with a potential role for hCdc14A. PMID:19390576

  20. Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

    NASA Astrophysics Data System (ADS)

    Fatehullah, A.; Sharma, S.; Newton, I. P.; Langlands, A. J.; Lay, H.; Nelson, S. A.; McMahon, R. K.; McIlvenny, N.; Appleton, P. L.; Cochran, S.; Näthke, I. S.

    2016-07-01

    Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic.

  1. Berberine Inhibits Intestinal Polyps Growth in Apc (min/+) Mice via Regulation of Macrophage Polarization

    PubMed Central

    Piao, Meiyu; Cao, Hailong; He, NaNa; Yang, Boli; Dong, Wenxiao; Xu, Mengque; Yan, Fang; Zhou, Bing

    2016-01-01

    Antitumor effect of berberine has been reported in a wide spectrum of cancer, however, the mechanisms of which are not fully understood. The aim of this study was to investigate the hypothesis that berberine suppresses tumorigenesis in the familial adenomatous polyposis (FAP) by regulating the macrophage polarization in Apc (min/+) mouse model. Berberine was given to Apc (min/+) mice for 12 weeks. Primary macrophages were isolated; after berberine treatment, the change in signaling cascade was determined. The total number and size of polyps were reduced remarkably in berberine group, compared with control group. A significant decrease in protein levels of F4/80, mannose receptor (MR), and COX-2 in stroma of intestinal polyps and an increase in the level of iNOS were observed after berberine treatment. The mRNA level of MR and Arg-1 in berberine group was significantly lower than those in IL-10 or IL-4 group, while no significant difference in mRNA levels of iNOS and CXCL10 was observed. The migration and invasiveness assays in vitro showed that berberine could reduce the capability of migration and invasiveness. These findings suggest that berberine attenuates intestinal tumorigenesis by inhibiting the migration and invasion of colorectal tumor cells via regulation of macrophage polarization. PMID:27493671

  2. Berberine Inhibits Intestinal Polyps Growth in Apc (min/+) Mice via Regulation of Macrophage Polarization.

    PubMed

    Piao, Meiyu; Cao, Hailong; He, NaNa; Yang, Boli; Dong, Wenxiao; Xu, Mengque; Yan, Fang; Zhou, Bing; Wang, Bangmao

    2016-01-01

    Antitumor effect of berberine has been reported in a wide spectrum of cancer, however, the mechanisms of which are not fully understood. The aim of this study was to investigate the hypothesis that berberine suppresses tumorigenesis in the familial adenomatous polyposis (FAP) by regulating the macrophage polarization in Apc (min/+) mouse model. Berberine was given to Apc (min/+) mice for 12 weeks. Primary macrophages were isolated; after berberine treatment, the change in signaling cascade was determined. The total number and size of polyps were reduced remarkably in berberine group, compared with control group. A significant decrease in protein levels of F4/80, mannose receptor (MR), and COX-2 in stroma of intestinal polyps and an increase in the level of iNOS were observed after berberine treatment. The mRNA level of MR and Arg-1 in berberine group was significantly lower than those in IL-10 or IL-4 group, while no significant difference in mRNA levels of iNOS and CXCL10 was observed. The migration and invasiveness assays in vitro showed that berberine could reduce the capability of migration and invasiveness. These findings suggest that berberine attenuates intestinal tumorigenesis by inhibiting the migration and invasion of colorectal tumor cells via regulation of macrophage polarization. PMID:27493671

  3. Post-translational glycoprotein modifications regulate colon cancer stem cells and colon adenoma progression in Apc(min/+) mice through altered Wnt receptor signaling.

    PubMed

    Guo, Huabei; Nagy, Tamas; Pierce, Michael

    2014-11-01

    Deletion of GnT-V (MGAT5), which synthesizes N-glycans with β(1,6)-branched glycans, reduced the compartment of cancer stem cells (CSC) in the her-2 mouse model of breast cancer, leading to delay of tumor onset. Because GnT-V levels are also commonly up-regulated in colon cancer, we investigated their regulation of colon CSC and adenoma development. Anchorage-independent cell growth and tumor formation induced by injection of colon tumor cells into NOD/SCID mice were positively associated with GnT-V levels, indicating regulation of proliferation and tumorigenicity. Using Apc(min/+) mice with different GnT-V backgrounds, knock-out of GnT-V had no significant effect on the number of adenoma/mouse, but adenoma size was significantly reduced and accompanied increased survival of Apc(min/+) mice with GnT-V deletion (p < 0.01), suggesting an inhibition in the progression of colon adenoma caused by deletion of GnT-V. Decreased expression levels of GnT-V down-regulated the population of colon (intestine) CSC, affecting their ability for self-renewal and tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target genes were positively associated with expression levels of GnT-V, indicating the regulation of canonical Wnt/β-catenin signaling. By overexpressing the Wnt receptor, FZD-7, in colon cancer cells, we found that FZD-7 receptors expressed N-linked β(1,6) branching, indicating that FZD-7 can be modified by GnT-V. The aberrant Wnt signaling observed after modulating GnT-V levels is likely to result from altered N-linked β(1,6) branching on FZD-7, thereby affecting Wnt signaling, the compartment of CSC, and tumor progression.

  4. A novel approach to assess the spontaneous gastrointestinal bleeding risk of antithrombotic agents using Apc(min/+) mice.

    PubMed

    Wei, Huijun; Shang, Jin; Keohane, CarolAnn; Wang, Min; Li, Qiu; Ni, Weihua; O'Neill, Kim; Chintala, Madhu

    2014-06-01

    Assessment of the bleeding risk of antithrombotic agents is usually performed in healthy animals with some form of vascular injury to peripheral organs to induce bleeding. However, bleeding observed in patients with currently marketed antithrombotic drugs is typically spontaneous in nature such as intracranial haemorrhage (ICH) and gastrointestinal (GI) bleeding, which happens most frequently on top of preexisting pathologies such as GI ulcerations and polyps. Apc(min/+) mice are reported to develop multiple adenomas through the entire intestinal tract and display progressive anaemia.In this study, we evaluated the potential utility of Apc(min/+) mice as a model for assessing spontaneous GI bleeding with antithrombotic agents. Apc(min/+) mice exhibited progressive blood loss starting at the age of nine weeks. Despite the increase in bleeding, Apc(min/+) mice were in a hypercoagulable state and displayed an age-dependent increase in thrombin generation and circulating fibrinogen as well as a significant decrease in clotting times. We evaluated the effect of warfarin, dabigatran etexilate, apixaban and clopidogrel in this model by administering them in diet or in the drinking water to mice for 1-4 weeks. All of these marketed drugs significantly increased GI bleeding in Apc(min/+) mice, but not in wild-type mice. Although different exposure profiles of these antithrombotic agents make it challenging to compare the bleeding risk of compounds, our results indicate that the Apc(min/+) mouse may be a sensitive preclinical model for assessing the spontaneous GI bleeding risk of novel antithrombotic agents. PMID:24430131

  5. Proteomics Suggests a Role for APC-Survivin in Response to Somatostatin Analog Treatment of Neuroendocrine Tumors

    PubMed Central

    Kjellin, Hanna; Hashemi, Jamileh; Barriuso, Jorge; Juhlin, C. Christofer; Lu, Ming; Höög, Anders; Pastrián, Laura G.; Lamarca, Angela; Soto, Victoria Heredia; Zedenius, Jan; Mendiola, Marta; Lehtiö, Janne; Kjellman, Magnus

    2016-01-01

    Context: Somatostatin analogs are established in the treatment of neuroendocrine tumors (NETs) including small intestinal NET; however, the molecular mechanisms are not well known. Here, we examined the direct effects of lanreotide in NET cell line models. Setting and Design: The cell lines HC45 and H727 were treated with 10nM lanreotide for different time periods and alterations of the proteome were analyzed by in-depth high-resolution isoelectric focusing tandem liquid chromatography-mass spectrometry. We next investigated whether the observed suppression of survivin was mediated by adenomatous polyposis coli (APC) and possible effects on tumor proliferation in vitro. Expression of survivin was assessed by immunohistochemistry in 112 NET cases and compared with patient outcome. Results: We quantified 6451 and 7801 proteins in HC45 and H727, respectively. After short time lanreotide treatment APC was increased and survivin reduced. Overexpression of APC in H727 cells decreased, and APC knock-down elevated the survivin level. The lanreotide regulation of APC-survivin could be suppressed by small interfering RNA against somatostatin receptor 2. Although lanreotide only gave slight inhibition of proliferation, targeting of survivin with the small molecule YM155 dramatically reduced proliferation. Moderate or high as compared with low or absent total survivin expression was associated with shorter progression-free survival, independent of tumor stage, grade, and localization. Conclusions: We report a proteome-wide analysis of changes in response to lanreotide in NET cell lines. This analysis suggests a connection between somatostatin analog, APC, and survivin levels. Survivin is a possible prognostic factor and a new potential therapeutic target in NETs. PMID:27459532

  6. Missense Polymorphisms in the Adenomatous Polyposis Coli Gene and Colorectal Cancer Risk

    PubMed Central

    Cleary, Sean P.; Kim, Hyeja; Croitoru, Marina E.; Redston, Mark; Knight, Julia A.; Gallinger, Steven; Gryfe, Robert

    2009-01-01

    PURPOSE Whereas truncating germline mutations of the adenomatous polyposis coli (APC) gene give rise to familial adenomatous polyposis, missense polymorphisms of APC may confer a weaker risk for colorectal cancer. METHODS We sequenced the entire open reading frame of the APC gene and tested for two common MYH mutations in a population-based series of patients with colorectal cancer and 5 to 99 adenomas. Missense adenomatous polyposis coli alterations identified in this colorectal cancer multiple-polyp population were analyzed in a population-based series of patients with colorectal cancer and healthy control subjects. RESULTS Germline APC or mutY human homologue (MYH) alterations were identified in 16 of 39 colorectal cancer-multiple polyp patients. Four missense APC gene alterations (S130G, E1317Q, Dl822V, G2502S) were observed in 13 individuals and 3 additional patients carried presumed pathogenic (APC Y94X, biallelic MYH Y165C and heterozygous MYH G382D) mutations. When independently assessed in 971 patients with colorectal cancer and 954 healthy control subjects, none of the identified missense APC alterations conferred a significantly increased risk for colorectal cancer, odds ratio (95 percent confidence intervals): S130G=3.1 (0.29–32.25), E1317Q= 1.08 (0.59–2.74), G2502S= 1 (0.65–1.63), D1822V (heterozygous)=0.79 (0.64–0.98), D1822V (homozygous) =0.82 (0.63–1.27). CONCLUSIONS Germline missense APC alterations observed in 33 percent of patients with multiple colorectal neoplasms seemed to play a limited role in colorectal cancer risk when independently assessed by a population-based, case-control analysis. PMID:18612690

  7. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  8. Artificial antigen presenting cell (aAPC) mediated activation and expansion of natural killer T cells.

    PubMed

    East, James E; Sun, Wenji; Webb, Tonya J

    2012-01-01

    Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells(1). Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules(2). NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity(3-6), and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d (7). The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines(8). Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines(9,10). In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients(11). However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells(12, 13). Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is

  9. Application of the microbiological method DEFT/APC to detect minimally processed vegetables treated with gamma radiation

    NASA Astrophysics Data System (ADS)

    Araújo, M. M.; Duarte, R. C.; Silva, P. V.; Marchioni, E.; Villavicencio, A. L. C. H.

    2009-07-01

    Marketing of minimally processed vegetables (MPV) are gaining impetus due to its convenience, freshness and apparent health effect. However, minimal processing does not reduce pathogenic microorganisms to safe levels. Food irradiation is used to extend the shelf life and to inactivate food-borne pathogens. In combination with minimal processing it could improve safety and quality of MPV. A microbiological screening method based on the use of direct epifluorescent filter technique (DEFT) and aerobic plate count (APC) has been established for the detection of irradiated foodstuffs. The aim of this study was to evaluate the applicability of this technique in detecting MPV irradiation. Samples from retail markets were irradiated with 0.5 and 1.0 kGy using a 60Co facility. In general, with a dose increment, DEFT counts remained similar independent of the irradiation while APC counts decreased gradually. The difference of the two counts gradually increased with dose increment in all samples. It could be suggested that a DEFT/APC difference over 2.0 log would be a criteria to judge if a MPV was treated by irradiation. The DEFT/APC method could be used satisfactorily as a screening method for indicating irradiation processing.

  10. Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens

    PubMed Central

    Rosskopf, Sandra; Jutz, Sabrina; Neunkirchner, Alina; Candia, Martín R.; Jahn-Schmid, Beatrice; Bohle, Barbara; Pickl, Winfried F.; Steinberger, Peter

    2016-01-01

    We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1142–153 and Art v 125–36, respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. PMID:27539532

  11. 77 FR 34455 - In the Matter of Aegis Assessments, Inc., APC Group, Inc., Aurelio Resource Corp., BioAuthorize...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-11

    ... From the Federal Register Online via the Government Publishing Office SECURITIES AND EXCHANGE COMMISSION In the Matter of Aegis Assessments, Inc., APC Group, Inc., Aurelio Resource Corp., BioAuthorize... securities of BioAuthorize Holdings, Inc. because it has not filed any periodic reports since the...

  12. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

    PubMed Central

    Nabti, Ibtissem; Bormann, Jenny; Kudo, Nobuaki R.

    2014-01-01

    Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes. PMID:24637322

  13. Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

    PubMed Central

    Min, Mingwei; Mevissen, Tycho E. T.; De Luca, Maria; Komander, David; Lindon, Catherine

    2015-01-01

    The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner. PMID:26446837

  14. Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

    PubMed Central

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E.; Rahaim, Ronald J.; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J.; Malumbres, Marcos; Roussel, Martine F.; Roush, William R.; Hatten, Mary E.; Ayad, Nagi G.

    2015-01-01

    SUMMARY Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of central nervous system progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1) ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell cycle exit in the developing central nervous system. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a new therapeutic target. PMID:25843713

  15. APC/CCdh1 controls CtIP stability during the cell cycle and in response to DNA damage

    PubMed Central

    Lafranchi, Lorenzo; de Boer, Harmen R; de Vries, Elisabeth GE; Ong, Shao-En; Sartori, Alessandro A; van Vugt, Marcel ATM

    2014-01-01

    Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/CCdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage-induced G2 arrest. However, it is currently unknown whether APC/CCdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA-damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA-end resection factor, as a novel APC/CCdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G1 and after DNA damage in G2. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA-end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/CCdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle- and DNA damage-dependent manner. PMID:25349192

  16. Creation of an engineered APC system to explore and optimize the presentation of immunodominant peptides of major allergens.

    PubMed

    Rosskopf, Sandra; Jutz, Sabrina; Neunkirchner, Alina; Candia, Martín R; Jahn-Schmid, Beatrice; Bohle, Barbara; Pickl, Winfried F; Steinberger, Peter

    2016-01-01

    We have generated engineered APC to present immunodominant peptides derived from the major aero-allergens of birch and mugwort pollen, Bet v 1142-153 and Art v 125-36, respectively. Jurkat-based T cell reporter lines expressing the cognate allergen-specific T cell receptors were used to read out the presentation of allergenic peptides on the engineered APC. Different modalities of peptide loading and presentation on MHC class II molecules were compared. Upon exogenous loading with allergenic peptides, the engineered APC elicited a dose-dependent response in the reporter T cells and the presence of chemical loading enhancers strongly increased reporter activation. Invariant chain-based MHC class II targeting strategies of endogenously expressed peptides resulted in stronger activation of the reporters than exogenous loading. Moreover, we used Bet v 1 as model allergen to study the ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. PMID:27539532

  17. Positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis in budding yeast.

    PubMed

    Hatano, Yuhki; Naoki, Koike; Suzuki, Asuka; Ushimaru, Takashi

    2016-10-01

    The mitotic inhibitor securin is degraded via the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdc20 after anaphase onset. This triggers activation of the mitotic protease separase and thereby sister chromatid separation. However, only a proportion of securin molecules are degraded at metaphase-anaphase transition and the remaining molecules are still present in anaphase. The roles of securin and separase in late mitosis remain elusive. Here, we show that securin still inhibits separase to repress mitotic exit in anaphase in budding yeast. APC/C-Cdh1-mediated securin degradation at telophase further liberated separase, which promotes Cdc14 release and mitotic exit. Separase executed these events via its proteolytic action and that in the Cdc14 early release (FEAR) network. Cdc14 release further activated APC/C-Cdh1 in the manner of a positive feedback loop. Thus, the positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis. This study shows the importance of the two-step degradation mode of securin and the role of separase in mitotic exit.

  18. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity.

    PubMed

    Nabti, Ibtissem; Marangos, Petros; Bormann, Jenny; Kudo, Nobuaki R; Carroll, John

    2014-03-17

    Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes. PMID:24637322

  19. Swelling behavior of O-alkylated APCS coals as examined by the EPR spin probe method

    SciTech Connect

    Ding, Ruisong; Tucker, D.; Kispert, L.D.

    1995-12-31

    Known O-alkylation procedures have been used to derivatize the carboxyl and hydroxyl groups in the APCS coals Lewiston-Stockton, Wyodak-Anderson, Beulah-Zap, Illinois {number_sign}6, Upper Freeport, and Pittsburgh {number_sign}8. In general the resulting decrease in hydrogen bonding reduced the cyclical variation in nitroxide spin probe retention observed for nonalkylated coals when small amounts (<1%) of pyridine are present in the toluene swelling solvent. An increase in spin probe retention by the O-alkylated coals relative to the underivatized coals indicates a more open arrangement in the coal due to a decrease in attractive forces, confirming that microporosity increases with increasing rank.

  20. The role of APC/C(Cdh1) in replication stress and origin of genomic instability.

    PubMed

    Greil, C; Krohs, J; Schnerch, D; Follo, M; Felthaus, J; Engelhardt, M; Wäsch, R

    2016-06-01

    It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations. PMID:26455319

  1. The role of APC/C(Cdh1) in replication stress and origin of genomic instability.

    PubMed

    Greil, C; Krohs, J; Schnerch, D; Follo, M; Felthaus, J; Engelhardt, M; Wäsch, R

    2016-06-01

    It has been proposed that the APC/C(Cdh1) functions as a tumor suppressor by maintaining genomic stability. However, the exact nature of genomic instability following loss of Cdh1 is unclear. Using biochemistry and live cell imaging of single cells we found that Cdh1 knockdown (kd) leads to strong nuclear stabilization of the substrates cyclin A and B and deregulated kinetics of DNA replication. Restoration of the Cdh1-dependent G2 DNA damage checkpoint did not result in G2 arrest but blocked cells in prometaphase, suggesting that these cells enter mitosis despite incomplete replication. This results in DNA double-strand breaks, anaphase bridges, cytokinesis defects and tetraploidization. Tetraploid cells are the source of supernumerary centrosomes following Cdh1-kd, leading to multipolar mitosis or centrosome clustering, in turn resulting in merotelic attachment and lagging chromosomes. Whereas some of these events cause apoptosis during mitosis, surviving cells may accumulate chromosomal aberrations.

  2. Homozygous patients with APC resistance may remain paucisymptomatic or asymptomatic during oral contraception.

    PubMed

    Girolami, A; Simioni, P; Girolami, B; Radossi, P

    1996-09-01

    The effect of oral contraceptive therapy was studied in five patients with homozygous activated protein C resistance. Patients with this congenital abnormality, in contrast to those with antithrombin, protein C or protein S deficiencies, showed only a mild thrombotic tendency. In fact, only two of six observations (one patient took the pill on two separate occasions many years apart) showed deep vein thrombosis. No patient had pulmonary embolism. Two additional patients had a superficial vein thrombosis of the legs. In two instances, a superficial vein thrombosis and a deep vein thrombosis, concomitant risk factors were present (immobilization and surgery for an ovarian cyst, respectively). However, compared with heterozygous for the same abnormality, the symptomatic homozygous patients with APC resistance appeared to develop thrombosis after a shorter period of oral contraception.

  3. Validity of APCS score as a risk prediction score for advanced colorectal neoplasia in Chinese asymptomatic subjects

    PubMed Central

    Li, Wenbin; Zhang, Lili; Hao, Jianyu; Wu, Yongdong; Lu, Di; Zhao, Haiying; Wang, Zhenjie; Xu, Tianming; Yang, Hong; Qian, Jiaming; Li, Jingnan

    2016-01-01

    Abstract The Asia-Pacific Colorectal Screening (APCS) score is a risk-stratification tool that helps predict the risk for advanced colorectal neoplasia (ACN) in asymptomatic Asian populations, but has not yet been assessed for its validity of use in Mainland China. The aim of the study was to assess the validity of APCS score in asymptomatic Chinese population, and to identify other risk factors associated with ACN. Asymptomatic subjects (N = 1010) who underwent colonoscopy screening between 2012 and 2014 in Beijing were enrolled. APCS scores based on questionnaires were used to stratify subjects into high, moderate, and average-risk tiers. Cochran–Armitage test for trend was used to assess the association between ACN and risk tiers. Univariate and multivariate logistic regression was performed with ACN as the outcome, adjusting for APCS score, body mass index, alcohol consumption, self-reported diabetes, and use of nonsteroidal anti-inflammatory drugs as independent variables. The average age was 53.5 (standard deviation 8.4) years. The prevalence of ACN was 4.1% overall, and in the high, moderate, and average-risk tiers, the prevalence was 8.8%, 2.83%, and 1.55%, respectively (P < 0.001). High-risk tier had 3.3 and 6.1-fold increased risk of ACN as compared with those in the moderate and average-risk tiers, respectively. In univariate analysis, high-risk tier, obesity, diabetes, and alcohol consumption were associated with ACN. In multivariate analysis, only high-risk tier was an independent predictor of ACN. The APCS score can effectively identify a subset of asymptomatic Chinese population at high risk for ACN. Further studies are required to identify other risk factors, and the acceptability of the score to the general population will need to be further examined. PMID:27741134

  4. Nek2A destruction marks APC/C activation at the prophase-to-prometaphase transition by spindle-checkpoint-restricted Cdc20.

    PubMed

    Boekhout, Michiel; Wolthuis, Rob

    2015-04-15

    Nek2 isoform A (Nek2A) is a presumed substrate of the anaphase-promoting complex/cyclosome containing Cdc20 (APC/C(Cdc20)). Nek2A, like cyclin A, is degraded in mitosis while the spindle checkpoint is active. Cyclin A prevents spindle checkpoint proteins from binding to Cdc20 and is recruited to the APC/C in prometaphase. We found that Nek2A and cyclin A avoid being stabilized by the spindle checkpoint in different ways. First, enhancing mitotic checkpoint complex (MCC) formation by nocodazole treatment inhibited the degradation of geminin and cyclin A, whereas Nek2A disappeared at a normal rate. Second, depleting Cdc20 effectively stabilized cyclin A but not Nek2A. Nevertheless, Nek2A destruction crucially depended on Cdc20 binding to the APC/C. Third, in contrast to cyclin A, Nek2A was recruited to the APC/C before the start of mitosis. Interestingly, the spindle checkpoint very effectively stabilized an APC/C-binding mutant of Nek2A, which required the Nek2A KEN box. Apparently, in cells, the spindle checkpoint primarily prevents Cdc20 from binding destruction motifs. Nek2A disappearance marks the prophase-to-prometaphase transition, when Cdc20, regardless of the spindle checkpoint, activates the APC/C. However, Mad2 depletion accelerated Nek2A destruction, showing that spindle checkpoint release further increases APC/C(Cdc20) catalytic activity. PMID:25673878

  5. Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests

    PubMed Central

    Syred, Heather M.; van der Sar, Sjaak; Patel, Hitesh; Moresco, James J.; Sarkeshik, Ali; Yates, John R.; Rappsilber, Juri; Hardwick, Kevin G.

    2016-01-01

    The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests. PMID:26882497

  6. Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C–Cdc20 complex

    PubMed Central

    Sedgwick, Garry G; Hayward, Daniel G; Di Fiore, Barbara; Pardo, Mercedes; Yu, Lu; Pines, Jonathon; Nilsson, Jakob

    2013-01-01

    The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C–Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C–Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC. PMID:23288039

  7. Self-association of the APC tumor suppressor is required for the assembly, stability, and activity of the Wnt signaling destruction complex

    PubMed Central

    Kunttas-Tatli, Ezgi; Roberts, David M.; McCartney, Brooke M.

    2014-01-01

    The tumor suppressor adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3β, and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles we termed the destructosome. Whereas APC functions in the complex through its ability to bind both β-cat and Axin, we hypothesize that APC proteins play an additional role in destructosome assembly through self-association. Here we show that a novel N-terminal coil, the APC self-association domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine. PMID:25208568

  8. Clustering of T cell ligands on artificial APC membranes influences T cell activation and protein kinase C theta translocation to the T cell plasma membrane.

    PubMed

    Giannoni, Francesca; Barnett, Joellen; Bi, Kun; Samodal, Rodrigo; Lanza, Paola; Marchese, Patrizia; Billetta, Rosario; Vita, Randi; Klein, Mark R; Prakken, Berent; Kwok, William W; Sercarz, Eli; Altman, Amnon; Albani, Salvatore

    2005-03-15

    T cell activation is associated with active clustering of relevant molecules in membrane microdomains defined as the supramolecular activation cluster. The contact area between these regions on the surface of T cells and APC is defined as the immunological synapse. It has been recently shown that preclustering of MHC-peptide complexes in membrane microdomains on the APC surface affects the efficiency of immune synapse formation and the related T cell activation. Disruption of such clusters may reduce the efficiency of stimulation. We describe here an entirely artificial system for Ag-specific, ex vivo stimulation of human polyclonal T cells (artificial APC (aAPC)). aAPC are based on artificial membrane bilayers containing discrete membrane microdomains encompassing T cell ligands (i.e., appropriate MHC-peptide complexes in association with costimulatory molecules). We show here that preclustering of T cell ligands triggered a degree of T cell activation significantly higher than the one achieved when we used either soluble tetramers or aAPC in which MHC-peptide complexes were uniformly distributed within artificial bilayer membranes. This increased efficiency in stimulation was mirrored by increased translocation from the cytoplasm to the membrane of protein kinase theta, a T cell signaling molecule that colocalizes with the TCR within the supramolecular activation cluster, thus indicating efficient engagement of T cell activation pathways. Engineered aAPC may have immediate application for basic and clinical immunology studies pertaining to modulation of T cells ex vivo.

  9. 1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum.

    SciTech Connect

    Cort, John R.; Swenson, Michael; Magnuson, Timothy S.

    2011-03-04

    We report the 1H, 13C, and 15N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, from the acidophilic gram-negative facultatively anaerobic metal-reducing alpha-proteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c2 and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21 N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on 13C- and 15N- labeled substrates.

  10. Increase in a distinct pulmonary macrophage subset possessing an antigen-presenting cell phenotype and in vitro APC activity following silica exposure

    SciTech Connect

    Migliaccio, Christopher T. . E-mail: christopher.migliaccio@umontana.edu; Hamilton, Raymond F.; Holian, Andrij

    2005-06-01

    Silica inhalation results in chronic lung inflammation and fibrosis. While the role of the alveolar macrophage (AM) is considered key to the effects of silica on lung pathology, the etiology is not completely understood. Evidence suggests an increase in antigen presenting cell (APC) activity as a contributing factor to this process, as well as potential roles for both AM and interstitial macrophages (IM) in silicosis. In order to study the effects of crystalline silica on the APC activity of pulmonary macrophages, mice were exposed intranasally and changes in pulmonary macrophage populations were assessed using flow cytometry. Following intranasal instillation of silica, a significant increase in the APC activity of AM was observed, as well as a significant increase in a subset of IM expressing classic APC markers (MHC class II, CD11c). In addition, an in vitro system using bone marrow-derived macrophages (BMDM) was generated to assess the effects of silica on the APC activity of macrophages in vitro. Data using BMDM in the in vitro APC assay demonstrated a significant increase in APC activity following silica exposure, but not following exposure to saline or a control particle (TiO{sub 2}). Using a combination of in vivo and in vitro experiments, the current study describes a significant increase in an interstitial macrophage subset with an APC phenotype, as well as an increase in the APC activity of both AM and BMDM, as a direct result of exposure to crystalline silica. These studies suggest a specific mechanism, macrophage subset activation, by which crystalline silica exposure results in chronic pulmonary inflammation and, eventually, fibrosis.

  11. Spatiotemporal organization of Aurora-B by APC/CCdh1 after mitosis coordinates cell spreading through FHOD1

    PubMed Central

    Floyd, Suzanne; Whiffin, Nicola; Gavilan, Maria P.; Kutscheidt, Stefan; De Luca, Maria; Marcozzi, Chiara; Min, Mingwei; Watkins, Johnathan; Chung, Kathryn; Fackler, Oliver T.; Lindon, Catherine

    2013-01-01

    Summary Spatiotemporal regulation of mitotic kinase activity underlies the extensive rearrangement of cellular components required for cell division. One highly dynamic mitotic kinase is Aurora-B (AurB), which has multiple roles defined by the changing localisation of the chromosome passenger complex (CPC) as cells progress through mitosis, including regulation of cytokinesis and abscission. Like other mitotic kinases, AurB is a target of the anaphase-promoting complex (APC/C) ubiquitin ligase during mitotic exit, but it is not known if APC/C-mediated destruction plays any specific role in controlling AurB activity. We have examined the contribution of the Cdh1 coactivator-associated APC/CCdh1 to the organization of AurB activity as cells exit mitosis and re-enter interphase. We report that APC/CCdh1-dependent proteolysis restricts a cell-cortex-associated pool of active AurB in space and time. In early G1 phase this pool of AurB is found at protrusions associated with cell spreading. AurB retention at the cortex depends on a formin, FHOD1, critically required to organize the cytoskeleton after division. We identify AurB phosphorylation sites in FHOD1 and show that phosphomutant FHOD1 is impaired in post-mitotic assembly of oriented actin cables. We propose that Cdh1 contributes to spatiotemporal organization of AurB activity and that organization of FHOD1 activity by AurB contributes to daughter cell spreading after mitosis. PMID:23613471

  12. Environmental impact of APC residues from municipal solid waste incineration: reuse assessment based on soil and surface water protection criteria.

    PubMed

    Quina, Margarida J; Bordado, João C M; Quinta-Ferreira, Rosa M

    2011-01-01

    Waste management and environmental protection are mandatory requirements of modern society. In our study, air pollution control (APC) residues from municipal solid waste incinerators (MSWI) were considered as a mixture of fly ash and fine particulate solids collected in scrubbers and fabric filters. These are hazardous wastes and require treatment before landfill. Although there are a number of treatment options, it is highly recommended to find practical applications rather than just dump them in landfill sites. In general, for using a construction material, beyond technical specifications also soil and surface water criteria may be used to ensure environmental protection. The Dutch Building Materials Decree (BMD) is a valuable tool in this respect and it was used to investigate which properties do not meet the threshold criteria so that APC residues can be further used as secondary building material. To this end, some scenarios were evaluated by considering release of inorganic species from unmoulded and moulded applications. The main conclusion is that the high amount of soluble salts makes the APC residues a building material prohibited in any of the conditions tested. In case of moulding materials, the limits of heavy metals are complied, and their use in Category 1 would be allowed. However, also in this case, the soluble salts lead to the classification of "building material not allowed". The treatments with phosphates or silicates are able to solve the problem of heavy metals, but difficulties with the soluble salts are still observed. This analysis suggests that for APC residues to comply with soil and surface water protection criteria to be further used as building material at least a pre-treating for removing soluble salts is absolutely required.

  13. A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

    PubMed Central

    Kaur, Jagdeep; Olkhova, Elena; Malviya, Viveka Nand; Grell, Ernst; Michel, Hartmut

    2014-01-01

    Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed. PMID:24257746

  14. The E3 Ligase APC/C-Cdh1 Is Required for Associative Fear Memory and Long-Term Potentiation in the Amygdala of Adult Mice

    ERIC Educational Resources Information Center

    Pick, Joseph E.; Malumbres, Marcos; Klann, Eric

    2013-01-01

    The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating…

  15. Liver fatty acid-binding protein (L-Fabp) modifies intestinal fatty acid composition and adenoma formation in ApcMin/+ mice.

    PubMed

    Dharmarajan, Sekhar; Newberry, Elizabeth P; Montenegro, Grace; Nalbantoglu, Ilke; Davis, Victoria R; Clanahan, Michael J; Blanc, Valerie; Xie, Yan; Luo, Jianyang; Fleshman, James W; Kennedy, Susan; Davidson, Nicholas O

    2013-10-01

    Evidence suggests a relationship between dietary fat intake, obesity, and colorectal cancer, implying a role for fatty acid metabolism in intestinal tumorigenesis that is incompletely understood. Liver fatty acid-binding protein (L-Fabp), a dominant intestinal fatty acid-binding protein, regulates intestinal fatty acid trafficking and metabolism, and L-Fabp deletion attenuates diet-induced obesity. Here, we examined whether changes in intestinal fatty acid metabolism following L-Fabp deletion modify adenoma development in Apc(Min)(/+) mice. Compound L-Fabp(-/-)Apc(Min)(/+) mice were generated and fed a 10% fat diet balanced equally between saturated, monounsaturated, and polyunsaturated fat. L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in adenoma number and total polyp area compared with Apc(Min)(/+)controls, reflecting a significant shift in distribution toward smaller polyps. Adenomas from L-Fabp(-/-)Apc(Min)(/+) mice exhibited reductions in cellular proliferation, high-grade dysplasia, and nuclear β-catenin translocation. Intestinal fatty acid content was increased in L-Fabp(-/-)Apc(Min)(/+) mice, and lipidomic profiling of intestinal mucosa revealed significant shifts to polyunsaturated fatty acid species with reduced saturated fatty acid species. L-Fabp(-/-)Apc(Min)(/+) mice also showed corresponding changes in mRNA expression of enzymes involved in fatty acid elongation and desaturation. Furthermore, adenomas from L-Fabp(-/-)Apc(Min)(/+) mice displayed significant reductions in mRNA abundance of nuclear hormone receptors involved in cellular proliferation and in enzymes involved in lipogenesis. These findings collectively implicate L-Fabp as an important genetic modifier of intestinal tumorigenesis, and identify fatty acid trafficking and metabolic compartmentalization as an important pathway linking dietary fat intake, obesity, and intestinal tumor formation.

  16. Clock genes and cancer.

    PubMed

    Wood, Patricia A; Yang, Xiaoming; Hrushesky, William J M

    2009-12-01

    Period genes ( Per2, Per1) are essential circadian clock genes. They also function as negative growth regulators. Per2 mutant mice show de novo and radiation-induced epithelial hyperplasia, tumors, and an abnormal DNA damage response. Human tumors show Period gene mutations or decreased expression. Other murine clock gene mutations are not associated with a tumor prone phenotype. Shift work and nocturnal light exposure are associated with circadian clock disruption and with increased cancer risk. The mechanisms responsible for the connection between the circadian clock and cancer are not well defined. We propose that circadian disruption per se is not uniformly tumor promoting and the mechanisms for tumor promotion by specific circadian clock disturbances will differ dependent upon the genes and pathways involved. We propose that Period clock gene mutations promote tumorigenesis by unique molecular pathways. Per2 and Per1 modulate beta-catenin and cell proliferation in colon and non-colon cancer cells. Per2 mutation increases intestinal beta-catenin levels and colon polyp formation. Per2 mutation also increases Apc(Min/+)-mediated intestinal and colonic polyp formation. Intestinal tumorigenesis per se may also alter clock function as a result of increased beta-catenin destabilizing PER2 protein. Levels and circadian rhythm of PER2 in Apc(Min/+) mouse intestine are markedly decreased, and selective abnormalities in intestinal clock gene and clock-controlled gene expression are seen. We propose that tumor promotion by loss of PERIOD clock proteins is unique to these clock genes as a result of altered beta-catenin signaling and DNA damage response. PERIOD proteins may offer new targets for cancer prevention and control.

  17. Stabilisation/solidification of APC residues from MSW incineration with hydraulic binders and chemical additives.

    PubMed

    Quina, Margarida J; Bordado, João C M; Quinta-Ferreira, Rosa M

    2014-01-15

    This study focuses on the stabilisation/solidification (S/S) treatment of air pollution control (APC) residues from municipal solid waste (MSW) incineration. Six formulations (T1-T6) were tested based on different cements as binders, for the immobilisation of pollutants and to prevent their entering into the environment at unacceptable rates. Soluble phosphates and silicates were considered in some cases to fix heavy metals. The performance of T1-T6 products was measured in terms of initial and final setting times, mechanical strength, total availability and leaching from S/S products. Two monolithic leaching tests were used to estimate emissions of pollutants over 48h and 64 days. The results showed that the setting time was reduced when soluble phosphates were used. Moreover, although all the treatments have met the threshold of 1MPa for unconfined compressive strength, this parameter was significantly reduced due to matrix dissolution during immersion. After three cycles of leaching, the limit of 10% for solubilisation was exceeded for all treatments with the exception of T5 (with phosphates). This study demonstrated that the S/S treatment used at the industrial level can be improved with respect to toxic heavy metals, by using soluble silicates or phosphates, but not regarding soluble salts.

  18. First military use of activated Factor VII in an APC-III pelvic fracture.

    PubMed

    Williams, D J; Thomas, G O R; Pambakian, S; Parker, P J

    2005-03-01

    A male soldier in shock with an APC-III pelvic fracture was flown to an Air Assault Surgical Group (AASG) of 16 Close Support Medical Regiment at Al Amarah in Iraq. A pelvic external fixator was applied and his condition stabilized. Ultrasound scanning (FAST) showed an absent bladder, and a spreading retroperitoneal haematoma combined with intra-abdominal, free blood. Unfortunately he continued to bleed and required transfusion with the unit's entire stock of type-specific blood. At emergency laparotomy, uncontrollable pelvic bleeding was encountered and the abdominal aorta required clamping above the iliac bifurcation. Branches of the right internal iliac artery were the source and this was ligated. Some bleeding continued post-operatively: administration of activated Factor VII was associated with a marked reduction in the oozing from his fixator pin-sites and an improvement in his pH from 7.1 to 7.3. In total, 25 units of blood were transfused, 8 of which were fresh whole blood donated by individual members of the AASG. The patient survived, returned to the UK, had his injuries reconstructed, and is currently undergoing rehabilitation. This case illustrates the benefits of forward resuscitation surgery in wartime and the need for a multidisciplinary approach to trauma care.

  19. Prdm5 suppresses Apc(Min)-driven intestinal adenomas and regulates monoacylglycerol lipase expression.

    PubMed

    Galli, G G; Multhaupt, H A; Carrara, M; de Lichtenberg, K H; Christensen, I B J; Linnemann, D; Santoni-Rugiu, E; Calogero, R A; Lund, A H

    2014-06-19

    PRDM proteins are tissue-specific transcription factors often deregulated in diseases, particularly in cancer where different members have been found to act as oncogenes or tumor suppressors. PRDM5 is a poorly characterized member of the PRDM family for which several studies have reported a high frequency of promoter hypermethylation in cancer types of gastrointestinal origin. We report here the characterization of Prdm5 knockout mice in the context of intestinal carcinogenesis. We demonstrate that loss of Prdm5 increases the number of adenomas throughout the murine small intestine on an Apc(Min) background. By using the genome-wide ChIP-seq (chromatin immunoprecipitation (ChIP) followed by DNA sequencing) and transcriptome analyses we identify loci encoding proteins involved in metabolic processes as prominent PRDM5 targets and characterize monoacylglycerol lipase (Mgll) as a direct PRDM5 target in human colon cancer cells and in Prdm5 mutant mouse intestines. Moreover, we report the downregulation of PRDM5 protein expression in human colon neoplastic lesions. In summary, our data provide the first causal link between Prdm5 loss and intestinal carcinogenesis, and uncover an extensive and novel PRDM5 target repertoire likely facilitating the tumor-suppressive functions of PRDM5.

  20. Task-based imaging of colon cancer in the ApcMin/+ mouse model

    NASA Astrophysics Data System (ADS)

    McNally, James B.; Kirkpatrick, Nathaniel D.; Hariri, Lida P.; Tumlinson, Alexandre R.; Besselsen, David G.; Gerner, Eugene W.; Utzinger, Urs; Barton, Jennifer K.

    2006-05-01

    Optical coherence tomography (OCT), laser-induced fluorescence (LIF), and laser-scanning confocal microscopy (LSCM) were used for the task of multimodal study of healthy and adenomatous mouse colon. The results from each modality were compared with histology, which served as the gold standard. The ApcMin/+ genetic mouse model of colon cancer was compared with wild-type mice. In addition, a special diet was used for the task of studying the origins of a 680 nm autofluorescent signal that was previously observed in colon. The study found close agreement among each of the modalities and with histology. All four modalities were capable of identifying diseased tissue accurately. The OCT and LSCM images provided complementary structural information about the tissue, while the autofluorescence signal measured by LIF and LSCM provided biochemical information. OCT and LIF were performed in vivo and nondestructively, while the LSCM and histology required extraction of the tissue. The magnitude of the 680 nm signal correlates with chlorophyll content in the mouse diet, suggesting that the autofluorescent compound is a dietary metabolite.

  1. Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin

    PubMed Central

    Shimizu, Satomi; Miyamoto, Shingo; Fujii, Gen; Nakanishi, Ruri; Onuma, Wakana; Ozaki, Yoshihiko; Fujimoto, Kyoko; Yano, Tomohiro; Mutoh, Michihiro

    2015-01-01

    Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis. PMID:26236099

  2. Regulation of mitochondrial morphology by APC/CCdh1-mediated control of Drp1 stability

    PubMed Central

    Horn, Sarah R.; Thomenius, Michael J.; Johnson, Erika Segear; Freel, Christopher D.; Wu, Judy Q.; Coloff, Jonathan L.; Yang, Chih-Sheng; Tang, Wanli; An, Jie; Ilkayeva, Olga R.; Rathmell, Jeffrey C.; Newgard, Christopher B.; Kornbluth, Sally

    2011-01-01

    Homeostatic maintenance of cellular mitochondria requires a dynamic balance between fission and fusion, and controlled changes in morphology are important for processes such as apoptosis and cellular division. Interphase mitochondria have been described as an interconnected network that fragments as cells enter mitosis, and this mitotic mitochondrial fragmentation is known to be regulated by the dynamin-related GTPase Drp1 (dynamin-related protein 1), a key component of the mitochondrial division machinery. Loss of Drp1 function and the subsequent failure of mitochondrial division during mitosis lead to incomplete cytokinesis and the unequal distribution of mitochondria into daughter cells. During mitotic exit and interphase, the mitochondrial network reforms. Here we demonstrate that changes in mitochondrial dynamics as cells exit mitosis are driven in part through ubiquitylation of Drp1, catalyzed by the APC/CCdh1 (anaphase-promoting complex/cyclosome and its coactivator Cdh1) E3 ubiquitin ligase complex. Importantly, inhibition of Cdh1-mediated Drp1 ubiquitylation and proteasomal degradation during interphase prevents the normal G1 phase regrowth of mitochondrial networks following cell division. PMID:21325626

  3. Stromal deletion of the APC tumor suppressor in mice triggers development of endometrial cancer

    PubMed Central

    Tanwar, Pradeep S.; Zhang, LiHua; Roberts, Drucilla J.; Teixeira, Jose M.

    2011-01-01

    The contribution of the stromal microenvironment to the progression of endometrial cancer (EC) has not been well explored. We have conditionally expressed a mutant allele of adenomatous polyposis coli (APCcKO) in murine uterine stroma cells to study its effect on uterine development and function. In addition to metrorrhagia, the mice develop complex atypical endometrial gland hyperplasia that progresses to endometrial carcinoma in situ and endometrial adenocarcinoma as evidenced by myometrial invasion. Stromal cells subjacent to the carcinoma cells express αSMA with fewer cells expressing PDGFR-α compared to normal stromal cells suggesting that the mutant stromal cells have acquired a more myofibroblastic phenotype, which have been described as cancer-associated fibroblasts and have been shown to induce carcinogenesis in other organ systems. Analyses of human EC specimens showed substantial αSMA expression in the stroma compared with normal endometrial stroma cells. We also show that APCcKO mutant uteri and human EC have decreased stromal levels of TGFβ and BMP activities and that the mutant uteri failed to respond to exogenous estradiol stimulation. The mutant stroma cells also had higher levels of VEGF and SDF signaling components and diminished expression of ERα and PR which is common in advanced stages of human EC and is an indicator of poor prognosis. Our results indicate that de novo mutation or loss of heterozygosity in stromal APC is sufficient to induce endometrial hyperplasia and endometrial carcinogenesis by mechanisms that are consistent with unopposed estrogen signaling in the endometrial epithelium. PMID:21363919

  4. Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

    PubMed Central

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P.; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  5. Uncovering the role of APC-Cdh1 in generating the dynamics of S-phase onset

    PubMed Central

    Yuan, Xi; Srividhya, Jeyaraman; De Luca, Thomas; Lee, Ju-hyong E.; Pomerening, Joseph R.

    2014-01-01

    Cdh1, a coactivator of the anaphase-promoting complex (APC), is a potential tumor suppressor. Cdh1 ablation promotes precocious S-phase entry, but it was unclear how this affects DNA replication dynamics while contributing to genomic instability and tumorigenesis. We find that Cdh1 depletion causes early S-phase onset in conjunction with increase in Rb/E2F1-mediated cyclin E1 expression, but reduced levels of cyclin E1 protein promote this transition. We hypothesize that this is due to a weakened cyclin-dependent kinase inhibitor (CKI)–cyclin-dependent kinase 2 positive-feedback loop, normally generated by APC-Cdh1–mediated proteolysis of Skp2. Indeed, Cdh1 depletion increases Skp2 abundance while diminishing levels of the CKI p27. This lowers the level of cyclin E1 needed for S-phase entry and delays cyclin E1 proteolysis during S-phase progression while corresponding to slowed replication fork movement and reduced frequency of termination events. In summary, using both experimental and computational approaches, we show that APC-Cdh1 establishes a stimulus–response relationship that promotes S phase by ensuring that proper levels of p27 accumulate during G1 phase, and defects in its activation accelerate the timing of S-phase onset while prolonging its progression. PMID:24356446

  6. Phosphorylation-triggered CUEDC2 degradation promotes UV-induced G1 arrest through APC/C(Cdh1) regulation.

    PubMed

    Zhang, Wei-Na; Zhou, Jie; Zhou, Tao; Li, Ai-Ling; Wang, Na; Xu, Jin-Jing; Chang, Yan; Man, Jiang-Hong; Pan, Xin; Li, Tao; Li, Wei-Hua; Mu, Rui; Liang, Bing; Chen, Liang; Jin, Bao-Feng; Xia, Qing; Gong, Wei-Li; Zhang, Xue-Min; Wang, Li; Li, Hui-Yan

    2013-07-01

    DNA damage triggers cell cycle arrest to provide a time window for DNA repair. Failure of arrest could lead to genomic instability and tumorigenesis. DNA damage-induced G1 arrest is generally achieved by the accumulation of Cyclin-dependent kinase inhibitor 1 (p21). However, p21 is degraded and does not play a role in UV-induced G1 arrest. The mechanism of UV-induced G1 arrest thus remains elusive. Here, we have identified a critical role for CUE domain-containing protein 2 (CUEDC2) in this process. CUEDC2 binds to and inhibits anaphase-promoting complex/cyclosome-Cdh1 (APC/C(Cdh1)), a critical ubiquitin ligase in G1 phase, thereby stabilizing Cyclin A and promoting G1-S transition. In response to UV irradiation, CUEDC2 undergoes ERK1/2-dependent phosphorylation and ubiquitin-dependent degradation, leading to APC/C(Cdh1)-mediated Cyclin A destruction, Cyclin-dependent kinase 2 inactivation, and G1 arrest. A nonphosphorylatable CUEDC2 mutant is resistant to UV-induced degradation. Expression of this stable mutant effectively overrides UV-induced G1-S block. These results establish CUEDC2 as an APC/C(Cdh1) inhibitor and indicate that regulated CUEDC2 degradation is critical for UV-induced G1 arrest.

  7. Killer artificial antigen presenting cells (KaAPC) for efficient in vitro depletion of human antigen-specific T cells.

    PubMed

    Schütz, Christian; Fleck, Martin; Schneck, Jonathan P; Oelke, Mathias

    2014-01-01

    Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion. PMID:25145915

  8. Aggregate material formulated with MSWI bottom ash and APC fly ash for use as secondary building material.

    PubMed

    del Valle-Zermeño, R; Formosa, J; Chimenos, J M; Martínez, M; Fernández, A I

    2013-03-01

    The main goal of this paper is to obtain a granular material formulated with Municipal Solid Waste Incineration (MSWI) bottom ash (BA) and air pollution control (APC) fly ash to be used as secondary building material. Previously, an optimum concrete mixture using both MSWI residues as aggregates was formulated. A compromise between the environmental behavior whilst maximizing the reuse of APC fly ash was considered and assessed. Unconfined compressive strength and abrasion resistance values were measured in order to evaluate the mechanical properties. From these results, the granular mixture was not suited for certain applications owing to the high BA/APC fly ash content and low cement percentages used to reduce the costs of the final product. Nevertheless, the leaching test performed showed that the concentrations of all heavy metals were below the limits established by the current Catalan legislation for their reutilization. Therefore, the material studied might be mainly used in embankments, where high mechanical properties are not needed and environmental safety is assured. PMID:23102641

  9. High dietary niacin may increase prostaglandin formation but does not increase tumor formation in ApcMin/+ mice.

    PubMed

    Kwong, Alan M; Tippin, Brigette L; Materi, Alicia M; Buslon, Virgilio S; French, Samuel W; Lin, Henry J

    2011-01-01

    High doses of niacin (nicotinic acid) used to treat dyslipidemias cause flushing, due to high levels of prostaglandin D(2) (PGD(2)). GPR109A, a G-protein coupled receptor, triggers the flushing in the skin. In addition to boosting PGD(2), niacin binding to GPR109A activates the entire prostanoid cascade. We found that GPR109A occurs throughout the gastrointestinal tract. Mice that alternated between a 1% niacin diet and a control diet had higher urinary prostaglandin E(2) (PGE(2)) metabolite levels when on niacin (2.8-fold increase; 95% confidence interval, 1.8-3.9). PGE(2) promotes tumors in the intestines, whereas PGD(2) may have an opposite effect, on the basis of our report showing that transgenic hematopoietic prostaglandin D synthase suppresses intestinal adenomas in Apc(Min/+) mice. To determine if either tumor growth or tumor suppression prevails, we fed Apc(Min/+) mice a 1% niacin diet and assessed tumor development. A 1% niacin diet did not affect the number of tumors scored histologically in Apc(Min/+) mice at 14 wk (33 mice on niacin, 33 controls). Although niacin stimulates production of various prostaglandins, our results support an interpretation that very high intakes of niacin are safe in relation to intestinal tumors in this model.

  10. Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model

    PubMed Central

    2011-01-01

    The proteomic effects of specific cancer-related mutations have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation by using an isogenic CRC cell culture model in which wildtype APC expression has been ectopically restored. Using LC–MS/MS label free shotgun proteomics, over 5000 proteins were identified in SW480Null (mutant APC) and SW480APC (APC restored). We observed 155 significantly differentially expressed proteins between the two cell lines, with 26 proteins showing opposite expression trends relative to gene expression measurements. Protein changes corresponded to previously characterized features of the APCNull phenotype: loss of cell adhesion proteins, increase in cell cycle regulators, alteration in Wnt signaling related proteins, and redistribution of β-catenin. Increased expression of RNA processing and isoprenoid biosynthetic proteins occurred in SW480Null cells. Therefore, shotgun proteomics reveals proteomic differences associated with a single gene change, including many novel differences that fall outside known target pathways. PMID:22103262

  11. In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC).

    PubMed

    Durai, Malarvizhi; Krueger, Christine; Ye, Zhaohui; Cheng, Linzhao; Mackensen, Andreas; Oelke, Mathias; Schneck, Jonathan P

    2009-02-01

    Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth. PMID:18563409

  12. Environmental impact of APC residues from municipal solid waste incineration: Reuse assessment based on soil and surface water protection criteria

    SciTech Connect

    Quina, Margarida J.; Bordado, Joao C.M.; Quinta-Ferreira, Rosa M.

    2011-09-15

    Highlights: > The Dutch Building Material Decree (BMD) was used to APC residues from MSWI. > BMD is a straightforward tool to calculate expectable loads to the environment of common pollutants. > Chloride load to the environment lead to classification of building material not allowed. > At least a pre-treatment (e.g. washing) is required in order to remove soluble salts. > The stabilization with phosphates or silicates eliminate the problem of heavy metals. - Abstract: Waste management and environmental protection are mandatory requirements of modern society. In our study, air pollution control (APC) residues from municipal solid waste incinerators (MSWI) were considered as a mixture of fly ash and fine particulate solids collected in scrubbers and fabric filters. These are hazardous wastes and require treatment before landfill. Although there are a number of treatment options, it is highly recommended to find practical applications rather than just dump them in landfill sites. In general, for using a construction material, beyond technical specifications also soil and surface water criteria may be used to ensure environmental protection. The Dutch Building Materials Decree (BMD) is a valuable tool in this respect and it was used to investigate which properties do not meet the threshold criteria so that APC residues can be further used as secondary building material. To this end, some scenarios were evaluated by considering release of inorganic species from unmoulded and moulded applications. The main conclusion is that the high amount of soluble salts makes the APC residues a building material prohibited in any of the conditions tested. In case of moulding materials, the limits of heavy metals are complied, and their use in Category 1 would be allowed. However, also in this case, the soluble salts lead to the classification of 'building material not allowed'. The treatments with phosphates or silicates are able to solve the problem of heavy metals, but

  13. Drosophila-Cdh1 (Rap/Fzr) a regulatory subunit of APC/C is required for synaptic morphology, synaptic transmission and locomotion.

    PubMed

    Wise, Alexandria; Schatoff, Emma; Flores, Julian; Hua, Shao-Ying; Ueda, Atsushi; Wu, Chun-Fang; Venkatesh, Tadmiri

    2013-11-01

    The assembly of functional synapses requires the orchestration of the synthesis and degradation of a multitude of proteins. Protein degradation and modification by the conserved ubiquitination pathway has emerged as a key cellular regulatory mechanism during nervous system development and function (Kwabe and Brose, 2011). The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase complex primarily characterized for its role in the regulation of mitosis (Peters, 2002). In recent years, a role for APC/C in nervous system development and function has been rapidly emerging (Stegmuller and Bonni, 2005; Li et al., 2008). In the mammalian central nervous system the activator subunit, APC/C-Cdh1, has been shown to be a regulator of axon growth and dendrite morphogenesis (Konishi et al., 2004). In the Drosophila peripheral nervous system (PNS), APC2, a ligase subunit of the APC/C complex has been shown to regulate synaptic bouton size and activity (van Roessel et al., 2004). To investigate the role of APC/C-Cdh1 at the synapse we examined loss-of-function mutants of Rap/Fzr (Retina aberrant in pattern/Fizzy related), a Drosophila homolog of the mammalian Cdh1 during the development of the larval neuromuscular junction in Drosophila. Our cell biological, ultrastructural, electrophysiological, and behavioral data showed that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function as well as locomotion defects. Data presented here show changes in size and morphology of synaptic boutons, and, muscle tissue organization. Electrophysiological experiments show that loss-of-function mutants exhibit increased frequency of spontaneous miniature synaptic potentials, indicating a higher rate of spontaneous synaptic vesicle fusion events. In addition, larval locomotion and peristaltic movement were also impaired. These findings suggest a role for Drosophila APC/C-Cdh1 mediated ubiquitination in regulating synaptic morphology

  14. Sex-dependent Differences in Intestinal Tumorigenesis Induced in Apc1638N/+ Mice by Exposure to {gamma} Rays

    SciTech Connect

    Trani, Daniela; Moon, Bo-Hyun; Kallakury, Bhaskar; Hartmann, Dan P.; Datta, Kamal; Fornace, Albert J.

    2013-01-01

    Purpose: The purpose of the present study was to assess the effect of 1 and 5 Gy radiation doses and to investigate the interplay of gender and radiation with regard to intestinal tumorigenesis in an adenomatous polyposis coli (APC) mutant mouse model. Methods and Materials: Apc1638N/+ female and male mice were exposed whole body to either 1 Gy or 5 Gy of {gamma} rays and euthanized when most of the treated mice became moribund. Small and large intestines were processed to determine tumor burden, distribution, and grade. Expression of proliferation marker Ki-67 and estrogen receptor (ER)-{alpha} were also assessed by immunohistochemistry. Results: We observed that, with both 1 Gy and 5 Gy of {gamma} rays, females displayed reduced susceptibility to radiation-induced intestinal tumorigenesis compared with males. As for radiation effect on small intestinal tumor progression, although no substantial differences were found in the relative frequency and degree of dysplasia of adenomas in irradiated animals compared with controls, invasive carcinomas were found in 1-Gy- and 5-Gy-irradiated animals. Radiation exposure was also shown to induce an increase in protein levels of proliferation marker Ki-67 and sex-hormone receptor ER-{alpha} in both non tumor mucosa and intestinal tumors from irradiated male mice. Conclusions: We observed important sex-dependent differences in susceptibility to radiation-induced intestinal tumorigenesis in Apc1638N/+ mutants. Furthermore, our data provide evidence that exposure to radiation doses as low as 1 Gy can induce a significant increase in intestinal tumor multiplicity as well as enhance tumor progression in vivo.

  15. Leaching of APC residues from secondary Pb metallurgy using single extraction tests: the mineralogical and the geochemical approach.

    PubMed

    Ettler, Vojtech; Mihaljevic, Martin; Sebek, Ondrej; Strnad, Ladislav

    2005-05-20

    Two air-pollution-control (APC) residues--one from flue gas cooling with alkaline water and one from deionized water cooling--from secondary lead metallurgy were submitted to two different standardized short-term leaching protocols: US EPA toxicity characteristic leaching procedure (TCLP) and static leaching according to Czech/European norm EN 12457-2. The experimental procedure was coupled with detailed mineralogical investigation of the solid material (SEM, XRPD) and speciation-solubility calculations using the PHREEQC-2 geochemical code. Both types of residues were considered as hazardous materials exhibiting substantial leaching of Pb (up to 7130 mg/l) and other inorganic contaminants. However, the APC residue produced by flue gas cooling with alkaline water (sample B) exhibits more favourable leaching and environmental characteristics than that produced by simple deionised water cooling (sample A). At pH < 5, primary caracolite (Na3Pb2(SO4)3Cl) and potassium lead chloride (KCl.2PbCl2) are completely or partially dissolved and transformed to residual anglesite (PbSO4), cotunnite (PbCl2) and laurionite (Pb(OH)Cl). At pH 5-6, anglesite is still the principal residual product, whereas at pH > 6, phosgenite (PbCl2.PbCO3) became the dominant secondary phase. The results are consistent with the mineralogical and geochemical studies focused on acidic forest soils highly polluted by smelter emissions, where anglesite, as a unique Pb-bearing phase, has been detected. From the technological point of view, the mixing of APC residue with alkaline water, followed by an increase in the suspension pH and equilibration with atmospheric CO2, may be used to ensure the precipitation of less soluble Pb carbonates, which are more easily recycled in the Pb recovery process in the metallurgical plant.

  16. A Cdh1-APC/FMRP Ubiquitin Signaling Link Drives mGluR-Dependent Synaptic Plasticity in the Mammalian Brain

    PubMed Central

    Huang, Ju; Ikeuchi, Yoshiho; Malumbres, Marcos; Bonni, Azad

    2015-01-01

    SUMMARY Deregulation of synaptic plasticity may contribute to the pathogenesis of developmental cognitive disorders. In particular, exaggerated mGluR-dependent LTD is featured in fragile X syndrome, but the mechanisms that regulate mGluR-LTD remain incompletely understood. We report that conditional knockout of Cdh1, the key regulatory subunit of the ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC), profoundly impairs mGluR-LTD in the hippocampus. Mechanistically, we find that Cdh1-APC operates in the cytoplasm to drive mGluR-LTD. We also identify the fragile X syndrome protein FMRP as a substrate of Cdh1-APC. Endogenous Cdh1-APC forms a complex with endogenous FMRP, and knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. Knockout of FMRP suppresses, and expression of an FMRP mutant protein that fails to interact with Cdh1 phenocopies, the Cdh1 knockout phenotype of impaired mGluR-LTD. These findings define Cdh1-APC and FMRP as components of a novel ubiquitin-signaling pathway that regulates mGluR-LTD in the brain. PMID:25913861

  17. High order overlay modeling and APC simulation with Zernike-Legendre polynomials

    NASA Astrophysics Data System (ADS)

    Ju, JawWuk; Kim, MinGyu; Lee, JuHan; Sherwin, Stuart; Hoo, George; Choi, DongSub; Lee, Dohwa; Jeon, Sanghuck; Lee, Kangsan; Tien, David; Pierson, Bill; Robinson, John C.; Levy, Ady; Smith, Mark D.

    2015-03-01

    Feedback control of overlay errors to the scanner is a well-established technique in semiconductor manufacturing [1]. Typically, overlay errors are measured, and then modeled by least-squares fitting to an overlay model. Overlay models are typically Cartesian polynomial functions of position within the wafer (Xw, Yw), and of position within the field (Xf, Yf). The coefficients from the data fit can then be fed back to the scanner to reduce overlay errors in future wafer exposures, usually via a historically weighted moving average. In this study, rather than using the standard Cartesian formulation, we examine overlay models using Zernike polynomials to represent the wafer-level terms, and Legendre polynomials to represent the field-level terms. Zernike and Legendre polynomials can be selected to have the same fitting capability as standard polynomials (e.g., second order in X and Y, or third order in X and Y). However, Zernike polynomials have the additional property of being orthogonal over the unit disk, which makes them appropriate for the wafer-level model, and Legendre polynomials are orthogonal over the unit square, which makes them appropriate for the field-level model. We show several benefits of Zernike/Legendre-based models in this investigation in an Advanced Process Control (APC) simulation using highly-sampled fab data. First, the orthogonality property leads to less interaction between the terms, which makes the lot-to-lot variation in the fitted coefficients smaller than when standard polynomials are used. Second, the fitting process itself is less coupled - fitting to a lower-order model, and then fitting the residuals to a higher order model gives very similar results as fitting all of the terms at once. This property makes fitting techniques such as dual pass or cascading [2] unnecessary, and greatly simplifies the options available for the model recipe. The Zernike/Legendre basis gives overlay performance (mean plus 3 sigma of the residuals

  18. MASTL(Greatwall) regulates DNA damage responses by coordinating mitotic entry after checkpoint recovery and APC/C activation

    PubMed Central

    Wong, Po Yee; Ma, Hoi Tang; Lee, Hyun-jung; Poon, Randy Y. C.

    2016-01-01

    The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation. PMID:26923777

  19. Timely Degradation of Wip1 Phosphatase by APC/C Activator Protein Cdh1 is Necessary for Normal Mitotic Progression.

    PubMed

    Jeong, Ho-Chang; Gil, Na-Yeon; Lee, Ho-Soo; Cho, Seung-Ju; Kim, Kyungtae; Chun, Kwang-Hoon; Cho, Hyeseong; Cha, Hyuk-Jin

    2015-08-01

    Wip1 belongs to the protein phosphatase C (PP2C) family, of which expression is up-regulated by a number of external stresses, and serves as a stress modulator in normal physiological conditions. When overexpressed, premature dephosphorylation of stress-mediators by Wip1 results in abrogation of tumor surveillance, thus Wip1 acts as an oncogene. Previously, the functional regulation of Wip1 in cell-cycle progression by counteracting cellular G1 and G2/M checkpoint activity in response to DNA damage was reported. However, other than in stress conditions, the function and regulatory mechanism of Wip1 has not been fully determined. Herein, we demonstrated that protein regulation of Wip1 occurs in a cell cycle-dependent manner, which is directly governed by APC/C(Cdh1) at the end of mitosis. In particular, we also showed evidence that Wip1 phosphatase activity is closely associated with its own protein stability, suggesting that reduced phosphatase activity of Wip1 during mitosis could trigger its degradation. Furthermore, to verify the physiological role of its phosphatase activity during mitosis, we established doxycycline-inducible cell models, including a Wip1 wild type (WT) and phosphatase dead mutant (Wip1 DA). When ectopically expressing Wip1 WT, we observed a delay in the transition from metaphase to anaphase. In conclusion, these studies show that mitotic degradation of Wip1 by APC/C(Cdh1) is important for normal mitotic progression.

  20. PP2A delays APC/C-dependent degradation of separase-associated but not free securin

    PubMed Central

    Hellmuth, Susanne; Böttger, Franziska; Pan, Cuiping; Mann, Matthias; Stemmann, Olaf

    2014-01-01

    The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2- or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpoint-mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase. PMID:24781523

  1. Reducing overlay sampling for APC-based correction per exposure by replacing measured data with computational prediction

    NASA Astrophysics Data System (ADS)

    Noyes, Ben F.; Mokaberi, Babak; Oh, Jong Hun; Kim, Hyun Sik; Sung, Jun Ha; Kea, Marc

    2016-03-01

    One of the keys to successful mass production of sub-20nm nodes in the semiconductor industry is the development of an overlay correction strategy that can meet specifications, reduce the number of layers that require dedicated chuck overlay, and minimize measurement time. Three important aspects of this strategy are: correction per exposure (CPE), integrated metrology (IM), and the prioritization of automated correction over manual subrecipes. The first and third aspects are accomplished through an APC system that uses measurements from production lots to generate CPE corrections that are dynamically applied to future lots. The drawback of this method is that production overlay sampling must be extremely high in order to provide the system with enough data to generate CPE. That drawback makes IM particularly difficult because of the throughput impact that can be created on expensive bottleneck photolithography process tools. The goal is to realize the cycle time and feedback benefits of IM coupled with the enhanced overlay correction capability of automated CPE without impacting process tool throughput. This paper will discuss the development of a system that sends measured data with reduced sampling via an optimized layout to the exposure tool's computational modelling platform to predict and create "upsampled" overlay data in a customizable output layout that is compatible with the fab user CPE APC system. The result is dynamic CPE without the burden of extensive measurement time, which leads to increased utilization of IM.

  2. Expression of APC protein during tongue malignant transformation in galectin-3-deficient mice challenged by the carcinogen 4-nitroquniline-n-oxide

    PubMed Central

    de Souza, Marcus Vinicius Rodrigues; Servato, João Paulo Silva; Loyola, Adriano Mota; Cardoso, Sérgio Vitorino; Chammas, Roger; Liu, Fu-Tong; Silva, Marcelo José Barbosa; de Faria, Paulo Rogério

    2014-01-01

    Galectin-3 (Gal3) has been implicated in the development of different tumors because of its involvement in the Wnt signaling pathway by promoting beta-catenin translocation into the nucleus. The APC protein, a negative regulator of this pathway, has been strongly implicated in the development of colon cancer, but still has an undetermined role in the formation of oral cancer. Therefore, this study aimed to evaluate the relationship between Gal3, the Wnt signaling pathway, and APC expression in dysplasias and carcinomas developed experimentally in mice. Sixty galectin-3-deficient (Gal3-/-) and 60 wild-type (Gal3+/+) mice were early employed to be treated with the carcinogen 4NQO for 16 weeks and killed at either week 16 or week 32. Tongues were removed, processed and embedded in paraffin blocks. Sections 5 μm thick were made, and then stained by H&E to establish the diagnosis of dysplasia and carcinoma. Sections of 2 μm thickness were made to detect APC expression in these lesions by immunohistochemistry. Oral carcinogenesis occurred in both groups of mice, but no statistical difference was reached. APC expression was exclusively seen in the cytoplasm of all lesions studied. In the intragroup analysis, the majority of dysplasias and carcinomas exhibiting higher APC immunoreactivity was observed in Gal3-/- mice compared to Gal3+/+ mice, but no significant difference was found. However, a statistical difference was only observed between dysplastic lesions from two mice. Our results showed that neither the absence of Gal3 nor the APC protein appears to play a role in malignant transformation of the tongue. PMID:25031746

  3. ACKR4 on Stromal Cells Scavenges CCL19 To Enable CCR7-Dependent Trafficking of APCs from Inflamed Skin to Lymph Nodes.

    PubMed

    Bryce, Steven A; Wilson, Ruairi A M; Tiplady, Eleanor M; Asquith, Darren L; Bromley, Shannon K; Luster, Andrew D; Graham, Gerard J; Nibbs, Robert J B

    2016-04-15

    Dermal dendritic cells and epidermal Langerhans cells are APCs that migrate from skin to draining lymph nodes (LN) to drive peripheral tolerance and adaptive immunity. Their migration requires the chemokine receptor CCR7, which directs egress from the skin via dermal lymphatic vessels and extravasation into the LN parenchyma from lymph in the subcapsular sinus. CCR7 is activated by two chemokines: CCL19 and CCL21. CCL21 alone is sufficient for the migration of APCs from skin to LN. CCL19 and CCL21 also bind atypical chemokine receptor (ACKR) 4. ACKR4-mediated CCL21 scavenging by lymphatic endothelial cells lining the subcapsular sinus ceiling stabilizes interfollicular CCL21 gradients that direct lymph-borne CCR7(+)APCs into the parenchyma of mouse LN. In this study, we show that ACKR4 also aids APC egress from mouse skin under steady-state and inflammatory conditions. ACKR4 plays a particularly prominent role during cutaneous inflammation when it facilitates Langerhans cell egress from skin and enables the accumulation of dermal dendritic cells in skin-draining LN. Stromal cells in mouse skin, predominantly keratinocytes and a subset of dermal lymphatic endothelial cells, express ACKR4 and are capable of ACKR4-dependent chemokine scavenging in situ. ACKR4-mediated scavenging of dermal-derived CCL19, rather than CCL21, is critical during inflammation, because the aberrant trafficking of skin-derived APCs inAckr4-deficient mice is completely rescued by genetic deletion ofCcl19 Thus, ACKR4 on stromal cells aids the egress of APCs from mouse skin, and, during inflammation, facilitates CCR7-dependent cell trafficking by scavenging CCL19.

  4. Role of RIS/APC for manufacturing RFG/LSD. [Refinery Information Systems/Advanced Process Control, ReFormulated Gasoline/Low Sulfur Diesels

    SciTech Connect

    Latour, P.R. )

    1994-01-01

    Revolutionary changes in quality specifications (number, complexity, uncertainty, economic sensitivity) for reformulated gasolines (RFG) and low-sulfur diesels (LSD) are being addressed by powerful, new, computer-integrated manufacturing technology for Refinery Information Systems and Advanced Process Control (RIS/APC). This paper shows how the five active RIS/APC functions: performance measurement, optimization, scheduling, control and integration are used to manufacture new, clean fuels competitively. With current industry spending for this field averaging 2 to 3 cents/bbl crude, many refineries can capture 50 to 100 cents/bbl if the technology is properly employed and sustained throughout refining operations, organizations, and businesses.

  5. Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human activated protein C (APC). Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops.

    PubMed

    Schmidt, Amy E; Padmanabhan, Kaillathe; Underwood, Matthew C; Bode, Wolfram; Mather, Timothy; Bajaj, S Paul

    2002-08-01

    The serine protease domain of activated protein C (APC) contains a Na+ and a Ca2+ site. However, the number and identity of the APC residues that coordinate to Na+ is not precisely known. Further, the functional link between the Na+ and the Ca2+ site is insufficiently defined, and their linkage to the substrate S1 site has not been studied. Here, we systematically investigate the functional significance of these two cation sites and their thermodynamic links to the S1 site. Kinetic data reveal that Na+ binds to the substrate-occupied APC with K(d) values of approximately 24 mm in the absence and approximately 6 mm in the presence of Ca2+. Sodium-occupied APC has approximately 100-fold increased catalytic efficiency ( approximately 4-fold decrease in K(m) and approximately 25-fold increase in k(cat)) in hydrolyzing S-2288 (H-d-Ile-Pro-Arg-p-nitroanilide) and Ca2+ further increases this k(cat) slightly ( approximately 1.2-fold). Ca2+ binds to the protease domain of APC with K(d) values of approximately 438 microm in the absence and approximately 105 microm in the presence of Na+. Ca2+ binding to the protease domain of APC does not affect K(m) but increases the k(cat) approximately 10-fold, and Na+ further increases this k(cat) approximately 3-fold and decreases the K(m) value approximately 3.7-fold. In agreement with the K(m) data, sodium-occupied APC has approximately 4-fold increased affinity in binding to p-aminobenzamidine (S1 probe). Crystallographically, the Ca2+ site in APC is similar to that in trypsin, and the Na+ site is similar to that in factor Xa but not thrombin. Collectively, the Na+ site is thermodynamically linked to the S1 site as well as to the protease domain Ca2+ site, whereas the Ca2+ site is only linked to the Na+ site. The significance of these findings is that under physiologic conditions, most of the APC will exist in Na2+-APC-Ca2+ form, which has 110-fold increased proteolytic activity. PMID:12029084

  6. Genetic Anthropology of the Colorectal Cancer–Susceptibility Allele APC I1307K: Evidence of Genetic Drift within the Ashkenazim

    PubMed Central

    Niell, Bethany L.; Long, Jeffrey C.; Rennert, Gad; Gruber, Stephen B.

    2003-01-01

    The adenomatous polyposis coli (APC) I1307K allele is found in 6% of the Ashkenazi Jewish population and in 1%–2% of Sephardi Jews; it confers a relative risk of 1.5–2.0 for colorectal cancer (CRC) on all carriers. Within the Ashkenazim, the existence of numerous high-prevalence mutations, including I1307K, has sparked controversy over whether genetic drift or selection is the underlying cause. For the present population-based case-control study of CRC in Israel, we tested whether selection has operated at I1307K. We also estimated the age of the I1307K allele, to understand its origin in the context of the Jewish diasporas and subsequent founder events. We genotyped 83 matched pairs, in which one or both members of the pair carried I1307K, at three microsatellites and two SNPs. Haplotypes were statistically constructed using PHASE software. Single-marker age estimates for I1307K were calculated using the approach described by Risch et al. A common progenitor haplotype spanned across APC I1307K from the centromeric marker D5S135 to the telomeric marker D5S346 and was observed in individuals of Ashkenazi, Sephardi, and Arab descent. The ancestor of modern I1307K alleles existed 87.9–118 generations ago (∼2,200–2,950 years ago). This age estimate indicates that I1307K existed at about the time of the beginning of the Jewish diaspora, explaining its presence in non-Ashkenazi populations. Our data do not indicate that selection operated at I1307K (D5S346, P=.114; D5S135, P=.373), providing compelling evidence that the high frequency of disease-susceptibility alleles in the Ashkenazim is due to genetic drift, not selection. This research underscores the importance of the migratory patterns of ancestral populations in the ethnic and geographic distribution of APC I1307K. PMID:14624392

  7. The influence of pH on the leaching behaviour of inorganic components from municipal solid waste APC residues

    SciTech Connect

    Quina, Margarida J. Bordado, Joao C.M.; Quinta-Ferreira, Rosa M.

    2009-09-15

    The influence of pH on the leaching behaviour of air pollution control (APC) residues produced in municipal solid waste incineration (MSWI) is addressed in this study. The residue is considered hazardous waste, and in accordance with their chemical properties, the leaching of contaminants into the environment is the main concern. Several leaching tests can be used for research studies or regulatory purposes, where a wide variety of conditions may be tested. Our work deals mainly with the leaching behaviour of toxic heavy metals (Pb, Cd, Zn, Cr, Ni, Cu) and inorganics associated with soluble salts (Na, K, Ca, Cl). The main goal is to obtain an overview of the leachability of APC residues produced in a Portuguese MSWI process. Among the different variables that may have influence on the leaching behaviour, pH of the leachant solution is the most important one, and was evaluated through pH static tests. The acid neutralization capacity (ANC) of the residue was also determined, which is in the range of 6.2-6.8 meq g{sup -1} (for pH = 7) and 10.1-11.6 meq g{sup -1} (for pH = 4). The analysis of the leaching behaviour is particularly important when the leaching is solubility controlled. The amphoteric behaviour of some elements was observed, namely for Pb and Zn, which is characterized through high solubilization at low and high pH and moderate or low solubility at neutral or moderate high pH. The solubility curves for Pb, Cd, Zn, Cr, Ni and Cu as a function of pH were obtained, which are very useful for predicting the leaching behaviour in different scenarios. The solubility of K and Na reveals to be nearly independent of the solution pH and the released amount is mainly availability-controlled. Moreover, the pH static test showed that Cl{sup -} is the most pH-independent species. The APC residue turns out to be a hazardous waste because of the high leaching of lead and chloride. On the other hand, leaching of elements like cadmium, nickel and copper is limited by the

  8. The influence of pH on the leaching behaviour of inorganic components from municipal solid waste APC residues.

    PubMed

    Quina, Margarida J; Bordado, João C M; Quinta-Ferreira, Rosa M

    2009-09-01

    The influence of pH on the leaching behaviour of air pollution control (APC) residues produced in municipal solid waste incineration (MSWI) is addressed in this study. The residue is considered hazardous waste, and in accordance with their chemical properties, the leaching of contaminants into the environment is the main concern. Several leaching tests can be used for research studies or regulatory purposes, where a wide variety of conditions may be tested. Our work deals mainly with the leaching behaviour of toxic heavy metals (Pb, Cd, Zn, Cr, Ni, Cu) and inorganics associated with soluble salts (Na, K, Ca, Cl). The main goal is to obtain an overview of the leachability of APC residues produced in a Portuguese MSWI process. Among the different variables that may have influence on the leaching behaviour, pH of the leachant solution is the most important one, and was evaluated through pH static tests. The acid neutralization capacity (ANC) of the residue was also determined, which is in the range of 6.2-6.8 meq g(-1) (for pH=7) and 10.1-11.6 meq g(-1) (for pH=4). The analysis of the leaching behaviour is particularly important when the leaching is solubility controlled. The amphoteric behaviour of some elements was observed, namely for Pb and Zn, which is characterized through high solubilization at low and high pH and moderate or low solubility at neutral or moderate high pH. The solubility curves for Pb, Cd, Zn, Cr, Ni and Cu as a function of pH were obtained, which are very useful for predicting the leaching behaviour in different scenarios. The solubility of K and Na reveals to be nearly independent of the solution pH and the released amount is mainly availability-controlled. Moreover, the pH static test showed that Cl(-) is the most pH-independent species. The APC residue turns out to be a hazardous waste because of the high leaching of lead and chloride. On the other hand, leaching of elements like cadmium, nickel and copper is limited by the high pH of the

  9. Brain energy metabolism in glutamate-receptor activation and excitotoxicity: role for APC/C-Cdh1 in the balance glycolysis/pentose phosphate pathway.

    PubMed

    Rodriguez-Rodriguez, Patricia; Almeida, Angeles; Bolaños, Juan P

    2013-04-01

    Recent advances in the field of brain energy metabolism strongly suggest that glutamate receptor-mediated neurotransmission is coupled with molecular signals that switch-on glucose utilization pathways to meet the high energetic requirements of neurons. Failure to adequately coordinate energy supply for neurotransmission ultimately results in a positive amplifying loop of receptor over-activation leading to neuronal death, a process known as excitotoxicity. In this review, we revisited current concepts in excitotoxic mechanisms, their involvement in energy substrate utilization, and the signaling pathways that coordinate both processes. In particular, we have focused on the novel role played by the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1, in cell metabolism. Our laboratory identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) -a key glycolytic-promoting enzyme- as an APC/C-Cdh1 substrate. Interestingly, APC/C-Cdh1 activity is inhibited by over-activation of glutamate receptors through a Ca(2+)-mediated mechanism. Furthermore, by inhibiting APC/C-Cdh1 activity, glutamate-receptors activation promotes PFKFB3 stabilization, leading to increased glycolysis and decreased pentose-phosphate pathway activity. This causes a loss in neuronal ability to regenerate glutathione, triggering oxidative stress and delayed excitotoxicity. Further investigation is critical to identify novel molecules responsible for the coupling of energy metabolism with glutamatergic neurotransmission and excitotoxicity, as well as to help developing new therapeutic strategies against neurodegeneration.

  10. A Panel of Artificial APCs Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Dominant and Subdominant Viral Epitopes for Adoptive Therapy1

    PubMed Central

    Hasan, Aisha N.; Kollen, Wouter J.; Trivedi, Deepa; Selvakumar, Annamalai; Dupont, Bo; Sadelain, Michel; O'Reilly, Richard J.

    2009-01-01

    Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous antigen-presenting cells (APCs) are often limited in supply. Here, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1 and LFA-3 that each stably express one of a series of 6 common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mers spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65 specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8+, IFNγ+ and exhibited HLA-restricted CMVpp65 specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in man. Sensitization with AAPCs also permitted expansion of IFNγ+ cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA disparate donors. PMID:19635907

  11. The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/CCdh1 ubiquitin ligase complex

    PubMed Central

    Bott, Laura C.; Salomons, Florian A.; Maric, Dragan; Liu, Yuhong; Merry, Diane; Fischbeck, Kenneth H.; Dantuma, Nico P.

    2016-01-01

    Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA. PMID:27312068

  12. A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts.

    PubMed

    Fuertes Marraco, Silvia A; Baumgaertner, Petra; Legat, Amandine; Rufer, Nathalie; Speiser, Daniel E

    2012-11-30

    Artificial antigen-presenting cells (aAPC) are widely used for both clinical and basic research applications, as cell-based or bead-based scaffolds, combining immune synapse components of interest. Adequate and controlled preparation of aAPCs is crucial for subsequent immunoassays. We reveal that certain proteins such as activatory anti-CD3 antibody can be out-competed by other proteins (e.g. inhibitory receptor ligands such as PDL1:Fc) during the coating of aAPC beads, under the usually performed coating procedures. This may be misleading, as we found that decreased CD8 T cell activity was not due to inhibitory receptor triggering but rather because of unexpectedly low anti-CD3 antibody density on the beads upon co-incubation with inhibitory receptor ligands. We propose an optimized protocol, and emphasize the need to quality-control the coating of proteins on aAPC beads prior to their use in immunoassays. PMID:22867742

  13. The SnRK2-APC/CTE regulatory module mediates the antagonistic action of gibberellic acid and abscisic acid pathways

    PubMed Central

    Lin, Qibing; Wu, Fuqing; Sheng, Peike; Zhang, Zhe; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Cheng, Zhijun; Wang, Jie; Wang, Haiyang; Wan, Jianmin

    2015-01-01

    Abscisic acid (ABA) and gibberellic acid (GA) antagonistically regulate many developmental processes and responses to biotic or abiotic stresses in higher plants. However, the molecular mechanism underlying this antagonism is still poorly understood. Here, we show that loss-of-function mutation in rice Tiller Enhancer (TE), an activator of the APC/CTE complex, causes hypersensitivity and hyposensitivity to ABA and GA, respectively. We find that TE physically interacts with ABA receptor OsPYL/RCARs and promotes their degradation by the proteasome. Genetic analysis also shows OsPYL/RCARs act downstream of TE in mediating ABA responses. Conversely, ABA inhibits APC/CTE activity by phosphorylating TE through activating the SNF1-related protein kinases (SnRK2s), which may interrupt the interaction between TE and OsPYL/RCARs and subsequently stabilize OsPYL/RCARs. In contrast, GA can reduce the level of SnRK2s and may promote APC/CTE-mediated degradation of OsPYL/RCARs. Thus, we propose that the SnRK2-APC/CTE regulatory module represents a regulatory hub underlying the antagonistic action of GA and ABA in plants. PMID:26272249

  14. Aβ Induces Excitotoxicity Mediated by APC/C-Cdh1 Depletion That Can Be Prevented by Glutaminase Inhibition Promoting Neuronal Survival

    PubMed Central

    Fuchsberger, T.; Martínez-Bellver, S.; Giraldo, E.; Teruel-Martí, V.; Lloret, A.; Viña, J.

    2016-01-01

    The E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated by the fizzy-related protein homolog/CDC20-like protein 1 (cdh1) in post-mitotic neurons. Growing evidence suggests that dysregulation of APC/C-Cdh1 is involved in neurodegenerative diseases. Here we show in neurons that oligomers of amyloid beta (Aβ), a peptide related to Alzheimer’s disease, cause proteasome-dependent degradation of cdh1. This leads to a subsequent increase in glutaminase (a degradation target of APC/C-Cdh1), which causes an elevation of glutamate levels and further intraneuronal Ca2+ dysregulation, resulting in neuronal apoptosis. Glutaminase inhibition prevents glutamate excitotoxicity and apoptosis in Aβ treated neurons. Furthermore, glutamate also decreases cdh1 and leads to accumulation of glutaminase, suggesting that there may be a positive feedback loop of cdh1 inactivation. We confirmed the main findings in vivo using microinjection of either Aβ or glutamate in the CA1 region of the rat hippocampus. We show here for the first time in vivo that both Aβ and glutamate cause nuclear exclusion of cdh1 and an increase in glutaminase. These results show that maintaining normal APC/C-Cdh1 activity may be a useful target in Alzheimer’s disease treatment. PMID:27514492

  15. Aβ Induces Excitotoxicity Mediated by APC/C-Cdh1 Depletion That Can Be Prevented by Glutaminase Inhibition Promoting Neuronal Survival.

    PubMed

    Fuchsberger, T; Martínez-Bellver, S; Giraldo, E; Teruel-Martí, V; Lloret, A; Viña, J

    2016-08-12

    The E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated by the fizzy-related protein homolog/CDC20-like protein 1 (cdh1) in post-mitotic neurons. Growing evidence suggests that dysregulation of APC/C-Cdh1 is involved in neurodegenerative diseases. Here we show in neurons that oligomers of amyloid beta (Aβ), a peptide related to Alzheimer's disease, cause proteasome-dependent degradation of cdh1. This leads to a subsequent increase in glutaminase (a degradation target of APC/C-Cdh1), which causes an elevation of glutamate levels and further intraneuronal Ca(2+) dysregulation, resulting in neuronal apoptosis. Glutaminase inhibition prevents glutamate excitotoxicity and apoptosis in Aβ treated neurons. Furthermore, glutamate also decreases cdh1 and leads to accumulation of glutaminase, suggesting that there may be a positive feedback loop of cdh1 inactivation. We confirmed the main findings in vivo using microinjection of either Aβ or glutamate in the CA1 region of the rat hippocampus. We show here for the first time in vivo that both Aβ and glutamate cause nuclear exclusion of cdh1 and an increase in glutaminase. These results show that maintaining normal APC/C-Cdh1 activity may be a useful target in Alzheimer's disease treatment.

  16. The polyglutamine-expanded androgen receptor responsible for spinal and bulbar muscular atrophy inhibits the APC/C(Cdh1) ubiquitin ligase complex.

    PubMed

    Bott, Laura C; Salomons, Florian A; Maric, Dragan; Liu, Yuhong; Merry, Diane; Fischbeck, Kenneth H; Dantuma, Nico P

    2016-06-17

    Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/C(Cdh1) complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/C(Cdh1) by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/C(Cdh1) and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA.

  17. Metals accumulations during thermal processing of sewage sludge - characterization of bottom ash and air pollution control (APC) residues

    NASA Astrophysics Data System (ADS)

    Kasina, Monika; Kowalski, Piotr R.; Michalik, Marek

    2016-04-01

    Due to increasing mass of sewage sludge, problems in its management have appeared. Over years sewage sludge was landfilled, however due to EU directives concerning environmental issues this option is no longer possible. This type of material is considered hazardous due to highly concentrated metals and harmful elements, toxic organic substances and biological components (e.g. parasites, microbes). Currently in Europe, incineration is considered to be the most reasonable method for sewage sludge treatment. As a result of sludge incineration significant amount of energy is recovered due to high calorific value of sewage sludge but bottom ash and APC residues are being produced. In this study we show the preliminary results of chemical and mineral analyses of both bottom ash and APC residues produced in fluidized bed boiler in sewage sludge incineration plant in Poland, with a special emphasis on metals which, as a part of incombustible fraction can accumulate in the residual materials after thermal processing. The bottom ash was a SiO2-P2O5-Fe2O3-CaO-Al2O3 dominated material. Main mineral phases identified in X-ray diffraction patterns were: quartz, feldspar, hematite, and phosphates (apatite and scholzite). The bottom ash was characterized by high content of Zn - 4472 mg kg‑1, Cu - 665.5 mg kg‑1, Pb - 138 mg kg‑1, Ni - 119.5 mg kg‑1, and interestingly high content of Au - 0.858 mg kg‑1 The APC residues composition was dominated by soluble phases which represent more than 90% of the material. The XRD patterns indicated thenardite, halite, anhydrite, calcite and apatite as main mineral phases. The removal of soluble phases by dissolution in deionised water caused a significant mass reduction (ca. 3% of material remained on the filters). Calcite, apatite and quartz were main identified phases. The content of metals in insoluble material is relatively high: Zn - 6326 mg kg‑1, Pb - 514.3 mg kg‑1, Cu - 476.6 mg kg‑1, Ni - 43.3 mg kg‑1. The content of Cd

  18. Metals accumulations during thermal processing of sewage sludge - characterization of bottom ash and air pollution control (APC) residues

    NASA Astrophysics Data System (ADS)

    Kasina, Monika; Kowalski, Piotr R.; Michalik, Marek

    2016-04-01

    Due to increasing mass of sewage sludge, problems in its management have appeared. Over years sewage sludge was landfilled, however due to EU directives concerning environmental issues this option is no longer possible. This type of material is considered hazardous due to highly concentrated metals and harmful elements, toxic organic substances and biological components (e.g. parasites, microbes). Currently in Europe, incineration is considered to be the most reasonable method for sewage sludge treatment. As a result of sludge incineration significant amount of energy is recovered due to high calorific value of sewage sludge but bottom ash and APC residues are being produced. In this study we show the preliminary results of chemical and mineral analyses of both bottom ash and APC residues produced in fluidized bed boiler in sewage sludge incineration plant in Poland, with a special emphasis on metals which, as a part of incombustible fraction can accumulate in the residual materials after thermal processing. The bottom ash was a SiO2-P2O5-Fe2O3-CaO-Al2O3 dominated material. Main mineral phases identified in X-ray diffraction patterns were: quartz, feldspar, hematite, and phosphates (apatite and scholzite). The bottom ash was characterized by high content of Zn - 4472 mg kg-1, Cu - 665.5 mg kg-1, Pb - 138 mg kg-1, Ni - 119.5 mg kg-1, and interestingly high content of Au - 0.858 mg kg-1 The APC residues composition was dominated by soluble phases which represent more than 90% of the material. The XRD patterns indicated thenardite, halite, anhydrite, calcite and apatite as main mineral phases. The removal of soluble phases by dissolution in deionised water caused a significant mass reduction (ca. 3% of material remained on the filters). Calcite, apatite and quartz were main identified phases. The content of metals in insoluble material is relatively high: Zn - 6326 mg kg-1, Pb - 514.3 mg kg-1, Cu - 476.6 mg kg-1, Ni - 43.3 mg kg-1. The content of Cd, As, Se and Hg was

  19. The viral enterprises in autoimmunity: conversion of target cells into de novo APCs is the presage to autoimmunity.

    PubMed

    Temajo, Norbert O; Howard, Neville

    2012-07-01

    An autoimmune disease (AD) occurs in a situation where an individual's protective immune system attacks and destroys the individual's own tissues and organ(s), causing a recognizable syndrome(s). The viruses feature in the triggering of autoimmune diseases in genetically primed individuals through generating a viral group of regulatory immediate early proteins (IE). The IE indulges in promiscuous regulations of the viral replications as well as of host intracellular proteins. But there are consequences in the IE controlling host cell protein regulations, which we suggest as: the IE titration of the transactivator protein, autoimmune regulator (AIRE), which causes abolition of central tolerance; and the IE titration of the repressor protein, FOXP3, which results in the breach of peripheral tolerance. Titrations of AIRE and FOXP3 allow the escape of autoreactive T cells into the (peripheral) circulation where they can reach and zero in on self-tissues. The AD-predisposing MHC-II-DR-DQ haplotypes probably play a crucial role in the shaping of the T cell repertoire intrathymically for the survival of budding autoreactive T cell receptors (TCRs). Finally, we suggest there is IE titration of the repressors, the histone deacetylases (HDACs), in target organ cells which then consequentially express de novo MHC-II molecules and become de novo non-professional antigen-presenting cells (APCs), able to present viral peptides to cognate TCRs, thereby enrolling themselves for apoptotic death: a destiny of all APCs in immune responses, in general. Extensive apoptotic destruction of organ cells leads to an autoimmune syndrome(s).

  20. C57BL/KsJ-db/db-ApcMin/+ Mice Exhibit an Increased Incidence of Intestinal Neoplasms

    PubMed Central

    Hata, Kazuya; Kubota, Masaya; Shimizu, Masahito; Moriwaki, Hisataka; Kuno, Toshiya; Tanaka, Takuji; Hara, Akira; Hirose, Yoshinobu

    2011-01-01

    The numbers of obese people and diabetic patients are ever increasing. Obesity and diabetes are high-risk conditions for chronic diseases, including certain types of cancer, such as colorectal cancer (CRC). The aim of this study was to develop a novel animal model in order to clarify the pathobiology of CRC development in obese and diabetic patients. We developed an animal model of obesity and colorectal cancer by breeding the C57BL/KsJ-db/db (db/db) mouse, an animal model of obesity and type II diabetes, and the C57BL/6J-ApcMin/+ (Min/+) mouse, a model of familial adenomatous polyposis. At 15 weeks of age, the N9 backcross generation of C57BL/KsJ-db/db-ApcMin/+ (db/db-Min/+) mice developed an increased incidence and multiplicity of adenomas in the intestinal tract when compared to the db/m-Min/+ and m/m-Min/+ mice. Blood biochemical profile showed significant increases in insulin (8.3-fold to 11.7-fold), cholesterol (1.2-fold to 1.7-fold), and triglyceride (1.2-fold to 1.3-fold) in the db/db-Min/+ mice, when compared to those of the db/m-Min/+ and m/m-Min/+ mice. Increases (1.4-fold to 2.6-fold) in RNA levels of insulin-like growth factor (IGF)-1, IRF-1R, and IGF-2 were also observed in the db/db- Min/+ mice. These results suggested that the IGFs, as well as hyperlipidemia and hyperinsulinemia, promoted adenoma formation in the db/db-Min/+ mice. Our results thus suggested that the db/db-Min/+ mice should be invaluable for studies on the pathogenesis of CRC in obese and diabetes patients and the therapy and prevention of CRC in these patients. PMID:22174655

  1. Chemoprevention of intestinal polyps in ApcMin/+ mice fed with western or balanced diets by drinking annurca apple polyphenol extract.

    PubMed

    Fini, Lucia; Piazzi, Giulia; Daoud, Yahya; Selgrad, Michael; Maegawa, Shinji; Garcia, Melissa; Fogliano, Vincenzo; Romano, Marco; Graziani, Giulia; Vitaglione, Paola; Carmack, Susanne W; Gasbarrini, Antonio; Genta, Robert M; Issa, Jean-Pierre; Boland, C Richard; Ricciardiello, Luigi

    2011-06-01

    The Western diet (WD) is associated with a higher incidence of colorectal cancer (CRC) than the Mediterranean diet. Polyphenols extracted from Annurca apple showed chemopreventive properties in CRC cells. A multifactorial, four-arm study by using wild-type (wt) and Apc(Min/+) mice was carried out to evaluate the effect on polyp number and growth of APE treatment (60 μmol/L) ad libitum in drinking water combined with a WD or a balanced diet (BD) for 12 weeks. Compared with APE treatment, we found a significant drop in body weight (P < 0.0001), severe rectal bleeding (P = 0.0076), presence of extraintestinal tumors, and poorer activity status (P = 0.0034) in water-drinking Apc(Min/+) mice, more remarkably in the WD arm. In the BD and WD groups, APE reduced polyp number (35% and 42%, respectively, P < 0.001) and growth (60% and 52%, respectively, P < 0.0001) in both colon and small intestine. Increased antioxidant activity was found in wt animals fed both diets and in Apc(Min/+) mice fed WD and drinking APE. Reduced lipid peroxidation was found in Apc(Min/+) mice drinking APE fed both diets and in wt mice fed WD. In normal mucosa, mice drinking water had lower global levels of DNA methylation than mice drinking APE. APE treatment is highly effective in reducing polyps in Apc(Min/+) mice and supports the concept that a mixture of phytochemicals, as they are naturally present in foods, represent a plausible chemopreventive agent for CRC, particularly in populations at high risk for colorectal neoplasia.

  2. ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002.

    PubMed

    Dong, Chunxia; Tang, Aihui; Zhao, Jindong; Mullineaux, Conrad W; Shen, Gaozhong; Bryant, Donald A

    2009-09-01

    Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.

  3. Cytotoxic minor histocompatibility antigen HA-1-specific CD8+ effector memory T cells: artificial APCs pave the way for clinical application by potent primary in vitro induction.

    PubMed

    Schilbach, Karin; Kerst, Gunter; Walter, Steffen; Eyrich, Matthias; Wernet, Dorothee; Handgretinger, Rupert; Xie, Weidong; Rammensee, Hans-Georg; Müller, Ingo; Bühring, Hans-Jörg; Niethammer, Dietrich

    2005-07-01

    Induction of cytotoxic T lymphocytes (CTLs) for treatment of relapsed leukemia after allogeneic stem-cell transplantation is hindered by the laborious and time-consuming procedure of generating dendritic cells for antigen presentation. Artificial antigen-presenting cells (aAPCs) offer the advantage of being readily available in sufficient numbers, thus allowing for a highly standardized in vitro induction of CTLs. We generated aAPCs coated with anti-CD28 antibody (Ab) and either high-density (HD) or low-density (LD) major histocompatibility complex (MHC) class I molecules loaded with HA-1(H), a nonapeptide derived from the hematopoiesis-restricted minor histocompatibility antigen HA-1. HD- and LD-aAPCs potently induced HA-1(H)-specific CD8+ CTLs from untouched CD8+ T cells of healthy donors. CTLs were subsequently purified by magnetic-activated cell sorting. HD- as well as LD-aAPC-induced CTLs exerted high HA-1H-specific cytotoxicity, resembled T(c)1 effector memory cells, survived a long time in vitro, and were expanded by a factor varying between 8.2 x 10(4) and 51 x 10(4). The T-cell receptor (TCR) repertoire of HA-1H tetramer-positive CTLs was oligoclonal with a prominent usage of Vbeta6. The TCR repertoire of tetramer-positive CTLs was distinct from and more restricted than that of tetramer-negative cells. These findings indicate that aAPCs are attractive tools for the ex vivo generation of HA-1H-specific CTLs suitable for immunotherapy of relapsed leukemia. PMID:15731181

  4. A GMCSF-neuroantigen fusion protein is a potent tolerogen in experimental autoimmune encephalomyelitis (EAE) that is associated with efficient targeting of neuroantigen to APC

    PubMed Central

    Blanchfield, J. Lori; Mannie, Mark D.

    2010-01-01

    Cytokine-NAg fusion proteins represent an emerging platform for specific targeting of self-antigen to particular APC subsets as a means to achieve antigen-specific immunological tolerance. This study focused on cytokine-NAg fusion proteins that targeted NAg to myeloid APC. Fusion proteins contained GM-CSF or the soluble extracellular domain of M-CSF as the N-terminal domain and the encephalitogenic 69–87 peptide of MBP as the C-terminal domain. GMCSF-NAg and MCSF-NAg fusion proteins were ∼1000-fold and 32-fold more potent than NAg in stimulating antigenic proliferation of MBP-specific T cells, respectively. The potentiated antigenic responses required cytokine-NAg covalent linkage and receptor-mediated uptake. That is, the respective cytokines did not potentiate antigenic responses when cytokine and NAg were added as separate molecules, and the potentiated responses were inhibited specifically by the respective free cytokine. Cytokine-dependent targeting of NAg was specific for particular subsets of APC. GMCSF-NAg and MCSF-NAg targeted NAg to DC and macrophages; conversely, IL4-NAg and IL2-NAg fusion proteins, respectively, induced an ∼1000-fold enhancement in NAg reactivity in the presence of B cell and T cell APC. GMCSF-NAg significantly attenuated severity of EAE when treatment was completed before encephalitogenic challenge or alternatively, when treatment was initiated after onset of EAE. MCSF-NAg also had significant tolerogenic activity, but GMCSF-NAg was substantially more efficacious as a tolerogen. Covalent GMCSF-NAg linkage was required for prevention and treatment of EAE. In conclusion, GMCSF-NAg was highly effective for targeting NAg to myeloid APC and was a potent, antigen-specific tolerogen in EAE. PMID:20007248

  5. Ambient PM exposure and DNA methylation in tumor suppressor genes: a cross-sectional study

    PubMed Central

    2011-01-01

    Exposure to ambient air particles matter (PM) has been associated with increased risk of lung cancer. Aberrant tumor suppressor gene promoter methylation has emerged as a promising biomarker for cancers, including lung cancer. Whether exposure to PM is associated with peripheral blood leukocyte (PBL) DNA methylation in tumor suppressor genes has not been evaluated. In 63 male healthy steel workers with well-characterized exposure to metal-rich particles nearby Brescia, Italy, we evaluated whether exposure to PM and metal components was associated with PBL DNA methylation in 4 tumor suppressor genes (i.e., APC, p16, p53 and RASSF1A). Blood samples were obtained on the 1st (baseline) and 4th day (post-exposure) of the same work week and DNA methylation was measured using pyrosequencing. A linear mixed model was used to examine the correlations of the exposure with promoter methylation levels. Mean promoter DNA methylation levels of APC or p16 were significantly higher in post-exposure samples compared to that in baseline samples (p-values = 0.005 for APC, and p-value = 0.006 for p16). By contrast, the mean levels of p53 or RASSF1A promoter methylation was decreased in post-exposure samples compared to that in baseline samples (p-value = 0.015 for p53; and p-value < 0.001 for RASSF1A). In post-exposure samples, APC methylation was positively associated with PM10 (β = 0.27, 95% CI: 0.13-0.40), and PM1 (β = 0.23, 95% CI: 0.09-0.38). In summary, ambient PM exposure was associated with PBL DNA methylation levels of tumor suppressor genes of APC, p16, p53 and RASSF1A, suggesting that such methylation alterations may reflect processes related to PM-induced lung carcinogenesis. PMID:21878113

  6. Methylation of multiple genes in hepatitis C virus associated hepatocellular carcinoma.

    PubMed

    Zekri, Abdel-Rahman N; Bahnasy, Abeer A; Shoeab, Fatma Elzahraa M; Mohamed, Waleed S; El-Dahshan, Dina H; Ali, Fahmey T; Sabry, Gilane M; Dasgupta, Nairajana; Daoud, Sayed S

    2014-01-01

    We studied promoter methylation (PM) of 11 genes in Peripheral Blood Lymphocytes (PBLs) and tissues of hepatitis C virus (HCV) associated hepatocellular carcinoma (HCC) and chronic hepatitis (CH) Egyptian patients. The present study included 31 HCC with their ANT, 38 CH and 13 normal hepatic tissue (NHT) samples. In all groups, PM of APC, FHIT, p15, p73, p14, p16, DAPK1, CDH1, RARβ, RASSF1A, O(6)MGMT was assessed by methylation-specific PCR (MSP). APC and O6-MGMT protein expression was assessed by immunohistochemistry (IHC) in the studied HCC and CH (20 samples each) as well as in a different HCC and CH set for confirmation of MSP results. PM was associated with progression from CH to HCC. Most genes showed high methylation frequency (MF) and the methylation index (MI) increased with disease progression. MF of p14, p73, RASSF1A, CDH1 and O(6)MGMT was significantly higher in HCC and their ANT. MF of APC was higher in CH. We reported high concordance between MF in HCC and their ANT, MF in PBL and CH tissues as well as between PM and protein expression of APC and O(6)MGMT. A panel of 4 genes (APC, p73, p14, O(6)MGMT) classifies the cases independently into HCC and CH with high accuracy (89.9%), sensitivity (83.9%) and specificity (94.7%). HCV infection may contribute to hepatocarcinogenesis through enhancing PM of multiple genes. PM of APC occurs early in the cascade while PM of p14, p73, RASSF1A, RARB, CDH1 and O(6)MGMT are late changes. A panel of APC, p73, p14, O6-MGMT could be used in monitoring CH patients for early detection of HCC. Also, we found that, the methylation status is not significantly affected by whether the tissue was from the liver or PBL, indicating the possibility of use PBL as indicator to genetic profile instead of liver tissue regardless the stage of disease. PMID:25685469

  7. Association of Genetic Variation in Genes Implicated in the β–Catenin Destruction Complex with Risk of Breast Cancer

    PubMed Central

    Wang, Xianshu; Goode, Ellen L.; Fredericksen, Zachary S.; Vierkant, Robert A.; Pankratz, V. Shane; Rider, David N.; Vachon, Celine M.; Cerhan, James R.; Olson, Janet E.; Couch, Fergus J.

    2009-01-01

    Aberrant Wnt/β-catenin signaling leading to nuclear accumulation of the oncogene product β-catenin is observed in a wide spectrum of human malignancies. The destruction complex in the Wnt/β-catenin pathway is critical for regulating the level of β-catenin in the cytoplasm and in the nucleus. Here we report a comprehensive study of the contribution of genetic variation in six genes encoding the β-catenin destruction complex (APC, AXIN1, AXIN2, CSNK1D, CSNK1E and GSK3B) to breast cancer using a Mayo Clinic Breast Cancer Case-Control Study. A total of 79 candidate functional and tagSNPs were genotyped in 798 invasive cases and 843 unaffected controls. Of these, rs454886 in the APC tumor suppressor gene was associated with increased breast cancer risk (per-allele odds ratio (OR) = 1.23; 95% CI, 1.05–1.43; Ptrend = 0.01). In addition, five SNPs in AXIN2 were associated with increased risk of breast cancer (Ptrend < 0.05). Haplotype-based tests identified significant associations between specific haplotypes in APC (P ≤ 0.03) and AXIN2 (P = 0.03) and breast cancer risk. Further characterization of the APC and AXIN2 variants suggested that AXIN2 rs4791171 was significantly associated with risk in premenopausal (Ptrend = 0.0002; q = 0.02) but not in postmenopausal women. The combination of our findings and numerous genetic and functional studies showing that APC and AXIN2 perform crucial tumor suppressor functions suggest that further investigation of the contribution of AXIN2 and APC SNPs to breast cancer risk are needed. PMID:18708403

  8. Common genetic variants in Wnt signaling pathway genes as potential prognostic biomarkers for colorectal cancer.

    PubMed

    Ting, Wen-Chien; Chen, Lu-Min; Pao, Jiunn-Bey; Yang, Ying-Pi; You, Bang-Jau; Chang, Ta-Yuan; Lan, Yu-Hsuan; Lee, Hong-Zin; Bao, Bo-Ying

    2013-01-01

    Compelling evidence has implicated the Wnt signaling pathway in the pathogenesis of colorectal cancer. We assessed the use of tag single nucleotide polymorphisms (tSNPs) in adenomatous polyposis coli (APC)/β-catenin (CTNNB1) genes to predict outcomes in patients with colorectal cancer. We selected and genotyped 10 tSNP to predict common variants across entire APC and CTNNB1 genes in 282 colorectal cancer patients. The associations of these tSNPs with distant metastasis-free survival and overall survival were evaluated by Kaplan-Meier analysis, Cox regression model, and survival tree analysis. The 5-year overall survival rate was 68.3%. Survival tree analysis identified a higher-order genetic interaction profile consisting of the APC rs565453, CTNNB1 2293303, and APC rs1816769 that was significantly associated with overall survival. The 5-year survival overall rates were 89.2%, 66.1%, and 58.8% for the low-, medium-, and high-risk genetic profiles, respectively (log-rank P = 0.001). After adjusting for possible confounders, including age, gender, carcinoembryonic antigen levels, tumor differentiation, stage, lymphovascular invasion, perineural invasion, and lymph node involvement, the genetic interaction profile remained significant. None of the studied SNPs were individually associated with distant metastasis-free survival and overall survival. Our results suggest that the genetic interaction profile among Wnt pathway SNPs might potentially increase the prognostic value in outcome prediction for colorectal cancer. PMID:23405266

  9. Distinguishing Lung Adenocarcinoma from Lung Squamous Cell Carcinoma by Two Hypomethylated and Three Hypermethylated Genes: A Meta-Analysis.

    PubMed

    Huang, Tao; Li, Jinyun; Zhang, Cheng; Hong, Qingxiao; Jiang, Danjie; Ye, Meng; Duan, Shiwei

    2016-01-01

    Significant differences in the aberrant methylation of genes exist among various histological types of non-small cell lung cancer (NSCLC), which includes adenocarcinoma (AC) and squamous cell carcinoma (SCC). Different chemotherapeutic regimens should be administered to the two NSCLC subtypes due to their unique genetic and epigenetic profiles. The purpose of this meta-analysis was to generate a list of differentially methylated genes between AC and SCC. Our meta-analysis encompassed 151 studies on 108 genes among 12946 AC and 10243 SCC patients. Our results showed two hypomethylated genes (CDKN2A and MGMT) and three hypermethylated genes (CDH13, RUNX3 and APC) in ACs compared with SCCs. In addition, our results showed that the pooled specificity and sensitivity values of CDH13 and APC were higher than those of CDKN2A, MGMT and RUNX3. Our findings might provide an alternative method to distinguish between the two NSCLC subtypes. PMID:26862903

  10. Palmatine from Mahonia bealei attenuates gut tumorigenesis in ApcMin/+ mice via inhibition of inflammatory cytokines.

    PubMed

    Ma, Wei-Kun; Li, Hui; Dong, Cui-Lan; He, Xin; Guo, Chang-Run; Zhang, Chun-Feng; Yu, Chun-Hao; Wang, Chong-Zhi; Yuan, Chun-Su

    2016-07-01

    Mahonia bealei is a Chinese folk medicine used to treat various ailments, in particular gastrointestinal inflammation‑related illnesses, and palmatine is one of its active constituents. In this study, ApcMin/+ mice, a genetically engineered model, were used to investigate the effects of palmatine on the initiation and progression of gut inflammation and tumorigenesis enhanced by a high‑fat diet. The in vitro antiproliferation and anti‑inflammation effects of palmatine were evaluated on HT‑29 and SW‑480 human colorectal cancer cell lines. The concentration‑related antiproliferative effects of palmatine on both cell lines (P<0.01) were observed. Palmatine significantly inhibited lipopolysaccharide‑induced increase in cytokine interleukin (IL)‑8 levels in the HT‑29 cells (P<0.01). In the in vivo studies with ApcMin/+ mice, after 10 or 20 mg/kg/day oral palmatine treatment, tumor numbers were significantly reduced in the small intestine and colon in a dose‑dependent manner (P<0.01 compared with the model group). The results were supported by tumor distribution data, body weight changes and organ index. The effect on survival was also dose‑dependent. Both the low‑ and high‑dose palmatine treatments significantly increased the life span of the mice (P<0.01). The gut histology from the model group showed a prominent adenomatous change along with inflammatory lesions. With palmatine treatment, however, the dysplastic changes were greatly reduced in the small intestine and colon tissue. Reverse transcription‑quantitative polymerase chain reaction analysis of interleukin (IL)‑1α, IL1‑β, IL‑8, granulocyte‑colony stimulating factor and granulocyte macrophage colony‑stimulating factor in the gut tissue showed that these inflammatory cytokines were reduced significantly following treatment (all P<0.01); serum cytokine levels were also decreased. Data suggests that palmatine has a clinical value in colorectal cancer therapeutics, and

  11. Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

    PubMed

    Lu, Jing; Chen, Baian; Li, Shengli; Sun, Quan

    2014-06-01

    Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease.

  12. Inhibition of epidermal growth factor receptor tyrosine kinase fails to suppress adenoma formation in ApcMin mice but induces duodenal injury.

    PubMed

    Ritland, S R; Gendler, S J; Burgart, L J; Fry, D W; Nelson, J M; Bridges, A J; Andress, L; Karnes, W E

    2000-09-01

    A highly selective, p.o. bioavailable irreversible inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, N-[4-(3-chloro4-fluorophenylamino)-quinazolin-6-yl]-ac rylamide (CFPQA), was evaluated for its ability to prevent intestinal adenoma formation in ApcMin mice. Ten-week continuous dietary exposure to CFPQA at doses sufficient to abolish intestinal EGFR tyrosine phosphorylation failed to affect intestinal tumor multiplicity or distribution but induced flat mucosal lesions in the duodenum characteristic of chronic injury. Intestinal trefoil factor, an intestinal peptide that mediates antiapoptotic effects through an EGFR-dependent mechanism, was notably absent in adenomas but was highly expressed in flat duodenal lesions. We conclude that chronic inhibition of EGFR tyrosine kinase by CFPQA does not prevent adenomas in ApcMin mice but may induce duodenal injury.

  13. Tryptase inhibitor APC 366 prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2 interactions in hepatic stellate cells.

    PubMed

    Lu, Jing; Chen, Baian; Li, Shengli; Sun, Quan

    2014-06-01

    Protease-activated receptor (PAR) 2 is a G-protein-coupled receptor that is activated by mast cell tryptase. PAR-2 activation augments profibrotic pathways through the induction of extracellular matrix proteins. PAR-2 is widely expressed in hepatic stellate cells (HSCs), but the role of tryptase/PAR-2 interaction in liver fibrosis is unclear. We studied the development of bile duct ligation (BDL)-induced hepatic fibrosis in rats treated with mast cell tryptase inhibitor APC 366, and showed that APC 366 reduced hepatic fibrosis scores, collagen content and serum biochemical parameters. Reduced fibrosis was associated with decreased expression of PAR-2 and α-smooth muscle actin (α-SMA). Our findings demonstrate that mast cell tryptase induces PAR-2 activation to augment HSC proliferation and promote hepatic fibrosis in rats. Treatment with tryptase antagonists may be a novel therapeutic approach to prevent fibrosis in patients with chronic liver disease. PMID:24735816

  14. Water quality assessment and apportionment of pollution sources using APCS-MLR and PMF receptor modeling techniques in three major rivers of South Florida.

    PubMed

    Haji Gholizadeh, Mohammad; Melesse, Assefa M; Reddi, Lakshmi

    2016-10-01

    In this study, principal component analysis (PCA), factor analysis (FA), and the absolute principal component score-multiple linear regression (APCS-MLR) receptor modeling technique were used to assess the water quality and identify and quantify the potential pollution sources affecting the water quality of three major rivers of South Florida. For this purpose, 15years (2000-2014) dataset of 12 water quality variables covering 16 monitoring stations, and approximately 35,000 observations was used. The PCA/FA method identified five and four potential pollution sources in wet and dry seasons, respectively, and the effective mechanisms, rules and causes were explained. The APCS-MLR apportioned their contributions to each water quality variable. Results showed that the point source pollution discharges from anthropogenic factors due to the discharge of agriculture waste and domestic and industrial wastewater were the major sources of river water contamination. Also, the studied variables were categorized into three groups of nutrients (total kjeldahl nitrogen, total phosphorus, total phosphate, and ammonia-N), water murkiness conducive parameters (total suspended solids, turbidity, and chlorophyll-a), and salt ions (magnesium, chloride, and sodium), and average contributions of different potential pollution sources to these categories were considered separately. The data matrix was also subjected to PMF receptor model using the EPA PMF-5.0 program and the two-way model described was performed for the PMF analyses. Comparison of the obtained results of PMF and APCS-MLR models showed that there were some significant differences in estimated contribution for each potential pollution source, especially in the wet season. Eventually, it was concluded that the APCS-MLR receptor modeling approach appears to be more physically plausible for the current study. It is believed that the results of apportionment could be very useful to the local authorities for the control and

  15. Water quality assessment and apportionment of pollution sources using APCS-MLR and PMF receptor modeling techniques in three major rivers of South Florida.

    PubMed

    Haji Gholizadeh, Mohammad; Melesse, Assefa M; Reddi, Lakshmi

    2016-10-01

    In this study, principal component analysis (PCA), factor analysis (FA), and the absolute principal component score-multiple linear regression (APCS-MLR) receptor modeling technique were used to assess the water quality and identify and quantify the potential pollution sources affecting the water quality of three major rivers of South Florida. For this purpose, 15years (2000-2014) dataset of 12 water quality variables covering 16 monitoring stations, and approximately 35,000 observations was used. The PCA/FA method identified five and four potential pollution sources in wet and dry seasons, respectively, and the effective mechanisms, rules and causes were explained. The APCS-MLR apportioned their contributions to each water quality variable. Results showed that the point source pollution discharges from anthropogenic factors due to the discharge of agriculture waste and domestic and industrial wastewater were the major sources of river water contamination. Also, the studied variables were categorized into three groups of nutrients (total kjeldahl nitrogen, total phosphorus, total phosphate, and ammonia-N), water murkiness conducive parameters (total suspended solids, turbidity, and chlorophyll-a), and salt ions (magnesium, chloride, and sodium), and average contributions of different potential pollution sources to these categories were considered separately. The data matrix was also subjected to PMF receptor model using the EPA PMF-5.0 program and the two-way model described was performed for the PMF analyses. Comparison of the obtained results of PMF and APCS-MLR models showed that there were some significant differences in estimated contribution for each potential pollution source, especially in the wet season. Eventually, it was concluded that the APCS-MLR receptor modeling approach appears to be more physically plausible for the current study. It is believed that the results of apportionment could be very useful to the local authorities for the control and

  16. Systems Biology Modeling of Five Pathways for Regulation and Potent Inhibition of the Anaphase-Promoting Complex (APC/C): Pivotal Roles for MCC and BubR1

    PubMed Central

    2015-01-01

    Abstract Correct DNA segregation is a fundamental process that ensures the precise and reliable inheritance of genomic information for the propagation of cell life. Eukaryotic cells have evolved a conserved surveillance control mechanism for DNA segregation named the Spindle Assembly Checkpoint (SAC).The SAC ensures that the sister chromatids of the duplicated genome are not separated and distributed to the spindle poles before all chromosomes have been properly linked to the microtubules of the mitotic spindle. Biochemically, the SAC delays cell cycle progression by preventing activation of the anaphase-promoting complex (APC/C) or cyclosome whose activation by Cdc20 is required for sister-chromatid separation; this marks the transition into anaphase. In response to activation of the checkpoint, various species control the activity of both APC/C and Cdc20. However, the underlying regulatory pathways remain largely elusive. In this study, five possible model variants of APC/C regulation were constructed, namely BubR1, Mad2, MCC, MCF2, and an all-pathways model variant. These models were validated with experimental data from the literature. A wide range of parameter values has been tested to find the critical values of the APC/C binding rate. The results show that all variants are able to capture the wild-type behavior of the APC/C. However, only one model variant, which included both MCC as well as BubR1 as potent inhibitors of the APC/C, was able to reproduce both wild-type and mutant type behavior of APC/C regulation. In conclusion, the presented work informs the regulation of fundamental processes such as SAC and APC/C in cell biology and has successfully distinguished between five competing dynamical models using a systems biology approach. The results attest that systems-level approaches are vital for molecular and cell biology. PMID:25871779

  17. Cell-cycle-dependent PC-PLC regulation by APC/C(Cdc20)-mediated ubiquitin-proteasome pathway.

    PubMed

    Fu, Da; Ma, Yushui; Wu, Wei; Zhu, Xuchao; Jia, Chengyou; Zhao, Qianlei; Zhang, Chunyi; Wu, Xing Zhong

    2009-07-01

    Phosphatidylcholine-specific phospholipase C (PC-PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC-PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC-PLC were co-immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH-7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC-PLC and Cdc20 were co-localized in the perinuclear endoplasmic reticulum region (the "juxtanuclear quality control" compartment, JUNQ). The expression level and activities of PC-PLC changed in a cell-cycle-dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC-PLC, and caused PC-PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC-PLC regulation in cell cycles is controlled by APC/C(Cdc20)-mediated UPP.

  18. Inhibition of intestinal tumorigenesis in Apc(min/+) mice by green tea polyphenols (polyphenon E) and individual catechins.

    PubMed

    Hao, Xingpei; Sun, Yuhai; Yang, Chung S; Bose, Mousumi; Lambert, Joshua D; Ju, Jihyeung; Lu, Gang; Lee, Mao-Jung; Park, Sunny; Husain, Ali; Wang, Steven

    2007-01-01

    In this work, we compared the cancer preventive activities of Polyphenon E (PPE), a standardized green tea polyphenol preparation given in diet versus drinking fluid as well as the activities of PPE versus individual catechins. We treated Apc(Min/+) mice for 9 wk with 0.08% (-)-epigallocatechin-3-gallate (EGCG), 0.08% (-)-epicatechin-3-gallate, or 0.12% PPE in drinking fluid or diet. Only 0.12% dietary PPE and 0.08% EGCG in drinking fluid significantly decreased tumor multiplicity (70% and 51%, respectively). Compared to PPE in drinking fluid, dietary PPE delivered twofold more EGCG to the small intestine. Immunohistochemistry showed that adenomas in groups treated with PPE and EGCG had decreased cell proliferation, Beta -catenin nuclear expression, and phospho-Akt levels; higher cleaved caspase-3 levels, and partially restored retinoid X receptor alpha expression. The results suggest that these molecular events contribute to the cancer prevention activity of EGCG and PPE. Furthermore, diet appears to be a better route of administration for PPE than drinking fluid.

  19. HCV RNA Activates APCs via TLR7/TLR8 While Virus Selectively Stimulates Macrophages Without Inducing Antiviral Responses

    PubMed Central

    Zhang, Yuwei; El-Far, Mohamed; Dupuy, Franck P.; Abdel-Hakeem, Mohamed S.; He, Zhong; Procopio, Francesco Andrea; Shi, Yu; Haddad, Elias K.; Ancuta, Petronela; Sekaly, Rafick-Pierre; Said, Elias A.

    2016-01-01

    The innate and adaptive immune systems fail to control HCV infection in the majority of infected individuals. HCV is an ssRNA virus, which suggests a role for Toll-like receptors (TLRs) 7 and 8 in initiating the anti-viral response. Here we demonstrate that HCV genomic RNA harbours specific sequences that initiate an anti-HCV immune response through TLR7 and TLR8 in various antigen presenting cells. Conversely, HCV particles are detected by macrophages, but not by monocytes and DCs, through a TLR7/8 dependent mechanism; this leads to chloroquine sensitive production of pro-inflammatory cytokines including IL-1β, while the antiviral type I Interferon response is not triggered in these cells. Antibodies to DC-SIGN, a c-type lectin selectively expressed by macrophages but not pDCs or mDCs, block the production of cytokines. Novel anti-HCV vaccination strategies should target the induction of TLR7/8 stimulation in APCs in order to establish potent immune responses against HCV. PMID:27385120

  20. Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.

    PubMed

    Bonnans, Caroline; Lohela, Marja; Werb, Zena

    2014-10-06

    Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. Apc(Min/+) mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

  1. ADAP–SLP-76 Binding Differentially Regulates Supramolecular Activation Cluster (SMAC) Formation Relative to T Cell–APC Conjugation

    PubMed Central

    Wang, Hongyan; McCann, Fiona E.; Gordan, John D.; Wu, Xiang; Raab, Monika; Malik, Talat H.; Davis, Daniel M.; Rudd, Christopher E.

    2004-01-01

    T cell–APC conjugation as mediated by leukocyte function-associated antigen-1 (LFA-1)–intercellular adhesion molecule (ICAM)-1 binding is followed by formation of the supramolecular activation cluster (SMAC) at the immunological synapse. The intracellular processes that regulate SMAC formation and its influence on T cell function are important questions to be addressed. Here, using a mutational approach, we demonstrate that binding of adaptor adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76 differentially regulates peripheral SMAC (pSMAC) formation relative to conjugation. Although mutation of the YDDV sites (termed M12) disrupted SLP-76 SH2 domain binding and prevented the ability of ADAP to increase conjugation and LFA-1 clustering, M12 acted selectively as a dominant negative (DN) inhibitor of pSMAC formation, an effect that was paralleled by a DN effect on interleukin-2 production. ADAP also colocalized with LFA-1 at the immunological synapse. Our findings identify ADAP–SLP-76 binding as a signaling event that differentially regulates SMAC formation, and support a role for SMAC formation in T cell cytokine production. PMID:15477347

  2. Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

    PubMed Central

    Werb, Zena

    2014-01-01

    Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment. PMID:25350573

  3. Pilot-scale road subbase made with granular material formulated with MSWI bottom ash and stabilized APC fly ash: environmental impact assessment.

    PubMed

    del Valle-Zermeño, R; Formosa, J; Prieto, M; Nadal, R; Niubó, M; Chimenos, J M

    2014-02-15

    A granular material (GM) to be used as road sub-base was formulated using 80% of weathered bottom ash (WBA) and 20% of mortar. The mortar was prepared separately and consisted in 50% APC and 50% of Portland cement. A pilot-scale study was carried on by constructing three roads in order to environmentally evaluate the performance of GM in a real scenario. By comparing the field results with those of the column experiments, the overestimations observed at laboratory scale can be explained by the potential mechanisms in which water enters into the road body and the pH of the media. An exception was observed in the case of Cu, whose concentration release at the test road was higher. The long-time of exposure at atmospheric conditions might have favoured oxidation of organic matter and therefore the leaching of this element. The results obtained showed that immobilization of all heavy metals and metalloids from APC is achieved by the pozzolanic effect of the cement mortar. This is, to the knowledge of the authors, the only pilot scale study that is considering reutilization of APC as a safe way to disposal.

  4. Pilot-scale road subbase made with granular material formulated with MSWI bottom ash and stabilized APC fly ash: environmental impact assessment.

    PubMed

    del Valle-Zermeño, R; Formosa, J; Prieto, M; Nadal, R; Niubó, M; Chimenos, J M

    2014-02-15

    A granular material (GM) to be used as road sub-base was formulated using 80% of weathered bottom ash (WBA) and 20% of mortar. The mortar was prepared separately and consisted in 50% APC and 50% of Portland cement. A pilot-scale study was carried on by constructing three roads in order to environmentally evaluate the performance of GM in a real scenario. By comparing the field results with those of the column experiments, the overestimations observed at laboratory scale can be explained by the potential mechanisms in which water enters into the road body and the pH of the media. An exception was observed in the case of Cu, whose concentration release at the test road was higher. The long-time of exposure at atmospheric conditions might have favoured oxidation of organic matter and therefore the leaching of this element. The results obtained showed that immobilization of all heavy metals and metalloids from APC is achieved by the pozzolanic effect of the cement mortar. This is, to the knowledge of the authors, the only pilot scale study that is considering reutilization of APC as a safe way to disposal. PMID:24394668

  5. The E3 ligase APC/C-Cdh1 is required for associative fear memory and long-term potentiation in the amygdala of adult mice.

    PubMed

    Pick, Joseph E; Malumbres, Marcos; Klann, Eric

    2012-12-14

    The anaphase promoting complex/cyclosome (APC/C) is an E3 ligase regulated by Cdh1. Beyond its role in controlling cell cycle progression, APC/C-Cdh1 has been detected in neurons and plays a role in long-lasting synaptic plasticity and long-term memory. Herein, we further examined the role of Cdh1 in synaptic plasticity and memory by generating knockout mice where Cdh1 was conditionally eliminated from the forebrain post-developmentally. Although spatial learning and memory in the Morris water maze (MWM) was normal, the Cdh1 conditional knockout (cKO) mice displayed enhanced reversal learning in the MWM and in a water-based Y maze. In addition, we found that the Cdh1 cKO mice had impaired associative fear memory and exhibited impaired long-term potentiation (LTP) in amygdala slices. Finally, we observed increased expression of Shank1 and NR2A expression in amygdalar slices from the Cdh1 cKO mice following the induction of LTP, suggesting a possible molecular mechanism underlying the behavioral and synaptic plasticity impairments displayed in these mice. Our findings are consistent with a role for the APC/C-Cdh1 in fear memory and synaptic plasticity in the amygdala.

  6. MPO-type single-mode multi-fiber connector: Low-loss and high-return-loss intermateability of APC-MPO connectors

    NASA Astrophysics Data System (ADS)

    Satake, Toshiaki; Nagasawa, Shinji; Hughes, Mike; Lutz, Sharon

    2011-01-01

    The electrical communication laboratory of NTT started the research of MT (Mechanically Transferable) connector in early 1980s. The initial goal was to realize a multi-fiber connector which can repeat low loss, stable, reliable and low-cost connections of subscriber optical fiber cable networks for more than 20 years period in the field. We review the multi-fiber alignment design with two guide pins, and following several technical improvements toward the final MT connector used in the commercial telecommunication networks. And then, we review development histories to reach to the low-loss, high-return-loss and reliable APC-MPO (Angled Physical Contact Multi-fiber Push On) connectors introduced in NTT COs and in Verizon's FTTH (Fiber To The Home) networks. In the latter half, we propose the low-loss intermateability design for connectors made by different suppliers in order to enable mass introductions into large scale systems. In addition we also describe an accurate connector loss presumption method for different lots' ferrules based on the MT ferrule dimension data before assembling the connectors. We believe with a wide intermateability of APC-MPO connector will increase its use in the fields. The APC-MPO connector manufactured based on the proposed design had low insertion losses of less than 0.25 dB at the same level of simplex connectors and the higher level of return losses higher than 65 dB.

  7. Cdk5-mediated inhibition of APC/C-Cdh1 switches on the cyclin D1-Cdk4-pRb pathway causing aberrant S-phase entry of postmitotic neurons

    PubMed Central

    Veas-Pérez de Tudela, Miguel; Maestre, Carolina; Delgado-Esteban, María; Bolaños, Juan P.; Almeida, Angeles

    2015-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells. To enter the S-phase, APC/C must be inactivated by phosphorylation of its cofactor, Cdh1. In post-mitotic cells such as neurons APC/C-Cdh1 complex is highly active and responsible for the continuous degradation of mitotic cyclins. However, the specific molecular pathway that determines neuronal cell cycle blockade in post-mitotic neurons is unknown. Here, we show that activation of glutamatergic receptors in rat cortical primary neurons endogenously triggers cyclin-dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1 leading to its cytoplasmic accumulation and disassembly from the APC3 core protein, causing APC/C inactivation. Conversely, pharmacological or genetic inhibition of Cdk5 promotes Cdh1 ubiquitination and proteasomal degradation. Furthermore, we show that Cdk5-mediated phosphorylation and inactivation of Cdh1 leads to p27 depletion, which switches on the cyclin D1-cyclin-dependent kinase-4 (Cdk4)-retinoblastoma protein (pRb) pathway to allow the S-phase entry of neurons. However, neurons do not proceed through the cell cycle and die by apoptosis. These results indicate that APC/C-Cdh1 actively suppresses an aberrant cell cycle entry and death of neurons, highlighting its critical function in neuroprotection. PMID:26658992

  8. Distinguishing between cancer driver and passenger gene alteration candidates via cross-species comparison: a pilot study

    PubMed Central

    2010-01-01

    Background We are developing a cross-species comparison strategy to distinguish between cancer driver- and passenger gene alteration candidates, by utilizing the difference in genomic location of orthologous genes between the human and other mammals. As an initial test of this strategy, we conducted a pilot study with human colorectal cancer (CRC) and its mouse model C57BL/6J ApcMin/+, focusing on human 5q22.2 and 18q21.1-q21.2. Methods We first performed bioinformatics analysis on the evolution of 5q22.2 and 18q21.1-q21.2 regions. Then, we performed exon-targeted sequencing, real time quantitative polymerase chain reaction (qPCR), and real time quantitative reverse transcriptase PCR (qRT-PCR) analyses on a number of genes of both regions with both human and mouse colon tumors. Results These two regions (5q22.2 and 18q21.1-q21.2) are frequently deleted in human CRCs and encode genuine colorectal tumor suppressors APC and SMAD4. They also encode genes such as MCC (mutated in colorectal cancer) with their role in CRC etiology unknown. We have discovered that both regions are evolutionarily unstable, resulting in genes that are clustered in each human region being found scattered at several distinct loci in the genome of many other species. For instance, APC and MCC are within 200 kb apart in human 5q22.2 but are 10 Mb apart in the mouse genome. Importantly, our analyses revealed that, while known CRC driver genes APC and SMAD4 were disrupted in both human colorectal tumors and tumors from ApcMin/+ mice, the questionable MCC gene was disrupted in human tumors but appeared to be intact in mouse tumors. Conclusions These results indicate that MCC may not actually play any causative role in early colorectal tumorigenesis. We also hypothesize that its disruption in human CRCs is likely a mere result of its close proximity to APC in the human genome. Expanding this pilot study to the entire genome may identify more questionable genes like MCC, facilitating the discovery of

  9. Irsogladine maleate, a gastric mucosal protectant, suppresses intestinal polyp development in Apc-mutant mice.

    PubMed

    Onuma, Wakana; Tomono, Susumu; Miyamoto, Shinngo; Fujii, Gen; Hamoya, Takahiro; Fujimoto, Kyoko; Miyoshi, Noriyuki; Fukai, Fumio; Wakabayashi, Keiji; Mutoh, Michihiro

    2016-02-23

    This study aimed to identify gastric mucosal protectants that suppress intestinal tumorigenesis in a mouse model. We chose six gastric mucosal protectants (ecabet sodium hydrate, irsogladine maleate, rebamipide, sofalcone, teprenone and troxipide) and examined their effects on the activity of oxidative stress-related transcriptional factors, including AP-1, NF-jB, NRF2, p53 and STAT3, in Caco-2 cells using a luciferase reporter gene assay. Among the six protectants, irsogladine maleate clearly inhibited NF-jB and AP-1 transcriptional activity. Furthermore, the chemopreventive property of irsogladine maleate was examined in a Min mouse model of familial adenomatous polyposis. Treatment with irsogladine maleate at doses of 5 and 50 ppm significantly reduced the number of intestinal polyps to 69% and 66% of the untreated control value, respectively. In these polyps, mRNA levels of the downstream targets of NF-jB, such as IL-1β and IL-6, were decreased by irsogladine maleate treatment. Moreover, the levels of oxidative stress-related markers, reactive carbonyl species, in the livers of Min mice were clearly decreased following the administration of irsogladine maleate. This study demonstrated that irsogladine maleate suppresses intestinal polyp formation in Min mice partly through the NF-jB signaling pathway, thus reducing oxidative stress.

  10. Irsogladine maleate, a gastric mucosal protectant, suppresses intestinal polyp development in Apc-mutant mice

    PubMed Central

    Onuma, Wakana; Tomono, Susumu; Miyamoto, Shinngo; Fujii, Gen; Hamoya, Takahiro; Fujimoto, Kyoko; Miyoshi, Noriyuki; Fukai, Fumio; Wakabayashi, Keiji; Mutoh, Michihiro

    2016-01-01

    This study aimed to identify gastric mucosal protectants that suppress intestinal tumorigenesis in a mouse model. We chose six gastric mucosal protectants (ecabet sodium hydrate, irsogladine maleate, rebamipide, sofalcone, teprenone and troxipide) and examined their effects on the activity of oxidative stress-related transcriptional factors, including AP-1, NF-jB, NRF2, p53 and STAT3, in Caco-2 cells using a luciferase reporter gene assay. Among the six protectants, irsogladine maleate clearly inhibited NF-jB and AP-1 transcriptional activity. Furthermore, the chemopreventive property of irsogladine maleate was examined in a Min mouse model of familial adenomatous polyposis. Treatment with irsogladine maleate at doses of 5 and 50 ppm significantly reduced the number of intestinal polyps to 69% and 66% of the untreated control value, respectively. In these polyps, mRNA levels of the downstream targets of NF-jB, such as IL-1β and IL-6, were decreased by irsogladine maleate treatment. Moreover, the levels of oxidative stress-related markers, reactive carbonyl species, in the livers of Min mice were clearly decreased following the administration of irsogladine maleate. This study demonstrated that irsogladine maleate suppresses intestinal polyp formation in Min mice partly through the NF-jB signaling pathway, thus reducing oxidative stress. PMID:26840084

  11. A rapid and scalable system for studying gene function in mice using conditional RNA interference

    PubMed Central

    Premsrirut, Prem K.; Dow, Lukas E.; Kim, Sang Yong; Camiolo, Matthew; Malone, Colin D.; Miething, Cornelius; Scuoppo, Claudio; Zuber, Johannes; Dickins, Ross A.; Kogan, Scott C.; Shroyer, Kenneth R.; Sordella, Raffaella; Hannon, Gregory J.; Lowe, Scott W.

    2011-01-01

    Summary RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene. PMID:21458673

  12. Hybrid biosynthetic gene therapy vector development and dual engineering capacity.

    PubMed

    Jones, Charles H; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P; Pfeifer, Blaine A

    2014-08-26

    Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

  13. Hybrid biosynthetic gene therapy vector development and dual engineering capacity

    PubMed Central

    Jones, Charles H.; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P.; Pfeifer, Blaine A.

    2014-01-01

    Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy. PMID:25114239

  14. Diet- and Genetically-Induced Obesity Differentially Affect the Fecal Microbiome and Metabolome in Apc1638N Mice

    PubMed Central

    Parnell, Laurence D.; Iyer, Lakshmanan K.; Liu, Zhenhua; Kane, Anne V.; Chen, C-Y. Oliver; Tai, Albert K.; Bowman, Thomas A.; Obin, Martin S.; Mason, Joel B.; Greenberg, Andrew S.; Choi, Sang-Woon; Selhub, Jacob; Paul, Ligi; Crott, Jimmy W.

    2015-01-01

    Obesity is a risk factor for colorectal cancer (CRC), and alterations in the colonic microbiome and metabolome may be mechanistically involved in this relationship. The relative contribution of diet and obesity per se are unclear. We compared the effect of diet- and genetically-induced obesity on the intestinal microbiome and metabolome in a mouse model of CRC. Apc1638N mice were made obese by either high fat (HF) feeding or the presence of the Leprdb/db (DbDb) mutation. Intestinal tumors were quantified and stool microbiome and metabolome were profiled. Genetic obesity, and to a lesser extent HF feeding, promoted intestinal tumorigenesis. Each induced distinct microbial patterns: taxa enriched in HF were mostly Firmicutes (6 of 8) while those enriched in DbDb were split between Firmicutes (7 of 12) and Proteobacteria (5 of 12). Parabecteroides distasonis was lower in tumor-bearing mice and its abundance was inversely associated with colonic Il1b production (p<0.05). HF and genetic obesity altered the abundance of 49 and 40 fecal metabolites respectively, with 5 in common. Of these 5, adenosine was also lower in obese and in tumor-bearing mice (p<0.05) and its concentration was inversely associated with colonic Il1b and Tnf production (p<0.05). HF and genetic obesity differentially alter the intestinal microbiome and metabolome. A depletion of adenosine and P.distasonis in tumor-bearing mice could play a mechanistic role in tumor formation. Adenosine and P. distasonis have previously been shown to be anti-inflammatory in the colon and we postulate their reduction could promote tumorigenesis by de-repressing inflammation. PMID:26284788

  15. Diet- and Genetically-Induced Obesity Differentially Affect the Fecal Microbiome and Metabolome in Apc1638N Mice.

    PubMed

    Pfalzer, Anna C; Nesbeth, Paula-Dene C; Parnell, Laurence D; Iyer, Lakshmanan K; Liu, Zhenhua; Kane, Anne V; Chen, C-Y Oliver; Tai, Albert K; Bowman, Thomas A; Obin, Martin S; Mason, Joel B; Greenberg, Andrew S; Choi, Sang-Woon; Selhub, Jacob; Paul, Ligi; Crott, Jimmy W

    2015-01-01

    Obesity is a risk factor for colorectal cancer (CRC), and alterations in the colonic microbiome and metabolome may be mechanistically involved in this relationship. The relative contribution of diet and obesity per se are unclear. We compared the effect of diet- and genetically-induced obesity on the intestinal microbiome and metabolome in a mouse model of CRC. Apc1638N mice were made obese by either high fat (HF) feeding or the presence of the Leprdb/db (DbDb) mutation. Intestinal tumors were quantified and stool microbiome and metabolome were profiled. Genetic obesity, and to a lesser extent HF feeding, promoted intestinal tumorigenesis. Each induced distinct microbial patterns: taxa enriched in HF were mostly Firmicutes (6 of 8) while those enriched in DbDb were split between Firmicutes (7 of 12) and Proteobacteria (5 of 12). Parabecteroides distasonis was lower in tumor-bearing mice and its abundance was inversely associated with colonic Il1b production (p<0.05). HF and genetic obesity altered the abundance of 49 and 40 fecal metabolites respectively, with 5 in common. Of these 5, adenosine was also lower in obese and in tumor-bearing mice (p<0.05) and its concentration was inversely associated with colonic Il1b and Tnf production (p<0.05). HF and genetic obesity differentially alter the intestinal microbiome and metabolome. A depletion of adenosine and P.distasonis in tumor-bearing mice could play a mechanistic role in tumor formation. Adenosine and P. distasonis have previously been shown to be anti-inflammatory in the colon and we postulate their reduction could promote tumorigenesis by de-repressing inflammation.

  16. Fine mapping of the human pentraxin gene region on chromosome 1q23

    SciTech Connect

    Walsh, M.T.; Whitehead, A.S.; Divane, A.

    1996-12-31

    The 1q21 to 25 region of human chromosome 1 contains genes which encode proteins with immune- and inflammation-associated functions. These include the pentraxin genes, for C-reactive protein (CRP), serum amyloid P(SAP) protein (APCS), and a CRP pseudogene (CRPP1). The region of chromosome 1 containing this cluster is syntenic with distal mouse chromosome 1. We constructed an approximately 1.4 megabase yeast artificial chromosome (YAC) contig with the pentraxin genes at its core. This four-YAC contig includes other genes with immune functions including the FCER1A gene, which encodes the {alpha}-subunit of the IgE high-affinity Fc receptor and the 1F1-16 gene, an interferon-{gamma}-induced gene. In addition, it contains the histone H3F2 and H4F2 genes and the gene for erythroid {alpha}-spectrin (SPTA1). The gene order is cen.-SPTA1-H4F2-H3F2-1F1-16-CRP-CRPP1-APCS-FCERIA-tel. The contig thus consists of a cluster of genes whose products either have immunological importance, bind DNA, or both. 68 refs., 3 figs., 2 tabs.

  17. Modified irinotecan and infusional 5-fluorouracil (mFOLFIRI) in patients with refractory advanced pancreas cancer (APC): a single-institution experience

    PubMed Central

    Bupathi, M.; Ahn, D. H.; Wu, C.; Ciombor, K. K.; Stephens, J. A.; Reardon, J.; Goldstein, D. A.; Bekaii-Saab, T.

    2016-01-01

    Pancreatic adenocarcinoma is the fourth leading cause of cancer death. Recently, MM-398 (nanoliposomal irinotecan) was shown to be associated with significant improvement in outcome measures with acceptable toxicities when combined with 5-fluorouracil (5-FU)/leucovorin (LV) compared to 5-FU/LV alone in patients failing one line of gemcitabine-based therapy. There is a paucity of data evaluating the role of irinotecan in combination with 5FU in advanced pancreas cancer (APC). We performed a retrospective analysis of all patients who received mFOLFIRI (minus bolus 5FU and LV). All patients with metastatic disease who had failed at least one line of gemcitabine-based therapy prior to receiving mFOLFIRI were included in this study. Descriptive statistics were used to assess the continuous variables and adverse events (AEs), and Kaplan–Meier methods were used to calculate the median progression-free survival (PFS) and overall survival (OS). Forty patients were included in this analysis. Patients received 1–5 lines of prior therapy (25 % with more than 3 lines of prior therapy). The mean age at diagnosis was 60, and 98 % had ECOG of 1. The mean CA 19-9 at the start of therapy was 33,169 U/ml. The median PFS was 2.59 months [95 % confidence interval (CI) (1.90, 3.54)], and OS was 4.75 months [95 % CI (3.14, 8.98)]. The most common AEs included fatigue (98 %), neuropathy (83 %), anorexia (68 %), nausea (60 %) and constipation (55 %). Grade 3 toxicities included fatigue (13 %) and rash (3 %). There were no observed grade 4 toxicities. In this single-institution retrospective analysis, mFOLFIRI was found to be both tolerable and relatively effective in a heavily pretreated patient population with APC. Future prospective studies should consider evaluating the role of mFOLFIRI in refractory APC. PMID:26995224

  18. Integration and automation of DoseMapper in a logic fab APC system: application for 45/40/28nm node

    NASA Astrophysics Data System (ADS)

    Le Gratiet, Bertrand; Salagnon, Christophe; de Caunes, Jean; Mikolajczak, Marc; Morin, Vincent; Chojnowski, Nicolas; Sundermann, Frank; Massin, Jean; Pelletier, Alice; Metz, Joel; Blancquaert, Yoann; Bouyssou, Regis; Pelissier, Arthur; Belmont, Olivier; Strapazzon, Anne; Phillips, Anna; Devoivre, Thierry; Bernard, Emilie; Batail, Estelle; Thevenon, Lionel; Bry, Benedicte; Bernard-Granger, Fabrice; Oumina, Ahmed; Baron, Marie-Pierre; Gueze, Didier

    2012-03-01

    The main difficulty related to DoseMapper correction is to generate an appropriate CD datacollection to feed DoseMapper and to generate DoseRecipe in a user friendly way, especially with a complex process mix. We could heavily measure the silicon and create, in feedback mode, the corresponding DoseRecipe. However, such approach in a logic fab becomes a heavy duty due to the number of different masks / product / processes. We have observed that process CD variability is significantly depending on systematic intrawafer and intrafield CD footprints that can be measured and applied has generic pre-correction for any new product/mask process in-line. The applied CD correction is based on a CD (intrafield: Mask + Straylight & intrawafer: Etch Bias) variability "model" handled by the FAB APC (Advanced Process Control). - Individual CD profile correction component are generated "off-line" (1) for Intrafield Mask via automatic CD extraction from a Reticle CD database (2) for Intrafield Straylight via a CD "model" (3) for Intrawafer Etch Bias via engineering input based on process monitoring. - These CD files are handled via the FAB APC/automation system which is remotely taking control of DoseMapper server via WEB services, so that CD profiles are generated "off-line" (before the lot is being processed) and stored in a profile database while DoseRecipes are created "real-time" on demand via the automation when the lot comes to the scanner to be processed. DoseRecipe and CD correction profiles management is done via the APC system. The automated DoseRecipe creation is now running since the beginning of 2011 contributing to bring both intrafield and intrawafer GATE CDu below 1nm 3sigma, for 45/40 & 28nm nodes.

  19. Source apportionment of ambient non-methane hydrocarbons in Hong Kong: application of a principal component analysis/absolute principal component scores (PCA/APCS) receptor model.

    PubMed

    Guo, H; Wang, T; Louie, P K K

    2004-06-01

    Receptor-oriented source apportionment models are often used to identify sources of ambient air pollutants and to estimate source contributions to air pollutant concentrations. In this study, a PCA/APCS model was applied to the data on non-methane hydrocarbons (NMHCs) measured from January to December 2001 at two sampling sites: Tsuen Wan (TW) and Central & Western (CW) Toxic Air Pollutants Monitoring Stations in Hong Kong. This multivariate method enables the identification of major air pollution sources along with the quantitative apportionment of each source to pollutant species. The PCA analysis identified four major pollution sources at TW site and five major sources at CW site. The extracted pollution sources included vehicular internal engine combustion with unburned fuel emissions, use of solvent particularly paints, liquefied petroleum gas (LPG) or natural gas leakage, and industrial, commercial and domestic sources such as solvents, decoration, fuel combustion, chemical factories and power plants. The results of APCS receptor model indicated that 39% and 48% of the total NMHCs mass concentrations measured at CW and TW were originated from vehicle emissions, respectively. 32% and 36.4% of the total NMHCs were emitted from the use of solvent and 11% and 19.4% were apportioned to the LPG or natural gas leakage, respectively. 5.2% and 9% of the total NMHCs mass concentrations were attributed to other industrial, commercial and domestic sources, respectively. It was also found that vehicle emissions and LPG or natural gas leakage were the main sources of C(3)-C(5) alkanes and C(3)-C(5) alkenes while aromatics were predominantly released from paints. Comparison of source contributions to ambient NMHCs at the two sites indicated that the contribution of LPG or natural gas at CW site was almost twice that at TW site. High correlation coefficients (R(2) > 0.8) between the measured and predicted values suggested that the PCA/APCS model was applicable for estimation

  20. Source apportionment of ambient non-methane hydrocarbons in Hong Kong: application of a principal component analysis/absolute principal component scores (PCA/APCS) receptor model.

    PubMed

    Guo, H; Wang, T; Louie, P K K

    2004-06-01

    Receptor-oriented source apportionment models are often used to identify sources of ambient air pollutants and to estimate source contributions to air pollutant concentrations. In this study, a PCA/APCS model was applied to the data on non-methane hydrocarbons (NMHCs) measured from January to December 2001 at two sampling sites: Tsuen Wan (TW) and Central & Western (CW) Toxic Air Pollutants Monitoring Stations in Hong Kong. This multivariate method enables the identification of major air pollution sources along with the quantitative apportionment of each source to pollutant species. The PCA analysis identified four major pollution sources at TW site and five major sources at CW site. The extracted pollution sources included vehicular internal engine combustion with unburned fuel emissions, use of solvent particularly paints, liquefied petroleum gas (LPG) or natural gas leakage, and industrial, commercial and domestic sources such as solvents, decoration, fuel combustion, chemical factories and power plants. The results of APCS receptor model indicated that 39% and 48% of the total NMHCs mass concentrations measured at CW and TW were originated from vehicle emissions, respectively. 32% and 36.4% of the total NMHCs were emitted from the use of solvent and 11% and 19.4% were apportioned to the LPG or natural gas leakage, respectively. 5.2% and 9% of the total NMHCs mass concentrations were attributed to other industrial, commercial and domestic sources, respectively. It was also found that vehicle emissions and LPG or natural gas leakage were the main sources of C(3)-C(5) alkanes and C(3)-C(5) alkenes while aromatics were predominantly released from paints. Comparison of source contributions to ambient NMHCs at the two sites indicated that the contribution of LPG or natural gas at CW site was almost twice that at TW site. High correlation coefficients (R(2) > 0.8) between the measured and predicted values suggested that the PCA/APCS model was applicable for estimation

  1. Identification of Aging-Associated Gene Expression Signatures That Precede Intestinal Tumorigenesis.

    PubMed

    Okuchi, Yoshihisa; Imajo, Masamichi; Mizuno, Rei; Kamioka, Yuji; Miyoshi, Hiroyuki; Taketo, Makoto Mark; Nagayama, Satoshi; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    Aging-associated alterations of cellular functions have been implicated in various disorders including cancers. Due to difficulties in identifying aging cells in living tissues, most studies have focused on aging-associated changes in whole tissues or certain cell pools. Thus, it remains unclear what kinds of alterations accumulate in each cell during aging. While analyzing several mouse lines expressing fluorescent proteins (FPs), we found that expression of FPs is gradually silenced in the intestinal epithelium during aging in units of single crypt composed of clonal stem cell progeny. The cells with low FP expression retained the wild-type Apc allele and the tissues composed of them did not exhibit any histological abnormality. Notably, the silencing of FPs was also observed in intestinal adenomas and the surrounding normal mucosae of Apc-mutant mice, and mediated by DNA methylation of the upstream promoter. Our genome-wide analysis then showed that the silencing of FPs reflects specific gene expression alterations during aging, and that these alterations occur in not only mouse adenomas but also human sporadic and hereditary (familial adenomatous polyposis) adenomas. Importantly, pharmacological inhibition of DNA methylation, which suppresses adenoma development in Apc-mutant mice, reverted the aging-associated silencing of FPs and gene expression alterations. These results identify aging-associated gene expression signatures that are heterogeneously induced by DNA methylation and precede intestinal tumorigenesis triggered by Apc inactivation, and suggest that pharmacological inhibition of the signature genes could be a novel strategy for the prevention and treatment of intestinal tumors. PMID:27589228

  2. Identification of Aging-Associated Gene Expression Signatures That Precede Intestinal Tumorigenesis

    PubMed Central

    Okuchi, Yoshihisa; Imajo, Masamichi; Mizuno, Rei; Kamioka, Yuji; Miyoshi, Hiroyuki; Taketo, Makoto Mark; Nagayama, Satoshi; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    Aging-associated alterations of cellular functions have been implicated in various disorders including cancers. Due to difficulties in identifying aging cells in living tissues, most studies have focused on aging-associated changes in whole tissues or certain cell pools. Thus, it remains unclear what kinds of alterations accumulate in each cell during aging. While analyzing several mouse lines expressing fluorescent proteins (FPs), we found that expression of FPs is gradually silenced in the intestinal epithelium during aging in units of single crypt composed of clonal stem cell progeny. The cells with low FP expression retained the wild-type Apc allele and the tissues composed of them did not exhibit any histological abnormality. Notably, the silencing of FPs was also observed in intestinal adenomas and the surrounding normal mucosae of Apc-mutant mice, and mediated by DNA methylation of the upstream promoter. Our genome-wide analysis then showed that the silencing of FPs reflects specific gene expression alterations during aging, and that these alterations occur in not only mouse adenomas but also human sporadic and hereditary (familial adenomatous polyposis) adenomas. Importantly, pharmacological inhibition of DNA methylation, which suppresses adenoma development in Apc-mutant mice, reverted the aging-associated silencing of FPs and gene expression alterations. These results identify aging-associated gene expression signatures that are heterogeneously induced by DNA methylation and precede intestinal tumorigenesis triggered by Apc inactivation, and suggest that pharmacological inhibition of the signature genes could be a novel strategy for the prevention and treatment of intestinal tumors. PMID:27589228

  3. Stockpile Stewardship: Los Alamos

    SciTech Connect

    McMillan, Charlie; Morgan, Nathanial; Goorley, Tom; Merrill, Frank; Funk, Dave; Korzekwa, Deniece; Laintz, Ken

    2012-01-26

    "Heritage of Science" is a short video that highlights the Stockpile Stewardship program at Los Alamos National Laboratory. Stockpile Stewardship was conceived in the early 1990s as a national science-based program that could assure the safety, security, and effectiveness of the U.S. nuclear deterrent without the need for full-scale underground nuclear testing. This video was produced by Los Alamos National Laboratory for screening at the Lab's Bradbury Science Museum in Los Alamos, NM and is narrated by science correspondent Miles O'Brien.

  4. Stockpile Stewardship: Los Alamos

    ScienceCinema

    McMillan, Charlie; Morgan, Nathanial; Goorley, Tom; Merrill, Frank; Funk, Dave; Korzekwa, Deniece; Laintz, Ken

    2016-07-12

    "Heritage of Science" is a short video that highlights the Stockpile Stewardship program at Los Alamos National Laboratory. Stockpile Stewardship was conceived in the early 1990s as a national science-based program that could assure the safety, security, and effectiveness of the U.S. nuclear deterrent without the need for full-scale underground nuclear testing. This video was produced by Los Alamos National Laboratory for screening at the Lab's Bradbury Science Museum in Los Alamos, NM and is narrated by science correspondent Miles O'Brien.

  5. A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony

    PubMed Central

    Whitfield, Zachary J.; Chisholm, Jennifer; Hawley, R. Scott; Orr-Weaver, Terry L.

    2013-01-01

    Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. We performed immunoprecipitation of Cortex followed by mass spectrometry, and identified the Polo kinase inhibitor Matrimony (Mtrm) as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. We found Mtrm protein levels to be reduced dramatically during the oocyte-to-embryo transition, and this downregulation did not take place in cort mutant eggs, consistent with Mtrm being a substrate of APCCort. We showed that Mtrm is subject to APCCort-mediated proteasomal degradation and have identified a putative APC/C recognition motif in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APCCort ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence. PMID:24019759

  6. The efficacy of recombinant human activated protein C (rhAPC) vs antithrombin III (at III) vs heparin, in the healing process of partial-thickness burns: a comparative study

    PubMed Central

    Kritikos, O.; Tsagarakis, M.; Tsoutsos, D.; Kittas, C.; Gorgoulis, V.; Papalois, A.; Giannopoulos, A.; Kakiopoulos, G.; Papadopoulos, O.

    2012-01-01

    Summary This is an experimental study regarding the positive effect of recombinant human activated protein C (rhAPC) in the healing process of partial-thickness burns, in comparison to antithrombin III and heparin. On a porcine model we induced superficial partial-thickness and deep partial-thickness burns and performed intravenous administration of the elements of study during the first 48 h. The progress of the condition of the injured tissues was evaluated by histopathological examination at specific time intervals. The results showed an improved healing response of the specimens treated with rhAPC compared to those treated with antithrombin III, heparin, and placebo. PMID:23233823

  7. Mutation analysis of 13 driver genes of colorectal cancer-related pathways in Taiwanese patients

    PubMed Central

    Chang, Yuli Christine; Chang, Jan-Gowth; Liu, Ta-Chih; Lin, Chien-Yu; Yang, Shu-Fen; Ho, Cheng-Mao; Chen, William Tzu-Liang; Chang, Ya-Sian

    2016-01-01

    AIM: To investigate the driver gene mutations associated with colorectal cancer (CRC) in the Taiwanese population. METHODS: In this study, 103 patients with CRC were evaluated. The samples consisted of 66 men and 37 women with a median age of 59 years and an age range of 26-86 years. We used high-resolution melting analysis (HRM) and direct DNA sequencing to characterize the mutations in 13 driver genes of CRC-related pathways. The HRM assays were conducted using the LightCycler® 480 Instrument provided with the software LightCycler® 480 Gene Scanning Software Version 1.5. We also compared the clinicopathological data of CRC patients with the driver gene mutation status. RESULTS: Of the 103 patients evaluated, 73.79% had mutations in one of the 13 driver genes. We discovered 18 novel mutations in APC, MLH1, MSH2, PMS2, SMAD4 and TP53 that have not been previously reported. Additionally, we found 16 de novo mutations in APC, BMPR1A, MLH1, MSH2, MSH6, MUTYH and PMS2 in cancerous tissues previously reported in the dbSNP database; however, these mutations could not be detected in peripheral blood cells. The APC mutation correlates with lymph node metastasis (34.69% vs 12.96%, P = 0.009) and cancer stage (34.78% vs 14.04%, P = 0.013). No association was observed between other driver gene mutations and clinicopathological features. Furthermore, having two or more driver gene mutations correlates with the degree of lymph node metastasis (42.86% vs 24.07%, P = 0.043). CONCLUSION: Our findings confirm the importance of 13 CRC-related pathway driver genes in the development of CRC in Taiwanese patients. PMID:26900293

  8. Modified irinotecan and infusional 5-fluorouracil (mFOLFIRI) in patients with refractory advanced pancreas cancer (APC): a single-institution experience.

    PubMed

    Bupathi, M; Ahn, D H; Wu, C; Ciombor, K K; Stephens, J A; Reardon, J; Goldstein, D A; Bekaii-Saab, T

    2016-04-01

    Pancreatic adenocarcinoma is the fourth leading cause of cancer death. Recently, MM-398 (nanoliposomal irinotecan) was shown to be associated with significant improvement in outcome measures with acceptable toxicities when combined with 5-fluorouracil (5-FU)/leucovorin (LV) compared to 5-FU/LV alone in patients failing one line of gemcitabine-based therapy. There is a paucity of data evaluating the role of irinotecan in combination with 5FU in advanced pancreas cancer (APC). We performed a retrospective analysis of all patients who received mFOLFIRI (minus bolus 5FU and LV). All patients with metastatic disease who had failed at least one line of gemcitabine-based therapy prior to receiving mFOLFIRI were included in this study. Descriptive statistics were used to assess the continuous variables and adverse events (AEs), and Kaplan-Meier methods were used to calculate the median progression-free survival (PFS) and overall survival (OS). Forty patients were included in this analysis. Patients received 1-5 lines of prior therapy (25 % with more than 3 lines of prior therapy). The mean age at diagnosis was 60, and 98 % had ECOG of 1. The mean CA 19-9 at the start of therapy was 33,169 U/ml. The median PFS was 2.59 months [95 % confidence interval (CI) (1.90, 3.54)], and OS was 4.75 months [95 % CI (3.14, 8.98)]. The most common AEs included fatigue (98 %), neuropathy (83 %), anorexia (68 %), nausea (60 %) and constipation (55 %). Grade 3 toxicities included fatigue (13 %) and rash (3 %). There were no observed grade 4 toxicities. In this single-institution retrospective analysis, mFOLFIRI was found to be both tolerable and relatively effective in a heavily pretreated patient population with APC. Future prospective studies should consider evaluating the role of mFOLFIRI in refractory APC. PMID:26995224

  9. Los Alamos offers Fellowships

    NASA Astrophysics Data System (ADS)

    Los Alamos National Laboratory in New Mexico is calling for applications for postdoctoral appointments and research fellowships. The positions are available in geoscience as well as other scientific disciplines.The laboratory, which is operated by the University of California for the Department of Energy, awards J. Robert Oppenheimer Research Fellowships to scientists that either have or will soon complete doctoral degrees. The appointments are for two years, are renewable for a third year, and carry a stipend of $51,865 per year. Potential applicants should send a resume or employment application and a statement of research goals to Carol M. Rich, Div. 89, Human Resources Development Division, MS P290, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 by mid-November.

  10. Evaluation and Analysis of SEASAT-A Scanning Multichannel Microwave Radiometer (SSMR) Antenna Pattern Correction (APC) Algorithm. Sub-task 4: Interim Mode T Sub B Versus Cross and Nominal Mode T Sub B

    NASA Technical Reports Server (NTRS)

    Kitzis, J. L.; Kitzis, S. N.

    1979-01-01

    The brightness temperature data produced by the SMMR Antenna Pattern Correction algorithm are evaluated. The evaluation consists of: (1) a direct comparison of the outputs of the interim, cross, and nominal APC modes; (2) a refinement of the previously determined cos beta estimates; and (3) a comparison of the world brightness temperature (T sub B) map with actual SMMR measurements.

  11. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  12. The zinc-binding region (ZBR) fragment of Emi2 can inhibit APC/C by targeting its association with the coactivator Cdc20 and UBE2C-mediated ubiquitylation

    PubMed Central

    Shoji, Shisako; Muto, Yutaka; Ikeda, Mariko; He, Fahu; Tsuda, Kengo; Ohsawa, Noboru; Akasaka, Ryogo; Terada, Takaho; Wakiyama, Motoaki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2014-01-01

    Anaphase-promoting complex or cyclosome (APC/C) is a multisubunit ubiquitin ligase E3 that targets cell-cycle regulators. Cdc20 is required for full activation of APC/C in M phase, and mediates substrate recognition. In vertebrates, Emi2/Erp1/FBXO43 inhibits APC/C-Cdc20, and functions as a cytostatic factor that causes long-term M phase arrest of mature oocytes. In this study, we found that a fragment corresponding to the zinc-binding region (ZBR) domain of Emi2 inhibits cell-cycle progression, and impairs the association of Cdc20 with the APC/C core complex in HEK293T cells. Furthermore, we revealed that the ZBR fragment of Emi2 inhibits in vitro ubiquitin chain elongation catalyzed by the APC/C cullin-RING ligase module, the ANAPC2–ANAPC11 subcomplex, in combination with the ubiquitin chain-initiating E2, E2C/UBE2C/UbcH10. Structural analyses revealed that the Emi2 ZBR domain uses different faces for the two mechanisms. Thus, the double-faced ZBR domain of Emi2 antagonizes the APC/C function by inhibiting both the binding with the coactivator Cdc20 and ubiquitylation mediated by the cullin-RING ligase module and E2C. In addition, the tail region between the ZBR domain and the C-terminal RL residues [the post-ZBR (PZ) region] interacts with the cullin subunit, ANAPC2. In the case of the ZBR fragment of the somatic paralogue of Emi2, Emi1/FBXO5, these inhibitory activities against cell division and ubiquitylation were not observed. Finally, we identified two sets of key residues in the Emi2 ZBR domain that selectively exert each of the dual Emi2-specific modes of APC/C inhibition, by their mutation in the Emi2 ZBR domain and their transplantation into the Emi1 ZBR domain. PMID:25161877

  13. Human Bubr1 Is a Mitotic Checkpoint Kinase That Monitors Cenp-E Functions at Kinetochores and Binds the Cyclosome/APC

    PubMed Central

    Chan, G.K.T.; Jablonski, S.A.; Sudakin, V.; Hittle, J.C.; Yen, T.J.

    1999-01-01

    Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386–396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300–303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727–735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E–dependent activities at the kinetochore and regulating cyclosome/APC activity. PMID:10477750

  14. Id1 Deficiency Protects against Tumor Formation in Apc(Min/+) Mice but Not in a Mouse Model of Colitis-Associated Colon Cancer.

    PubMed

    Zhang, Ning; Subbaramaiah, Kotha; Yantiss, Rhonda K; Zhou, Xi Kathy; Chin, Yvette; Benezra, Robert; Dannenberg, Andrew J

    2015-04-01

    Different mechanisms contribute to the development of sporadic, hereditary and colitis-associated colorectal cancer. Inhibitor of DNA binding/differentiation (Id) proteins act as dominant-negative antagonists of basic helix-loop-helix transcription factors. Id1 is a promising target for cancer therapy, but little is known about its role in the development of colon cancer. We used immunohistochemistry to demonstrate that Id1 is overexpressed in human colorectal adenomas and carcinomas, whether sporadic or syndromic. Furthermore, elevated Id1 levels were found in dysplasia and colon cancer arising in patients with inflammatory bowel disease. Because levels of PGE2 are also elevated in both colitis and colorectal neoplasia, we determined whether PGE2 could induce Id1. PGE2 via EP4 stimulated protein kinase A activity resulting in enhanced pCREB-mediated Id1 transcription in human colonocytes. To determine the role of Id1 in carcinogenesis, two mouse models were used. Consistent with the findings in humans, Id1 was overexpressed in tumors arising in both Apc(Min) (/+) mice, a model of familial adenomatous polyposis, and in experimental colitis-associated colorectal neoplasia. Id1 deficiency led to significant decrease in the number of intestinal tumors in Apc(Min) (/+) mice and prolonged survival. In contrast, Id1 deficiency did not affect the number or size of tumors in the model of colitis-associated colorectal neoplasia, likely due to exacerbation of colitis associated with Id1 loss. Collectively, these results suggest that Id1 plays a role in gastrointestinal carcinogenesis. Our findings also highlight the need for different strategies to reduce the risk of colitis-associated colorectal cancer compared with sporadic or hereditary colorectal cancer.

  15. The Los Alamos primer

    SciTech Connect

    Serber, R.

    1992-01-01

    This book contains the 1943 lecture notes of Robert Serber. Serber was a protege of J. Robert Oppenheimer and member of the team that built the first atomic bomb - reveal what the Los Alamos scientists knew, and did not know, about the terrifying weapon they were building.

  16. Los Angeles, CA.

    NASA Technical Reports Server (NTRS)

    1982-01-01

    A general view of the greater Los Angeles region of California (34,0N, 118.5W) showing the coastline, Vandenberg AFB, the south end of the Central Valley and the dry lake beds at Edwards AFB, landing site of the space shuttle.

  17. A recellularized human colon model identifies cancer driver genes.

    PubMed

    Chen, Huanhuan Joyce; Wei, Zhubo; Sun, Jian; Bhattacharya, Asmita; Savage, David J; Serda, Rita; Mackeyev, Yuri; Curley, Steven A; Bu, Pengcheng; Wang, Lihua; Chen, Shuibing; Cohen-Gould, Leona; Huang, Emina; Shen, Xiling; Lipkin, Steven M; Copeland, Neal G; Jenkins, Nancy A; Shuler, Michael L

    2016-08-01

    Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology. PMID:27398792

  18. Enhanced hypoxic preconditioning by isoflurane: signaling gene expression and requirement of intracellular Ca2+ and inositol triphosphate receptors

    PubMed Central

    Bickler, Philip E.; Fahlman, Christian S.

    2012-01-01

    Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca2+ and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day-old rats were preconditioned with hypoxia (95% N2, 5% CO2 for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O2 and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 hr after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca2+ in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca2+ buffer BAPTA-AM, the IP3 receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64–86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca2+ chelation and RNA silencing of IP3 receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP3 receptors and intracellular Ca2+. PMID:20434434

  19. Enhanced hypoxic preconditioning by isoflurane: signaling gene expression and requirement of intracellular Ca2+ and inositol triphosphate receptors.

    PubMed

    Bickler, Philip E; Fahlman, Christian S

    2010-06-22

    Neurons preconditioned with non-injurious hypoxia or the anesthetic isoflurane express different genes but are equally protected against severe hypoxia/ischemia. We hypothesized that neuroprotection would be augmented when preconditioning with isoflurane and hypoxic preconditioning are combined. We also tested if preconditioning requires intracellular Ca(2+) and the inositol triphosphate receptor, and if gene expression is similar in single agent and combined preconditioning. Hippocampal slice cultures prepared from 9 day old rats were preconditioned with hypoxia (95% N(2), 5% CO(2) for 15 min, HPC), 1% isoflurane for 15 min (APC) or their combination (CPC) for 15 min. A day later cultures were deprived of O(2) and glucose (OGD) to produce neuronal injury. Cell death was assessed 48 h after OGD. mRNA encoding 119 signal transduction genes was quantified with cDNA micro arrays. Intracellular Ca(2+) in CA1 region was measured with fura-2 during preconditioning. The cell-permeable Ca(2+) buffer BAPTA-AM, the IP(3) receptor antagonist Xestospongin C and RNA silencing were used to investigate preconditioning mechanisms. CPC decreased CA1, CA3 and dentate region death by 64-86% following OGD, more than HPC or APC alone (P<0.01). Gene expression following CPC was an amalgam of gene expression in HPC and APC, with simultaneous increases in growth/development and survival/apoptosis regulation genes. Intracellular Ca(2+) chelation and RNA silencing of IP(3) receptors prevented preconditioning neuroprotection and gene responses. We conclude that combined isoflurane-hypoxia preconditioning augments neuroprotection compared to single agents in immature rat hippocampal slice cultures. The mechanism involves genes for growth, development, apoptosis regulation and cell survival as well as IP(3) receptors and intracellular Ca(2+).

  20. Los Angeles, CA, USA

    NASA Technical Reports Server (NTRS)

    1991-01-01

    This rare cloud and smog free view of Los Angeles, CA (34.0N, 118.5W) is a result of strong Santa Ana winds blowing from the east. Both cultural and natural features are well displayed and all of the major streets, highways and freeways can be traced in their entirety throughout the city as well as the major business and commercial sections. On the eastern edge of the scene, the San Andreas fault cuts across from southeast to northwest.

  1. Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis

    PubMed Central

    Jirholt, Pernilla; Turesson, Olof; Wing, Kajsa; Holmdahl, Rikard; Kihlberg, Jan; Stern, Anna; Mårtensson, Inga-Lill; Henningsson, Louise; Gustafsson, Kenth; Gjertsson, Inger

    2016-01-01

    Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases. PMID:27159398

  2. Detection of a Tumor Suppressor Gene Variant Predisposing to Colorectal Cancer in an 18th Century Hungarian Mummy

    PubMed Central

    Feldman, Michal; Hershkovitz, Israel; Sklan, Ella H.; Kahila Bar-Gal, Gila; Pap, Ildikó; Szikossy, Ildikó; Rosin-Arbesfeld, Rina

    2016-01-01

    Mutations of the Adenomatous polyposis coli (APC) gene are common and strongly associated with the development of colorectal adenomas and carcinomas. While extensively studied in modern populations, reports on visceral tumors in ancient populations are scarce. To the best of our knowledge, genetic characterization of mutations associated with colorectal cancer in ancient specimens has not yet been described. In this study we have sequenced hotspots for mutations in the APC gene isolated from 18th century naturally preserved human Hungarian mummies. While wild type APC sequences were found in two mummies, we discovered the E1317Q missense mutation, known to be a colorectal cancer predisposing mutation, in a large intestine tissue of an 18th century mummy. Our data suggests that this genetic predisposition to cancer already existed in the pre-industrialization era. This study calls for similar investigations of ancient specimens from different periods and geographical locations to be conducted and shared for the purpose of obtaining a larger scale analysis that will shed light on past cancer epidemiology and on cancer evolution. PMID:26863316

  3. Ex vivo expansion of polyclonal and antigen-specific cytotoxic T lymphocytes by artificial APCs expressing ligands for the T-cell receptor, CD28 and 4-1BB.

    PubMed

    Maus, Marcela V; Thomas, Anna K; Leonard, Debra G B; Allman, David; Addya, Kathakali; Schlienger, Katia; Riley, James L; June, Carl H

    2002-02-01

    The ex vivo priming and expansion of human cytotoxic T lymphocytes (CTLs) has potential for use in immunotherapy applications for cancer and infectious diseases. To overcome the difficulty in obtaining sufficient numbers of CTLs, we have developed artificial antigen-presenting cells (aAPCs) expressing ligands for the T-cell receptor (TCR) and the CD28 and 4-1BB co-stimulatory surface molecules. These aAPCs reproducibly activate and rapidly expand polyclonal or antigen-specific CD8(+) T cells. The starting repertoire of CD8+ T cells was preserved during culture. Furthermore, apoptosis of cultured CD8(+) T cells was diminished by this approach. This approach may have important therapeutic implications for adoptive immunotherapy. PMID:11821859

  4. Evaluation and analysis of Seasat-A Scanning multichannel Microwave radiometer (SMMR) Antenna Pattern Correction (APC) algorithm. Sub-task 2: T sub B measured vs. T sub B calculated comparison results

    NASA Technical Reports Server (NTRS)

    Kitzis, J. L.; Kitzis, S. N.

    1979-01-01

    Interim Antenna Pattern Correction (APC) brightness temperature measurements for all ten SMMR channels are compared with calculated values generated from surface truth data. Plots and associated statistics are presented for the available points of coincidence between SMMR and surface truth measurements acquired for the Gulf of Alaska SEASAT Experiment. The most important conclusions of the study deal with the apparent existence of different instrument biases for each SMMR channel, and their variation across the scan.

  5. Colon tumors with the simultaneous induction of driver mutations in APC, KRAS, and PIK3CA still progress through the adenoma-to-carcinoma sequence

    PubMed Central

    Hadac, Jamie N.; Leystra, Alyssa A.; Olson, Terrah J. Paul; Maher, Molly E.; Payne, Susan N; Yueh, Alexander E.; Schwartz, Alexander R.; Albrecht, Dawn M.; Clipson, Linda; Pasch, Cheri A.; Matkowskyj, Kristina A.; Halberg, Richard B.; Deming, Dustin A.

    2015-01-01

    Human colorectal cancers often possess multiple mutations, including 3–6 driver mutations per tumor. The timing of when these mutations occur during tumor development and progression continues to be debated. More advanced lesions carry a greater number of driver mutations, indicating that colon tumors might progress from adenomas to carcinomas through the stepwise accumulation of mutations following tumor initiation. However, mutations that have been implicated in tumor progression have been identified in normal-appearing epithelial cells of the colon, leaving the possibility that these mutations might be present prior to the initiation of tumorigenesis. We utilized mouse models of colon cancer to investigate whether tumorigenesis still occurs through the adenoma-to-carcinoma sequence when multiple mutations are present at the time of tumor initiation. To create a model in which tumors could concomitantly possess mutations in Apc, Kras, and Pik3ca, we developed a novel minimally invasive technique to administer an adenovirus expressing Cre recombinase to a focal region of the colon. Here we demonstrate that the presence of these additional driver mutations at the time of tumor initiation results in increased tumor multiplicity and an increased rate of progression to invasive adenocarcinomas. These cancers can even metastasize to retroperitoneal lymph nodes or the liver. However, despite having as many as three concomitant driver mutations at the time of initiation, these tumors still proceed through the adenoma-to-carcinoma sequence. PMID:26276752

  6. Azathioprine Suppresses Ezrin-Radixin-Moesin-Dependent T Cell-APC Conjugation through Inhibition of Vav Guanosine Exchange Activity on Rac Proteins

    PubMed Central

    Poppe, Daniela; Tiede, Imke; Fritz, Gerhard; Becker, Christoph; Bartsch, Brigitte; Wirtz, Stefan; Strand, Dennis; Tanaka, Shinya; Galle, Peter R.; Bustelo, Xosé R.; Neurath, Markus F.

    2007-01-01

    We have shown recently that the azathioprine metabolite 6-Thio-GTP causes immunosuppression by blockade of GTPase activation in T lymphocytes. In the present study, we describe a new molecular mechanism by which 6-Thio-GTP blocks GTPase activation. Although 6-Thio-GTP could bind to various small GTPases, it specifically blocked activation of Rac1 and Rac2 but not of closely related Rho family members such as Cdc42 and RhoA in primary T cells upon stimulation with αCD28 or fibronectin. Binding of 6-Thio-GTP to Rac1 did not suppress Rac effector coupling directly but blocked Vav1 exchange activity upon 6-Thio-GTP hydrolysis, suggesting that 6-Thio-GTP loading leads to accumulation of 6-Thio-GDP-loaded, inactive Rac proteins over time by inhibiting Vav activity. In the absence of apoptosis, blockade of Vav-mediated Rac1 activation led to a blockade of ezrin-radixin-moesin dephosphorylation in primary T cells and suppression of T cell-APC conjugation. Azathioprine-generated 6-Thio-GTP thus prevents the development of an effective immune response via blockade of Vav activity on Rac proteins. These findings provide novel insights into the immunosuppressive effects of azathioprine and suggest that antagonists of the Vav-Rac signaling pathway may be useful for suppression of T cell-dependent pathogenic immune responses. PMID:16365460

  7. Diverse HLA-I Peptide Repertoires of the APC Lines MUTZ3-Derived Immature and Mature Dendritic Cells and THP1-Derived Macrophages.

    PubMed

    Nyambura, Lydon Wainaina; Jarmalavicius, Saulius; Baleeiro, Renato Brito; Walden, Peter

    2016-09-15

    Dendritic cells (DCs) and macrophages are specialized APCs that process and present self-Ags for induction of tolerance and foreign Ags to initiate T cell-mediated immunity. Related to differentiation states they have specific phenotypes and functions. However, the impact of these differentiations on Ag processing and presentation remains poorly defined. To gain insight into this, we analyzed and compared the HLA-I peptidomes of MUTZ3-derived human immature and mature DC lines and THP1-derived macrophages by liquid chromatography tandem mass spectrometry. We found that the HLA-I peptidomes were heterogeneous and individualized and were dominated by nonapeptides with similar HLA-I binding affinities and anchor residues. MUTZ3-derived DCs and THP1-derived macrophages were able to sample peptides from source proteins of almost all subcellular locations and were involved in various cellular functions in similar proportion, with preference to proteins involved in cell communication, signal transduction, protein metabolism, and transcription factor/regulator activity. PMID:27543614

  8. Stress-strain behaviour of IM7/977-2 and IM7/APC2 carbon fibre composites at low and high strain rates

    NASA Astrophysics Data System (ADS)

    Parry, D. J.; Al-Hazmi, F. S.

    2003-09-01

    The dynamic compressive behaviour of two IM7 carbon fibre composite systems has been studied at room temperature at strain rates from about 10^{-3} s^{-1} to 10^3 s^{-1}. A Hopkinson bar method was used for high rates and a conventional screw machine for low rates. One composite system (IM7/977-2) has a thermoset epoxy resin matrix toughened with thermoplastic material; the other system (IM7/APC2) has a thermoplastic PEEK matrix. Three types of fibre configuration were used for each system: (a) longitudinal unidirectional, 0° (LU), (b) transverse unidirectional, 90° (TU), and (c) transverse isotropic, (0°/90°/±45°)s (TI). At low rates, both systems showed similar stress-strain behaviour. For the LU types, the fracture stresses were about 790 MPa, while for the TU types the values were about 235 MPa. For the Tl specimens, the maximum available stress of 950 MPa was insufficient for fracture. At high rates, the fracture stress was about 25% greater than the quasistatic values for both LU types, and about 30% higher for both TU types. The major difference between the two systems was with the TI types: the energy density to fracture was about 65 MJ m^{-3} for the epoxy composite, while it was about 40 MJ m^{-3} for the PEEK composite.

  9. Synthesis, characterization, molecular modeling and antioxidant activity of (1E,5E)-1,5-bis(1-(pyridin-2-yl)ethylidene)carbonohydrazide (H2APC) and its zinc(II), cadmium(II) and mercury(II) complexes

    NASA Astrophysics Data System (ADS)

    El-Gammal, O. A.; Abu El-Reash, G. M.; Ghazy, S. E.; Radwan, A. H.

    2012-08-01

    A new series of Zn(II), Cd(II) and Hg(II) complexes of (1E,5E)-1,5-bis(1-(pyridin-2-yl)ethylidene)carbonohydrazide (H2APC) have been prepared and characterized by elemental analyses, spectral (IR, UV-visible, mass and 1H NMR) as well as magnetic and thermal measurements. The data revealed that the ligand acts a monobasic hexadentate, neutral tri- and monodentate in Zn(II), Cd(II) and Hg(II) complexes, respectively. An octahedral geometry is proposed for Zn(II) complex, a trigonal bi-pyramid for Cd(II) complex and a tetrahedral one for Hg(II) complex. The bond length, bond angle, HOMO, LUMO and charges on the atoms have been calculated to confirm the geometry of the ligand and the investigated complexes using material studio program. Kinetic parameters were determined for each thermal degradation stage of some complexes using Coats-Redfern and Horowitz-Metzger methods. The antioxidant, anti-hemolytic, and cytotoxic activities of the compounds have been screened. H2APC showed moderate antioxidant activity using ABTS and DPPH methods. With respect to erythrocyte hemolysis and in vitro Ehrlich ascites assay, H2APC exhibited the potent antioxidative activity followed by Cd(II) and Zn(II) complexes while Hg(II) complex showed very weak activity.

  10. Los Alamos National Laboratory

    SciTech Connect

    Dogliani, Harold O

    2011-01-19

    The purpose of the briefing is to describe general laboratory technical capabilities to be used for various groups such as military cadets or university faculty/students and post docs to recruit into a variety of Los Alamos programs. Discussed are: (1) development and application of high leverage science to enable effeictive, predictable and reliability outcomes; (2) deter, detect, characterize, reverse and prevent the proliferation of weapons of mass destruction and their use by adversaries and terrorists; (3) modeling and simulation to define complex processes, predict outcomes, and develop effective prevention, response, and remediation strategies; (4) energetic materials and hydrodynamic testing to develop materials for precise delivery of focused energy; (5) materials cience focused on fundamental understanding of materials behaviors, their quantum-molecular properties, and their dynamic responses, and (6) bio-science to rapidly detect and characterize pathogens, to develop vaccines and prophylactic remedies, and to develop attribution forensics.

  11. Los biocombustibles y el futuro

    NASA Video Gallery

    ¿Cómo podremos utilizar los biocombustibles en el futuro? La ingeniera aeroespacial de la NASA, Diana Centeno Gómez nos explica el futuro de los biocombustibles y cómo un día podrías trabajar con d...

  12. Suppressive effects of the NADPH oxidase inhibitor apocynin on intestinal tumorigenesis in obese KK-A(y) and Apc mutant Min mice.

    PubMed

    Komiya, Masami; Fujii, Gen; Miyamoto, Shingo; Takahashi, Mami; Ishigamori, Rikako; Onuma, Wakana; Ishino, Kousuke; Totsuka, Yukari; Fujimoto, Kyoko; Mutoh, Michihiro

    2015-11-01

    Obesity is a risk factor for colorectal cancer. The accumulation of abdominal fat tissue causes abundant reactive oxygen species production through the activation of NADPH oxidase due to excessive insulin stimulation. The enzyme NADPH oxidase catalyzes the production of reactive oxygen species and evokes the initiation and progression of tumorigenesis. Apocynin is an NADPH oxidase inhibitor that blocks the formation of the NADPH oxidase complex (active form). In this study, we investigated the effects of apocynin on the development of azoxymethane-induced colonic aberrant crypt foci in obese KK-A(y) mice and on the development of intestinal polyps in Apc mutant Min mice. Six-week-old KK-A(y) mice were injected with azoxymethane (200 μg/mouse once per week for 3 weeks) and given 250 mg/L apocynin or 500 mg/L apocynin in their drinking water for 7 weeks. Six-week-old Min mice were also treated with 500 mg/L apocynin for 6 weeks. Treatment with apocynin reduced the number of colorectal aberrant crypt foci in KK-A(y) mice by 21% and the number of intestinal polyps in Min mice by 40% compared with untreated mice. Both groups of mice tended to show improved oxidation of serum low-density lipoprotein and 8-oxo-2'-deoxyguanosine adducts in their adipose tissues. In addition, the inducible nitric oxide synthase mRNA levels in polyp tissues decreased. Moreover, apocynin was shown to suppress nuclear factor-κB transcriptional activity in vitro. These results suggest that apocynin and other NADPH oxidase inhibitors may be effective colorectal cancer chemopreventive agents.

  13. Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

    PubMed Central

    Huisman, Sander A; Bijman-Lagcher, Wendy; IJzermans, Jan NM; Smits, Ron; de Bruin, Ron WF

    2015-01-01

    Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients. PMID:25955194

  14. UV exposure inhibits intestinal tumor growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet.

    PubMed

    Rebel, Heggert; der Spek, Celia Dingemanse-van; Salvatori, Daniela; van Leeuwen, Johannes P T M; Robanus-Maandag, Els C; de Gruijl, Frank R

    2015-01-15

    Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV-induced vitamin D. FabplCre;Apc(15lox/+) mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: mean ± SD from 7.7 ± 1.9 in controls to 75 ± 15 nmol/l with vitamin D supplementation (no gender difference), and to 31 ± 13 nmol/l in males and 85 ± 17 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D-supplemented group (130 ± 25 mm(2), p = 0.018) and the UV-exposed group (88 ± 9 mm(2), p < 0.0005; no gender differences) compared to the control group (202 ± 23 mm(2)). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (p = 0.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.

  15. Ethanol-Induced Mast Cell-Mediated Inflammation Leads to Increased Susceptibility of Intestinal Tumorigenesis in the APC Δ468 Min Mouse Model of Colon Cancer

    PubMed Central

    Wimberly, Andre L.; Forsyth, Christopher B.; Khan, Mohammad Wasim; Pemberton, Alan; Khazaie, Khashayarsha; Keshavarzian, Ali

    2013-01-01

    Background Chronic and frequent ethanol (EtOH) intake has been associated with an increased incidence of several types of cancers including breast, mouth, throat, esophageal, stomach and colorectal (CRC). The underlying mechanism of this deleterious carcinogenic effect of alcohol has not been clearly established but inflammation may be one unifying feature of these cancers. We have recently shown that intestinal mast cells play a central role in intestinal carcinogenesis. In this study, we tested our hypothesis that mast cell-mediated inflammation is one underlying mechanism by which chronic alcohol promotes intestinal tumorigenesis. Methods APC Δ468 mice were fed either an alcohol containing Nanji liquid diet or isocaloric dextrose containing Nanji diet for 10 weeks and then sacrificed to collect small and large intestine samples. Assessments of tumor number and size as well as mast cell number and mast cell activity and histology score for invasion were compared between Control (dextrose fed) and Alcohol fed APCΔ468 mice. The effect of alcohol on mast cell mediated tumor migration was also assessed using an in vitro migration assay. Results Alcohol feeding increased both polyp number and size within both the small and large intestines of APCΔ468 mice. Only alcohol fed mice showed evidence of tumor invasion. Chronic alcohol feeding also resulted in an increased mast cell number and activity in tumor stroma and invading borders. In vitro migration assay showed that alcohol significantly increases mast cell mediated tumor migration in vitro. Conclusions Our data show that chronic alcohol intake promotes: (1) intestinal tumorigenesis and tumor invasion in genetically susceptible mice; (2) increases in polyp associated mast cells; (3) mast cell mediated tumor migration in vitro. Both our in vivo and in vitro studies suggest that mast cell mediated inflammation could be one mechanism by which alcohol promotes carcinogenesis. PMID:23320800

  16. TGF-β activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

    PubMed

    Pavlides, Savvas C; Lecanda, Jon; Daubriac, Julien; Pandya, Unnati M; Gama, Patricia; Blank, Stephanie; Mittal, Khushbakhat; Shukla, Pratibha; Gold, Leslie I

    2016-01-01

    We previously reported that aberrant TGF-β/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-β signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-β increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-β-mediated inhibition of proliferation. Protein synthesis was not required for TGF-β-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-β-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-β signaling.

  17. NARSTO EPA SS LOS ANGELES APS DATA

    Atmospheric Science Data Center

    2014-04-25

    NARSTO EPA SS LOS ANGELES APS DATA Project Title:  NARSTO Discipline:  ... Platform:  Ground Station Instrument:  APS - Aerodynamic Particle Sizer Location:  Los Angeles, ... Data Guide Documents:  Los Angeles APS Guide Los Angeles Project Plan  (PDF) Los Angeles ...

  18. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  19. ASC platforms at Los Alamos.

    SciTech Connect

    Shaw, S. R.

    2004-01-01

    This talk describes the history, current state, and future plans for ASC computational and data storage service at Los Alamos. The of the systems and services described is limited to those installed in and managed by Group CCN-7.

  20. Los Alamos National Laboratory Overview

    SciTech Connect

    Neu, Mary

    2010-06-02

    Mary Neu, Associate Director for Chemistry, Life and Earth Sciences at Los Alamos National Laboratory, delivers opening remarks at the "Sequencing, Finishing, Analysis in the Future" meeting in Santa Fe, NM

  1. HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

    PubMed

    Muthumani, Karuppiah; Shedlock, Devon J; Choo, Daniel K; Fagone, Paolo; Kawalekar, Omkar U; Goodman, Jonathan; Bian, Chaoran B; Ramanathan, Aarti A; Atman, Parikh; Tebas, Pablo; Chattergoon, Michael A; Choo, Andrew Y; Weiner, David B

    2011-09-15

    Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

  2. La inserción en el mercado laboral de los inmigrantes latinos en España y en los Estados Unidos: Diferencias por país de origen y estatus legal

    PubMed Central

    Connor, Phillip; Massey, Douglas

    2013-01-01

    Resumen Este artículo compara los resultados económicos entre los inmigrantes latinoamericanos en España y Estados Unidos. Detectamos un efecto de selección por el que la mayoría de los inmigrantes latinoamericanos en España proceden de Sudamérica de un entorno de clases medias, mientras la mayoría de los inmigrantes que van a los Estados Unidos son centroamericanos de clase baja. Este efecto de selección explica las diferencias transnacionales en la probabilidad de empleo, logro ocupacional y salarios obtenidos. A pesar de las diferencias en los orígenes y las características de los latinoamericanos en ambos países, los factores demográficos, humanos y de capital social parecen operar de forma similar en ambos países; y cuando los modelos se estiman separadamente por estatus legal, descubrimos que los efectos se acentúan más entre los inmigrantes irregulares cuando se los compara con los regulares, especialmente en Estados Unidos. PMID:24532857

  3. Promoter methylation status of tumor suppressor genes and inhibition of expression of DNA methyltransferase 1 in non-small cell lung cancer.

    PubMed

    Liu, Bangqing; Song, Jianfei; Luan, Jiaqiang; Sun, Xiaolin; Bai, Jian; Wang, Haiyong; Li, Angui; Zhang, Lifei; Feng, Xiaoyan; Du, Zhenzong

    2016-08-01

    DNA methylation is an epigenetic DNA modification catalyzed by DNA methyltransferase 1 (DNMT1). The purpose of this study was to investigate DNMT1 gene and protein expression and the effects of methylation status on tumor suppressor genes in human non-small cell lung cancer (NSCLC) cell lines grown in vitro and in vivo Human lung adenocarcinoma cell lines, A549 and H838, were grown in vitro and inoculated subcutaneously into nude mice to form tumors and were then treated with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, with and without treatment with the benzamide histone deacetylase inhibitor, entinostat (MS-275). DNMT1 protein expression was quantified by Western blot. Promoter methylation status of tumor suppressor genes (RASSF1A, ASC, APC, MGMT, CDH13, DAPK, ECAD, P16, and GATA4) was evaluated by methylation-specific polymerase chain reaction. Methylation status of the tumor suppressor genes was regulated by the DNMT1 gene, with the decrease of DNMT1 expression following DNA methylation treatment. Demethylation of tumor suppressor genes (APC, ASC, and RASSF1A) restored tumor growth in nude mice. The results of this study support a role for methylation of DNA as a potential epigenetic clinical biomarker of prognosis or response to therapy and for DNMT1 as a potential therapeutic target in NSCLC. PMID:27190263

  4. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  5. Mayo de Los Capomos, Sinaloa (Mayo of Los Capomos, Sinaloa).

    ERIC Educational Resources Information Center

    Freeze, Ray A.

    This document is one of 17 volumes on indigenous Mexican languages and is the result of a project undertaken by the Archivo de Lenguas Indigenas de Mexico. This volume contains information on Mayo, an indigenous language of Mexico spoken in Los Capomos, in the state of Sinaloa. The objective of collecting such a representative sampling of the…

  6. Los NIH anuncian el lanzamiento de los estudios ALCHEMIST

    Cancer.gov

    Los Estudios sobre la Secuenciación e Identificación de Marcadores para el Mejoramiento de la Terapia Adjuvante para el Cáncer de Pulmón, ALCHEMIST, identificarán a pacientes con cáncer de pulmón en estadio inicial cuyos tumores tienen cambios genéticos.

  7. Construction and Characterization of Haemophilus ducreyi Lipooligosaccharide (LOS) Mutants Defective in Expression of Heptosyltransferase III and β1,4-Glucosyltransferase: Identification of LOS Glycoforms Containing Lactosamine Repeats

    PubMed Central

    Filiatrault, Melanie J.; Gibson, Bradford W.; Schilling, Birgit; Sun, Shuhua; Munson, Robert S.; Campagnari, Anthony A.

    2000-01-01

    To begin to understand the role of the lipooligosaccharide (LOS) molecule in chancroid infections, we constructed mutants defective in expression of glycosyltransferase genes. Pyocin lysis and immunoscreening was used to identify a LOS mutant of Haemophilus ducreyi 35000. This mutant, HD35000R, produced a LOS molecule that lacked the monoclonal antibody 3F11 epitope and migrated with an increased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Structural studies indicated that the principal LOS glycoform contains lipid A, Kdo, and two of the three core heptose residues. HD35000R was transformed with a plasmid library of H. ducreyi 35000 DNA, and a clone producing the wild-type LOS was identified. Sequence analysis of the plasmid insert revealed one open reading frame (ORF) that encodes a protein with homology to the WaaQ (heptosyltransferase III) of Escherichia coli. A second ORF had homology to the LgtF (glucosyltransferase) of Neisseria meningitidis. Individual isogenic mutants lacking expression of the putative H. ducreyi heptosyltransferase III, the putative glucosyltransferase, and both glycosyltransferases were constructed and characterized. Each mutant was complemented with the representative wild-type genes in trans to restore expression of parental LOS and confirm the function of each enzyme. Matrix-assisted laser desorption ionization mass spectrometry and SDS-PAGE analysis identified several unique LOS glycoforms containing di-, tri-, and poly-N-acetyllactosamine repeats added to the terminal region of the main LOS branch synthesized by the heptosyltransferase III mutant. These novel H. ducreyi mutants provide important tools for studying the regulation of LOS assembly and biosynthesis. PMID:10816485

  8. Quantitative DNA Methylation Analysis of Candidate Genes in Cervical Cancer

    PubMed Central

    Siegel, Erin M.; Riggs, Bridget M.; Delmas, Amber L.; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D.

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97–1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated. PMID:25826459

  9. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    PubMed

    Siegel, Erin M; Riggs, Bridget M; Delmas, Amber L; Koch, Abby; Hakam, Ardeshir; Brown, Kevin D

    2015-01-01

    Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  10. Distribución en gran escala de los cúmulos globulares en Fornax

    NASA Astrophysics Data System (ADS)

    Ostrov, P. G.

    Para analizar los cúmulos globulares azules y rojos de NGC 1399 asociados con NGC 1399 en particular, o si los cúmulos azules representaban un sistema asociado con el cúmulo de Fornax en general, se obtuvieron imágenes CCD de gran formato con el telescopio de 4m del CTIO, en las bandas C y T1. Se describe el método empleado y lo encontrado.

  11. Palace Revolt in Los Angeles?

    ERIC Educational Resources Information Center

    Fuller, Bruce

    2010-01-01

    Antonio Villaraigosa, the mayor of Los Angeles, comes alive when recalling his start in local politics--as a labor organizer agitating for reform inside decrepit and overcrowded schools. In his quest to turn around the schools, the mayor has united working-class Latino parents, civil rights leaders, and big-money Democrats to challenge union…

  12. Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene.

    PubMed

    Qadri, Syed M; Mahmud, Hasan; Lang, Elisabeth; Gu, Shuchen; Bobbala, Diwakar; Zelenak, Christine; Jilani, Kashif; Siegfried, Alexandra; Föller, Michael; Lang, Florian

    2012-05-01

    Loss-of-function mutations in human adenomatous polyposis coli (APC) lead to multiple colonic adenomatous polyps eventually resulting in colonic carcinoma. Similarly, heterozygous mice carrying defective APC (apc(Min/+)) suffer from intestinal tumours. The animals further suffer from anaemia, which in theory could result from accelerated eryptosis, a suicidal erythrocyte death triggered by enhanced cytosolic Ca(2+) activity and characterized by cell membrane scrambling and cell shrinkage. To explore, whether APC-deficiency enhances eryptosis, we estimated cell membrane scrambling from annexin V binding, cell size from forward scatter and cytosolic ATP utilizing luciferin-luciferase in isolated erythrocytes from apc(Min/+) mice and wild-type mice (apc(+/+)). Clearance of circulating erythrocytes was estimated by carboxyfluorescein-diacetate-succinimidyl-ester labelling. As a result, apc(Min/+) mice were anaemic despite reticulocytosis. Cytosolic ATP was significantly lower and annexin V binding significantly higher in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Glucose depletion enhanced annexin V binding, an effect significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Extracellular Ca(2+) removal or inhibition of Ca(2+) entry with amiloride (1 mM) blunted the increase but did not abrogate the genotype differences of annexin V binding following glucose depletion. Stimulation of Ca(2+) -entry by treatment with Ca(2+) -ionophore ionomycin (10 μM) increased annexin V binding, an effect again significantly more pronounced in apc(Min/+) erythrocytes than in apc(+/+) erythrocytes. Following retrieval and injection into the circulation of the same mice, apc(Min/+) erythrocytes were more rapidly cleared from circulating blood than apc(+/+) erythrocytes. Most labelled erythrocytes were trapped in the spleen, which was significantly enlarged in apc(Min/+) mice. The observations point to accelerated eryptosis and subsequent

  13. Los Angeles and Its Mistress Machine

    ERIC Educational Resources Information Center

    Marx, Wesley

    1973-01-01

    Los Angeles city has acute air pollution problems because of lack of an adequate mass transit system and the type of local industries. Air pollution in Los Angeles has affected agricultural production, vegetation, and public health in nearby areas. (PS)

  14. Los Alamos Critical Assemblies Facility

    SciTech Connect

    Malenfant, R.E.

    1981-06-01

    The Critical Assemblies Facility of the Los Alamos National Laboratory has been in existence for thirty-five years. In that period, many thousands of measurements have been made on assemblies of /sup 235/U, /sup 233/U, and /sup 239/Pu in various configurations, including the nitrate, sulfate, fluoride, carbide, and oxide chemical compositions and the solid, liquid, and gaseous states. The present complex of eleven operating machines is described, and typical applications are presented.

  15. Yeast-Derived Particulate β-Glucan Treatment Subverts the Suppression of Myeloid-Derived Suppressor Cells (MDSC) by Inducing Polymorphonuclear MDSC Apoptosis and Monocytic MDSC Differentiation to APC in Cancer.

    PubMed

    Albeituni, Sabrin H; Ding, Chuanlin; Liu, Min; Hu, Xiaoling; Luo, Fengling; Kloecker, Goetz; Bousamra, Michael; Zhang, Huang-ge; Yan, Jun

    2016-03-01

    Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. In this study, we demonstrated that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived whole β-glucan particles (WGP; a ligand to engage and activate dectin-1, oral treatment in vivo) significantly decreased tumor weight and splenomegaly in tumor-bearing mice with reduced accumulation of polymorphonuclear MDSC but not monocytic MDSC (M-MDSC), and decreased polymorphonuclear MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiated into F4/80(+)CD11c(+) cells in vitro that served as potent APC to induce Ag-specific CD4(+) and CD8(+) T cell responses in a dectin-1-dependent manner. Additionally, Erk1/2 phosphorylation was required for the acquisition of APC properties in M-MDSC. Moreover, WGP-treated M-MDSC differentiated into CD11c(+) cells in vivo with high MHC class II expression and induced decreased tumor burden when inoculated s.c. with Lewis lung carcinoma cells. This effect was dependent on the dectin-1 receptor. Strikingly, patients with non-small cell lung carcinoma that had received WGP treatment for 10-14 d prior to any other treatment had a decreased frequency of CD14(-)HLA-DR(-)CD11b(+)CD33(+) MDSC in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.

  16. Olive oil prevents benzo(a)pyrene [B(a)P]-induced colon carcinogenesis through altered B(a)P metabolism and decreased oxidative damage in Apc(Min) mouse model.

    PubMed

    Banks, Leah D; Amoah, Priscilla; Niaz, Mohammad S; Washington, Mary K; Adunyah, Samuel E; Ramesh, Aramandla

    2016-02-01

    Colon cancer ranks third in cancer-related mortalities in the United States. Many studies have investigated factors that contribute to colon cancer in which dietary and environmental factors have been shown to play an integral role in the etiology of this disease. Specifically, human dietary intake of environmental carcinogens such as polycyclic aromatic hydrocarbons has generated interest in looking at how it exerts its effects in gastrointestinal carcinogenesis. Therefore, the objective of this study was to investigate the preventative effects of olive oil on benzo(a)pyrene [B(a)P]-induced colon carcinogenesis in adult Apc(Min) mice. Mice were assigned to a control (n=8) or treatment group (n=8) consisting of 25, 50 and 100-μg B(a)P/kg body weight (bw) dissolved in tricaprylin [B(a)P-only group] or olive oil daily via oral gavage for 60 days. Our studies showed that Apc(Min) mice exposed to B(a)P developed a significantly higher number (P<0.05) of larger dysplastic adenomas compared to those exposed to B(a)P + olive oil. Treatment of mice with B(a)P and olive oil significantly altered (P<0.05) the expression of drug-metabolizing enzymes in both the colon and liver tissues. However, only GST activity was significantly higher (P<0.05) in the liver of mice treated with 50- and 100-μg B(a)P/kg bw + olive oil. Lastly, olive oil promoted rapid detoxification of B(a)P by decreasing its organic metabolite concentrations and also decreasing the extent of DNA damage to colon and liver tissues (P<0.05). These results suggest that olive oil has a protective effect against B(a)P-induced colon tumors.

  17. Initial formal toxicity evaluation of APC-2, a novel fluorescent tracer agent for real-time measurement of glomerular filtration rate in preparation for a first-in-man clinical trial

    NASA Astrophysics Data System (ADS)

    Bugaj, Joseph E.; Dorshow, Richard B.

    2014-03-01

    The fluorescent tracer agent 2,5-bis[N-(1-carboxy-2-hydroxy)]carbamoyl-3,6-diaminopyrazine, designated APC-2, has been developed with properties and attributes necessary for use as a direct measure of glomerular filtration rate (GFR). Comparison to known standard exogenous GFR agents in animal models has demonstrated an excellent correlation. A clinical trial to demonstrate this same correlation in humans is in preparation. A battery of formal toxicity tests necessary for regulatory clearance to proceed with a clinical trial has been recently completed on this new fluorescent tracer agent. These include single dose toxicity studies in rats and dogs to determine overall toxicity and toxicokinetics of the compound. Blood compatibility, mutation assay, chromosomal aberration assay, and several other assays were also completed. Toxicity assessments were based on mortality, clinical signs, body weight, food consumption and anatomical pathology. Blood samples were collected to assess pharmacokinetic parameters including half-life, area under the curve, and clearance. Urine samples were collected to assess distribution. Doses of up to 200-300 times the estimated human dose were administered. No test-article related effects were noted on body weight, food consumption, ophthalmic observations and no abnormal pathology was seen in either macroscopic or microscopic evaluations of any organs or tissues. All animals survived to scheduled sacrifice. Transient discoloration of skin and urine was noted at the higher dose levels in both species as expected from a highly fluorescent compound and was not considered pathological. Thus initial toxicology studies of this new fluorescent tracer agent APC-2 have resulted in no demonstrable pathological test article concerns.

  18. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TENTH FLOOR SOUTH WING CAFETERIA FOOD LINE, FACING NORTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  19. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-FOURTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  20. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-SIXTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING SOUTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  1. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-SIXTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  2. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-FOURTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING SOUTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  3. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-SIXTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING NORTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  4. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-FOURTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING EAST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  5. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL TWENTY-SIXTH FLOOR EXISTING CONDITIONS SHOWING STRUCTURE AND BRACING, FACING SOUTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  6. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL TENTH FLOOR NORTH OFFICE WING SHOWING RADIATOR AND WINDOW, FACING EAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  7. John Ash, ALA., Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, ALA., Photographer August 1997. VIEW OF LOS ANGELES CITY HALL NINTH FLOOR NORTH OFFICE WING SHOWING PARTITIONS, WINDOWS AND RADIATOR, FACING SOUTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  8. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL NINTH FLOOR NORTH OFFICE WING SHOWING PARTITIONS, WINDOWS AND RADIATOR, FACING EAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  9. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR BREAK ROOM OFF OF ORIGINAL ART COMMISSION ROOMS, FACING NORTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  10. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR BREAK ROOM OFF OF ORIGINAL ART COMMISSION ROOMS, FACING SOUTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  11. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIRST FLOOR DOORS TO THE CITY CLERK AND TAX & PERMIT DIVISION OFFICES, FACING NORTH. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  12. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR SHOWING ROTUNDA COLUMN CAPITALS AND LIGHT FIXTURES, FACING SOUTHEAST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  13. Bruce D. Judd, FAIA, Photographer August 1997. DETAIL OF LOS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Bruce D. Judd, FAIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL NORTH ENTRANCE SHOWING DOORS AND LIGHT FIXTURES, FACING SOUTH. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  14. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL TENTH FLOOR SOUTH WING CAFETERIA LIGHT FIXTURE, FACING SOUTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  15. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR SHOWING ROTUNDA COLUMN CAPITALS AND LIGHT FIXTURES, FACING SOUTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  16. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR SHOWING ROTUNDA STANDING LIGHT FIXTURE, FACING NORTHEAST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  17. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL SOUTH ENTRY SHOWING DOORS AND LIGHT FIXTURES, FACING NORTH. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  18. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL EAST ENTRANCE PORCH SHOWING VAULTS AND LIGHT FIXTURE, FACING UP. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  19. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL WEST ENTRANCE COURTYARD SHOWING BRONZE DOORS, LIGHT FIXTURES AND GRILLS, FACING EAST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  20. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR CITY COUNCIL CHAMBERS SHOWING COLUMN, ARCADE AND SPECTATOR SEATING, FACING SOUTHWEST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  1. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR CITY COUNCIL CHAMBERS SHOWING PODIUM AND SEATING, FACING SOUTH. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  2. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL THIRD FLOOR CITY COUNCIL CHAMBERS SHOWING COLUMN, ARCADE AND SPECTATOR SEATING, FACING SOUTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  3. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL SEVENTH FLOOR CORRIDOR NEAR ELEVATOR LOBBY SHOWING FIRE HOSE CABINET, FACING EAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  4. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL FOURTEENTH FLOOR FIRE HOSE NEAR NORTHEAST STAIR, FACING EAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  5. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR NORTH OFFICE AREA, FACING NORTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  6. John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL SEVENTH FLOOR SOUTH OFFICE AREA SHOWING RADIATOR AND WINDOWS, FACING EAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  7. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR WEST OFFICE AREA THAT WAS ORIGINAL ART COMMISSION ROOMS, FACING NORTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  8. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR NORTH OFFICE AREA, FACING NORTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  9. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA SHOWING BEAM AND COLUMN CONNECTION NEAR NORTHWEST CORNER, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  10. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA SHOWING DEMOLITION OF SOUTH WALL, FACING SOUTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  11. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA SHOWING BEAM AND COLUMN CONNECTION NEAR SOUTHEAST CORNER, FACING SOUTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  12. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  13. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL SEVENTH FLOOR SOUTH OFFICE AREA SHOWING WOOD AND GLASS PARTITIONS, FACING SOUTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  14. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FOURTEENTH FLOOR, SERVICE AREA DOOR NEAR ELEVATOR LOBBY, FACING SOUTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  15. Bruce D. Judd, FAIA, Photographer August 1997. DETAIL OF LOS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Bruce D. Judd, FAIA, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL SIXTH FLOOR NORTH OFFICE AREA WINDOW, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  16. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA, FACING NORTHEAST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  17. John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    John Ash, AIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL SEVENTH FLOOR SOUTH OFFICE AREA SHOWING STRUCTURAL PIERS AND FLORESCENT LIGHTS, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  18. Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. DETAIL OF LOS ANGELES CITY HALL NINETEENTH FLOOR MAIN OFFICE AREA SHOWING DEMOLITION OF WEST WALL, FACING WEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  19. Bruce D. Judd, FAIA, Photographer August 1997. VIEW OF LOS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Bruce D. Judd, FAIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL ELEVENTH FLOOR KITCHEN OF EXECUTIVE DINING AREA SHOWING ARCHED STRUCTURE, FACING NORTHWEST - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  20. Bruce D. Judd, FAIA, Photographer August 1997. VIEW OF LOS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Bruce D. Judd, FAIA, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL SIXTH FLOOR NORTH OFFICE AREA, FACING NORTH - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  1. Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Monica Griesbach, Photographer August 1997. VIEW OF LOS ANGELES CITY HALL FIFTH FLOOR WEST OFFICE AREA THAT WAS ORIGINAL ART COMMISSION ROOMS, FACING NORTHWEST. - Los Angeles City Hall, 200 North Spring Street, Los Angeles, Los Angeles County, CA

  2. Los Alamos Science: Number 16

    SciTech Connect

    Cooper, N.G.

    1988-01-01

    It was an unusually stimulating day and a half at Los Alamos when two Nobel Laureates in physiology, a leading paleontologist, and a leading bio-astrophysicist came together to discuss ''Unsolved Problems in the Science of Life,'' the topic of the second in a series of special meetings sponsored by the Fellows of the Laboratory. Just like the first one on ''Creativity in Science,'' this colloquium took us into a broader arena of ideas and viewpoints than is our usual daily fare. To contemplate the evolution and mysteries of intelligent life from the speakers' diverse points of view at one time, in one place was indeed a rare experience.

  3. High Prevalence and Clinical Relevance of Genes Affected by Chromosomal Breaks in Colorectal Cancer

    PubMed Central

    van den Broek, Evert; Dijkstra, Maurits J. J.; Krijgsman, Oscar; Sie, Daoud; Haan, Josien C.; Traets, Joleen J. H.; van de Wiel, Mark A.; Nagtegaal, Iris D.; Punt, Cornelis J. A.; Carvalho, Beatriz; Ylstra, Bauke; Abeln, Sanne; Meijer, Gerrit A.; Fijneman, Remond J. A.

    2015-01-01

    Background Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes. Methods Primary CRC samples of patients with metastatic disease from CAIRO and CAIRO2 clinical trials were previously characterized by array-comparative genomic hybridization. These data were now used to determine the prevalence of CNA-associated chromosomal breaks within genes across 352 CRC samples. In addition, mutation status of the commonly affected APC, TP53, KRAS, PIK3CA, FBXW7, SMAD4, BRAF and NRAS genes was determined for 204 CRC samples by targeted massive parallel sequencing. Clinical relevance was assessed upon stratification of patients based on gene mutations and gene breakpoints that were observed in >3% of CRC cases. Results In total, 748 genes were identified that were recurrently affected by chromosomal breaks (FDR <0.1). MACROD2 was affected in 41% of CRC samples and another 169 genes showed breakpoints in >3% of cases, indicating that prevalence of gene breakpoints is comparable to the prevalence of well-known gene point mutations. Patient stratification based on gene breakpoints and point mutations revealed one CRC subtype with very poor prognosis. Conclusions We conclude that CNA-associated chromosomal breaks within genes represent a highly prevalent and clinically relevant subset of SVs in CRC. PMID:26375816

  4. Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer.

    PubMed

    Hoque, Mohammad Obaidul; Begum, Shahnaz; Topaloglu, Ozlem; Jeronimo, Carmen; Mambo, Elizabeth; Westra, William H; Califano, J A; Sidransky, David

    2004-08-01

    Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-beta2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-beta2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.

  5. Los Alamos opacity web page

    SciTech Connect

    Magee, N.H. Jr.; Clark, R.E.H.

    1998-02-01

    The Los Alamos opacity data base is now available on the World Wide Web at http://t4.lanl.gov. The data base contains both the original Astrophysical Opacity Library distributed worldwide in the 1980`s (for historical reference) and the new improved opacities from the Light Element Detailed Configuration OPacity (LEDCOP) code. Users can access the opacity data using the multigroup opacity code TOPS to obtain Rosseland and Planck gray opacities, group mean opacities over selected energy ranges, the monochromatic absorption coefficients and the average ionization over a wide range of temperatures and densities. As described in this paper, these quantities are available for all of the elements presently on the data base and TOPS will provide the same quantities for any arbitrary mixture of these elements.

  6. Los Alamos PC estimating system

    SciTech Connect

    Stutz, R.A.; Lemon, G.D.

    1987-01-01

    The Los Alamos Cost Estimating System (QUEST) is being converted to run on IBM personal computers. This very extensive estimating system is capable of supporting cost estimators from many different and varied fields. QUEST does not dictate any fixed method for estimating. QUEST supports many styles and levels of detail estimating. QUEST can be used with or without data bases. This system allows the estimator to provide reports based on levels of detail defined by combining work breakdown structures. QUEST provides a set of tools for doing any type of estimate without forcing the estimator to use any given method. The level of detail in the estimate can be mixed based on the amount of information known about different parts of the project. The system can support many different data bases simultaneously. Estimators can modify any cost in any data base.

  7. Gene doping.

    PubMed

    Harridge, Stephen D R; Velloso, Cristiana P

    2008-01-01

    Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities.

  8. Epigenetic regulation of human DCLK-1 gene during colon-carcinogenesis: clinical and mechanistic implications

    PubMed Central

    O’Connell, Malaney; Shubhashish, Sarkar

    2016-01-01

    Colorectal carcinogenesis is a multi-step process. While ~25% of colorectal cancers (CRCs) arise in patients with a family history (genetic predisposition), ~75% of CRCs are due to age-associated accumulation of epigenetic alterations which can result in the suppression of key tumor suppressor genes leading to mutations and activation of oncogenic pathways. Sporadic colon-carcinogenesis is facilitated by many molecular pathways of genomic instability which include chromosomal instability (CIN), micro-satellite instability (MSI) and CpG island methylator phenotype (CIMP), leading towards loss of homeostasis and onset of neoplastic transformation. The unopposed activation of Wnt/β-catenin pathways, either due to loss of APC function or up-regulation of related stimulatory pathways, results in unopposed hyperproliferation of colonic crypts, considered the single most important risk factor for colon carcinogenesis. Hypermethylation of CpG islands within the promoters of specific genes can potentially inactivate DNA repair genes and/or critical tumor suppressor genes. Recently, CpG methylation of the 5’ promoter of human (h) DCLK1 gene was reported in many human epithelial cancers, including colorectal cancers (CRCs), resulting in the loss of expression of the canonical long isoform of DCLK1 (DCLK1-L) in hCRCs. Instead, a shorter isoform of DCLK1 (DCLK1-S) was discovered to be expressed in hCRCs, from an alternate β promoter of DCLKL1-gene; the clinical and biological implications of these novel findings, in relation to recent publications is discussed. PMID:27777940

  9. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

    PubMed

    March, H Nikki; Rust, Alistair G; Wright, Nicholas A; ten Hoeve, Jelle; de Ridder, Jeroen; Eldridge, Matthew; van der Weyden, Louise; Berns, Anton; Gadiot, Jules; Uren, Anthony; Kemp, Richard; Arends, Mark J; Wessels, Lodewyk F A; Winton, Douglas J; Adams, David J

    2011-12-01

    The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development. PMID:22057237

  10. Whole-exome sequencing identifies rare pathogenic variants in new predisposition genes for familial colorectal cancer

    PubMed Central

    Esteban-Jurado, Clara; Vila-Casadesús, Maria; Garre, Pilar; Lozano, Juan José; Pristoupilova, Anna; Beltran, Sergi; Muñoz, Jenifer; Ocaña, Teresa; Balaguer, Francesc; López-Cerón, Maria; Cuatrecasas, Miriam; Franch-Expósito, Sebastià; Piqué, Josep M.; Castells, Antoni; Carracedo, Angel; Ruiz-Ponte, Clara; Abulí, Anna; Bessa, Xavier; Andreu, Montserrat; Bujanda, Luis; Caldés, Trinidad; Castellví-Bel, Sergi

    2015-01-01

    Purpose: Colorectal cancer is an important cause of mortality in the developed world. Hereditary forms are due to germ-line mutations in APC, MUTYH, and the mismatch repair genes, but many cases present familial aggregation but an unknown inherited cause. The hypothesis of rare high-penetrance mutations in new genes is a likely explanation for the underlying predisposition in some of these familial cases. Methods: Exome sequencing was performed in 43 patients with colorectal cancer from 29 families with strong disease aggregation without mutations in known hereditary colorectal cancer genes. Data analysis selected only very rare variants (0–0.1%), producing a putative loss of function and located in genes with a role compatible with cancer. Variants in genes previously involved in hereditary colorectal cancer or nearby previous colorectal cancer genome-wide association study hits were also chosen. Results: Twenty-eight final candidate variants were selected and validated by Sanger sequencing. Correct family segregation and somatic studies were used to categorize the most interesting variants in CDKN1B, XRCC4, EPHX1, NFKBIZ, SMARCA4, and BARD1. Conclusion: We identified new potential colorectal cancer predisposition variants in genes that have a role in cancer predisposition and are involved in DNA repair and the cell cycle, which supports their putative involvement in germ-line predisposition to this neoplasm. PMID:25058500

  11. Investigadores simulan los beneficios reales de los exámenes de detección

    Cancer.gov

    Artículo sobre los esfuerzos de la Red de Modelado de Intervención y Vigilancia del Cáncer (CISNET) del NCI para determinar los beneficios reales de los exámenes de detección del cáncer en la población general.

  12. Significant Silence in Elena Garro's "Los Perros"

    ERIC Educational Resources Information Center

    Burke, Jessica

    2010-01-01

    Elena Garro's one-act play "Los perros" (1958) confronts the difficult issue of sexual violence in rural Mexico, a problem that persists today. The characters struggle with the social reality of rape, alluding to the threat of sexual violence while avoiding addressing it directly. While words are granted an almost magical power in "Los perros",…

  13. NARSTO EPA SS LOS ANGELES SMPS DATA

    Atmospheric Science Data Center

    2014-04-25

    NARSTO EPA SS LOS ANGELES SMPS DATA Project Title:  NARSTO Discipline:  ... Particle Properties Particulate Matter Order Data:  ASDC Order Tool:   Order Data Guide Documents:  ... Earth Related Data:  Environmental Protection Agency Supersites Los Angeles, California SCAR-B ...

  14. NARSTO EPA SS LOS ANGELES PARTISOL DATA

    Atmospheric Science Data Center

    2014-05-07

    NARSTO EPA SS LOS ANGELES PARTISOL DATA Project Title:  NARSTO Discipline:  ... Properties Trace Elements Trace Metals Order Data:  Reverb:  Order Data Guide Documents:  ... Earth Related Data:  Environmental Protection Agency Supersites Los Angeles, California SCAR-B ...

  15. Vacunas contra los virus del papiloma humano

    Cancer.gov

    Una hoja informativa acerca de las vacunas contra los virus del papiloma humano (VPH) para prevenir infecciones con ciertos tipos de VPH, los cuales son la causa principal del cáncer de cuello del útero o cérvix.

  16. Trouble Brewing in Los Angeles. Policy Brief

    ERIC Educational Resources Information Center

    Buck, Stuart

    2010-01-01

    The city of Los Angeles will face enormous budgetary pressures from the growing deficits in public pensions, both at a state and local level. In this policy brief, the author estimates that Los Angeles faces a total $152.6 billion liability for pensions that are underfunded--including $49.1 billion for the city pension systems, $2.4 billion for…

  17. A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19.

    PubMed

    Singh, Harjeet; Huls, Helen; Kebriaei, Partow; Cooper, Laurence J N

    2014-01-01

    The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing methods to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CARs) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR(+) T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential. PMID:24329797

  18. A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19

    PubMed Central

    Singh, Harjeet; Huls, Helen; Cooper, Laurence JN

    2014-01-01

    Summary The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing approaches to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CAR) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase system improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR+ T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential. PMID:24329797

  19. A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19.

    PubMed

    Singh, Harjeet; Huls, Helen; Kebriaei, Partow; Cooper, Laurence J N

    2014-01-01

    The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing methods to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CARs) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR(+) T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential.

  20. Los Alamos Laser Eye Investigation.

    SciTech Connect

    Odom, C. R.

    2005-01-01

    A student working in a laser laboratory at Los Alamos National Laboratory sustained a serious retinal injury to her left eye when she attempted to view suspended particles in a partially evacuated target chamber. The principle investigator was using the white light from the flash lamp of a Class 4 Nd:YAG laser to illuminate the particles. Since the Q-switch was thought to be disabled at the time of the accident, the principal investigator assumed it would be safe to view the particles without wearing laser eye protection. The Laboratory Director appointed a team to investigate the accident and to report back to him the events and conditions leading up to the accident, equipment malfunctions, safety management causal factors, supervisory and management action/inaction, adequacy of institutional processes and procedures, emergency and notification response, effectiveness of corrective actions and lessons learned from previous similar events, and recommendations for human and institutional safety improvements. The team interviewed personnel, reviewed documents, and characterized systems and conditions in the laser laboratory during an intense six week investigation. The team determined that the direct and primary failures leading to this accident were, respectively, the principle investigator's unsafe work practices and the institution's inadequate monitoring of worker performance. This paper describes the details of the investigation, the human and institutional failures, and the recommendations for improving the laser safety program.

  1. Trichoderma genes

    SciTech Connect

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  2. [Gene therapy].

    PubMed

    Rodríguez-Fragoso, L

    1997-01-01

    In the last years there has been much progress in our understanding of molecular mechanisms in the pathogenesis of disease. In this review we provide an overview of gene therapy, its most actualized techniques for gene delivery, and we give specific examples of laboratory and clinical achievements to date. The development of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. As a result, concepts and methods that would have been considered purely science fiction 50 years ago are now used in the treatment of diseases. The widespread application of gene therapy technology to many diseases is already breaking down the traditional boundaries of modern medicine. However, despite its progress, several key technical drawbacks need to be overcome before gene therapy can be used safely and effectively in clinical settings. Technological developments, particularly in the areas of gene delivery and cell transplantation, will be critical for the successful practice of gene therapy.

  3. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  4. Expression analysis of eight amphioxus genes involved in the Wnt/β-catenin signaling pathway

    PubMed Central

    WANG, Jing; LI, Guang; QIAN, Guang-Hui; HUA, Jun-Hao; WANG, Yi-Quan

    2016-01-01

    The Wnt/β-catenin signaling pathway plays a crucial role in the embryonic development of metazoans. Although the pathway has been studied extensively in many model animals, its function in amphioxus, the most primitive chordate, remains largely uncharacterized. To obtain basic data for functional analysis, we identified and isolated seven genes (Lrp5/6, Dvl, APC, CkIα, CkIδ, Gsk3β, and Gro) of the Wnt/β-catenin signaling pathway from the amphioxus (Branchiostoma floridae) genome. Phylogenetic analysis revealed that amphioxus had fewer members of each gene family than that found in vertebrates. Whole-mount in situ hybridization showed that the genes were maternally expressed and broadly distributed throughout the whole embryo at the cleavage and blastula stages. Among them, Dvl was expressed asymmetrically towards the animal pole, while the others were evenly distributed in all blastomeres. At the mid-gastrula stage, the genes were specifically expressed in the primitive endomesoderm, but displayed different patterns. When the embryo developed into the neurula stage, the gene expressions were mainly detected in either paraxial somites or the tail bud. With the development of the embryo, the expression levels further decreased gradually and remained only in some pharyngeal regions or the tail bud at the larva stage. Our results suggest that the Wnt/β-catenin pathway might be involved in amphioxus somite formation and posterior growth, but not in endomesoderm specification. PMID:27265651

  5. Selectively Reduced Intracellular Proliferation of Salmonella enterica Serovar Typhimurium within APCs Limits Antigen Presentation and Development of a Rapid CD8 T Cell Response1

    PubMed Central

    Albaghdadi, Homam; Robinson, Nirmal; Finlay, Brett; Krishnan, Lakshmi; Sad, Subash

    2014-01-01

    Ag presentation to CD8+ T cells commences immediately after infection, which facilitates their rapid expansion and control of pathogen. This paradigm is not followed during infection with virulent Salmonella enterica serovar Typhimurium (ST), an intracellular bacterium that causes mortality in susceptible C57BL/6J mice within 7 days and a chronic infection in resistant mice (129 × 1SvJ). Infection of mice with OVA-expressing ST results in the development of a CD8+ T cell response that is detectable only after the second week of infection despite the early detectable bacterial burden. The mechanism behind the delayed CD8+ T cell activation was evaluated, and it was found that dendritic cells/macrophages or mice infected with ST-OVA failed to present Ag to OVA-specific CD8+ T cells. Lack of early Ag presentation was not rescued when mice or dendritic cells/macrophages were infected with an attenuated aroA mutant of ST or with mutants having defective Salmonella pathogenicity island I/II genes. Although extracellular ST proliferated extensively, the replication of ST was highly muted once inside macrophages. This muted intracellular proliferation of ST resulted in the generation of poor levels of intracellular Ag and peptide-MHC complex on the surface of dendritic cells. Additional experiments revealed that ST did not actively inhibit Ag presentation, rather it inhibited the uptake of another intracellular pathogen, Listeria monocytogenes, thereby causing inhibition of Ag presentation against L. monocytogenes. Taken together, this study reveals a dichotomy in the proliferation of ST and indicates that selectively reduced intra-cellular proliferation of virulent pathogens may be an important mechanism of immune evasion. PMID:19692639

  6. 82. FIRST AND SECOND AQUEDUCTS LOOKING NORTHEAST Los Angeles ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    82. FIRST AND SECOND AQUEDUCTS LOOKING NORTHEAST - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  7. 87. AQUEDUCT IN COVERED CONDUIT LOOKING NORTHWEST Los Angeles ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    87. AQUEDUCT IN COVERED CONDUIT LOOKING NORTHWEST - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  8. 47. LINED SECTION OF AQUEDUCT LOOKING NORTHWEST, COTTONWOOD Los ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    47. LINED SECTION OF AQUEDUCT LOOKING NORTHWEST, COTTONWOOD - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  9. 78. SECOND AQUEDUCT LOOKING SOUTH NEAR PINETREE SIPHON Los ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    78. SECOND AQUEDUCT LOOKING SOUTH NEAR PINETREE SIPHON - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  10. 84. LA AQUEDUCT CROSSING CALIFORNIA AQUEDUCT LOOKING NORTH Los ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    84. LA AQUEDUCT CROSSING CALIFORNIA AQUEDUCT LOOKING NORTH - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  11. 40. PLEASANT VALLEY RESERVOIR DAM LOOKING NORTHWEST Los Angeles ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    40. PLEASANT VALLEY RESERVOIR DAM LOOKING NORTHWEST - Los Angeles Aqueduct, From Lee Vining Intake (Mammoth Lakes) to Van Norman Reservoir Complex (San Fernando Valley), Los Angeles, Los Angeles County, CA

  12. Publications of Los Alamos research 1988

    SciTech Connect

    Varjabedian, K.; Dussart, S.A.; McClary, W.J.; Rich, J.A.

    1989-12-01

    This bibliography lists unclassified publications of work done at the Los Alamos National Laboratory for 1988. The entries, which are subdivided by broad subject categories, are cross-referenced with an author index and a numeric index.

  13. Shuttle Endeavour Flyover of Los Angeles Landmarks

    NASA Video Gallery

    Space shuttle Endeavour atop NASA's Shuttle Carrier Aircraft flew over many Los Angeles area landmarks on its final ferry flight Sept. 21, 2012, including the Coliseum, the Hollywood Sign, Griffith...

  14. New Rad Lab for Los Alamos

    SciTech Connect

    2008-08-06

    The topping out ceremony for a key construction stage in the Los Alamos National Laboratory's newest facility, the Radiological Laboratory Utility & Office Building. This is part of the National Nu...  

  15. Environmental surveillance at Los Alamos during 1994

    SciTech Connect

    1996-07-01

    This report describes environmental monitoring activities at Los Alamos National Laboratory for 1994. Data were collected to assess external penetrating radiation, airborne emissions, liquid effluents, radioactivity of environmental materials and food stuffs, and environmental compliance.

  16. New Rad Lab for Los Alamos

    ScienceCinema

    None

    2016-07-12

    The topping out ceremony for a key construction stage in the Los Alamos National Laboratory's newest facility, the Radiological Laboratory Utility & Office Building. This is part of the National Nu...  

  17. Edward Teller Returns to LOS Alamos

    NASA Astrophysics Data System (ADS)

    Hecker, Siegfried S.

    2010-01-01

    I was asked to share some reflections of Edward Teller's return to Los Alamos during my directorship. I met Teller late in his life. My comments focus on that time and they will be mostly in the form of stories of my interactions and those of my colleagues with Teller. Although the focus of this symposium is on Teller's contributions to science, at Los Alamos it was never possible to separate Teller's science from policy and controversy ...

  18. Internship at Los Alamos National Laboratory

    SciTech Connect

    Dunham, Ryan Q.

    2012-07-11

    Los Alamos National Laboratory (LANL) is located in Los Alamos, New Mexico. It provides support for our country's nuclear weapon stockpile as well as many other scientific research projects. I am an Undergraduate Student Intern in the Systems Design and Analysis group within the Nuclear Nonproliferation division of the Global Security directorate at LANL. I have been tasked with data analysis and modeling of particles in a fluidized bed system for the capture of carbon dioxide from power plant flue gas.

  19. Los Alamos Space Weather Summer School

    NASA Astrophysics Data System (ADS)

    Koller, J.

    2011-12-01

    Los Alamos National Lab recently initiated a new summer school specializing on space science, space weather, and instrumentation. The school is geared towards graduate level students and has been established to bring graduate students together with internationally recognized scientists at the Los Alamos National Lab. Students are receiving a prestigious Vela Fellowship to cover relocation expenses and cost of living for the duration of their stay in Los Alamos. For two months students have the opportunity to attend science lectures given by distinguished researchers at LANL. Topics are related to space weather research including plasma physics, radiation belts, numerical modeling, solar wind physics, spacecraft charging, and instrumentation. Students are also working closely with a Los Alamos mentor on exciting space weather science topics with access to Los Alamos GPS and geosynchronous data. The summer school concludes with project presentations by the students in a technical forum. The program is designed for graduate students currently enrolled at US Universities and open to all nationalities. We are presenting an overview of this exciting new program funded by IGPP (Institute of Geophysics and Planetary Physics), the Global Security Directorate, and the Directorate for Science, Technology and Engineering at Los Alamos National Lab.

  20. Single Gene Prognostic Biomarkers in Ovarian Cancer: A Meta-Analysis

    PubMed Central

    Willis, Scooter; Villalobos, Victor M.; Gevaert, Olivier; Abramovitz, Mark; Williams, Casey; Sikic, Branimir I.; Leyland-Jones, Brian

    2016-01-01

    Purpose To discover novel prognostic biomarkers in ovarian serous carcinomas. Methods A meta-analysis of all single genes probes in the TCGA and HAS ovarian cohorts was performed to identify possible biomarkers using Cox regression as a continuous variable for overall survival. Genes were ranked by p-value using Stouffer’s method and selected for statistical significance with a false discovery rate (FDR) <.05 using the Benjamini-Hochberg method. Results Twelve genes with high mRNA expression were prognostic of poor outcome with an FDR <.05 (AXL, APC, RAB11FIP5, C19orf2, CYBRD1, PINK1, LRRN3, AQP1, DES, XRCC4, BCHE, and ASAP3). Twenty genes with low mRNA expression were prognostic of poor outcome with an FDR <.05 (LRIG1, SLC33A1, NUCB2, POLD3, ESR2, GOLPH3, XBP1, PAXIP1, CYB561, POLA2, CDH1, GMNN, SLC37A4, FAM174B, AGR2, SDR39U1, MAGT1, GJB1, SDF2L1, and C9orf82). Conclusion A meta-analysis of all single genes identified thirty-two candidate biomarkers for their possible role in ovarian serous carcinoma. These genes can provide insight into the drivers or regulators of ovarian cancer and should be evaluated in future studies. Genes with high expression indicating poor outcome are possible therapeutic targets with known antagonists or inhibitors. Additionally, the genes could be combined into a prognostic multi-gene signature and tested in future ovarian cohorts. PMID:26886260

  1. The biology of novel animal genes: Mouse APEX gene knockout

    SciTech Connect

    MacInnes, M.; Altherr, M.R.; Ludwig, D.; Pedersen, R.; Mold, C.

    1997-07-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The controlled breeding of novel genes into mice, including the gene knockout (KO), or conversely by adding back transgenes provide powerful genetic technologies that together suffice to determine in large part the biological role(s) of novel genes. Inbred mouse remains the best understood and most useful mammalian experimental system available for tackling the biology of novel genes. The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE), is involved in a key step in the repair of spontaneous and induced AP sites in DNA. Efficient repair of these lesions is imperative to prevent the stable incorporation of mutations into the cellular genome which may lead to cell death or transformation. Loss or modulation of base excison repair activity in vivo may elevate the spontaneous mutation rate in cells, and may lead to a substantial increase in the incidence of cancer. Despite extensive biochemical analysis, however, the significance of these individual APE functions in vivo has not been elucidated. Mouse embryonic stem (ES) cells heterozygous for a deletion mutation in APE have been generated and whole animals containing the APE mutation have been derived from these ES cells. Animals homozygous for the APE null mutation die early in gestation, underscoring the biological significance of this DNA repair gene.

  2. La masa de los grandes impactores

    NASA Astrophysics Data System (ADS)

    Parisi, M. G.; Brunini, A.

    Los planetas han sido formados fundamentalmente acretando masa a través de colisiones con planetesimales sólidos. La masa más grande de la distribución de planetesimales y las masas máxima y mínima de los impactores, han sido calculadas usando los valores actuales del período y de la inclinación de los planetas (Lissauer & Safronov 1991; Parisi & Brunini 1996). Recientes investigaciones han mostrado, que las órbitas de los planetas gigantes no han sufrido variaciones con el tiempo, siendo su movimiento regular durante su evolución a partir de la finalización de la etapa de acreción (Laskar 1990, 1994). Por lo tanto, la eccentricidad actual de los planetas gigantes se puede utilizar para imponer una cota máxima a las masas y velocidades orbitales de los grandes impactores. Mediante un simple modelo dinámico, y considerando lo arriba mencionado, obtenemos la cota superior para la masa del planetesimal más grande que impactó a cada planeta gigante al final de su etapa de acreción. El resultado más importante de este trabajo es la estimación de la masa máxima permitida para impactar a Júpiter, la cúal es ~ 1.136 × 10 -1, siendo en el caso de Neptuno ~ 3.99 × 10 -2 (expresada en unidades de la masa final de cada planeta). Además, fue posible obtener la velocidad orbital máxima permitida para los impactores como una función de su masa, para cada planeta. Las cotas obtenidas para la masa y velocidad de los impactores de Saturno y Urano (en unidades de la masa y velocidad final de cada planeta respectivamente) son casi las mismas que las obtenidas para Júpiter debido a que estos tres planetas poseen similar eccentricidad actual. Nuestros resultados están en buen acuerdo con los obtenidos por Lissauer & Safronov (1991). Estas cotas podrían ser utilizadas para obtener la distribución de planetesimales en el Sistema Solar primitivo.

  3. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins.

    PubMed

    Meyer, Matthias; Arsuaga, Juan-Luis; de Filippo, Cesare; Nagel, Sarah; Aximu-Petri, Ayinuer; Nickel, Birgit; Martínez, Ignacio; Gracia, Ana; Bermúdez de Castro, José María; Carbonell, Eudald; Viola, Bence; Kelso, Janet; Prüfer, Kay; Pääbo, Svante

    2016-03-24

    A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago. An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals. However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history. PMID:26976447

  4. Nuclear DNA sequences from the Middle Pleistocene Sima de los Huesos hominins.

    PubMed

    Meyer, Matthias; Arsuaga, Juan-Luis; de Filippo, Cesare; Nagel, Sarah; Aximu-Petri, Ayinuer; Nickel, Birgit; Martínez, Ignacio; Gracia, Ana; Bermúdez de Castro, José María; Carbonell, Eudald; Viola, Bence; Kelso, Janet; Prüfer, Kay; Pääbo, Svante

    2016-03-24

    A unique assemblage of 28 hominin individuals, found in Sima de los Huesos in the Sierra de Atapuerca in Spain, has recently been dated to approximately 430,000 years ago. An interesting question is how these Middle Pleistocene hominins were related to those who lived in the Late Pleistocene epoch, in particular to Neanderthals in western Eurasia and to Denisovans, a sister group of Neanderthals so far known only from southern Siberia. While the Sima de los Huesos hominins share some derived morphological features with Neanderthals, the mitochondrial genome retrieved from one individual from Sima de los Huesos is more closely related to the mitochondrial DNA of Denisovans than to that of Neanderthals. However, since the mitochondrial DNA does not reveal the full picture of relationships among populations, we have investigated DNA preservation in several individuals found at Sima de los Huesos. Here we recover nuclear DNA sequences from two specimens, which show that the Sima de los Huesos hominins were related to Neanderthals rather than to Denisovans, indicating that the population divergence between Neanderthals and Denisovans predates 430,000 years ago. A mitochondrial DNA recovered from one of the specimens shares the previously described relationship to Denisovan mitochondrial DNAs, suggesting, among other possibilities, that the mitochondrial DNA gene pool of Neanderthals turned over later in their history.

  5. Nutritionally driven differential gene expression leads to heterochronic brain development in honeybee castes.

    PubMed

    Moda, Lívia Maria; Vieira, Joseana; Guimarães Freire, Anna Cláudia; Bonatti, Vanessa; Bomtorin, Ana Durvalina; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino

    2013-01-01

    The differential feeding regimes experienced by the queen and worker larvae of the honeybee Apis mellifera shape a complex endocrine response cascade that ultimately gives rise to differences in brain morphologies. Brain development analyzed at the morphological level from the third (L3) through fifth (L5) larval instars revealed an asynchrony between queens and workers. In the feeding phase of the last larval instar (L5F), two well-formed structures, pedunculi and calyces, are identifiable in the mushroom bodies of queens, both of which are not present in workers until a later phase (spinning phase, L5S). Genome-wide expression analyses and normalized transcript expression experiments monitoring specific genes revealed that this differential brain development starts earlier, during L3. Analyzing brains from L3 through L5S1 larvae, we identified 21 genes with caste-specific transcription patterns (e.g., APC-4, GlcAT-P, fax, kr-h1 and shot), which encode proteins that are potentially involved in the development of brain tissues through controlling the cell proliferation rate (APC4, kr-h1) and fasciculation (GlcAT-P, fax, and shot). Shot, whose expression is known to be required for axon extension and cell proliferation, was found to be transcribed at significantly higher levels in L4 queens compared with worker larvae. Moreover, the protein encoded by this gene was immunolocalized to the cytoplasm of cells near the antennal lobe neuropiles and proximal to the Kenyon cells in the brains of L4 queens. In conclusion, during the larval period, the brains of queens are larger and develop more rapidly than workers' brains, which represents a developmental heterochrony reflecting the effect of the differential feeding regime of the two castes on nervous system development. Furthermore, this differential development is characterized by caste-specific transcriptional profiles of a set of genes, thus pointing to a link between differential nutrition and differential

  6. Nutritionally driven differential gene expression leads to heterochronic brain development in honeybee castes.

    PubMed

    Moda, Lívia Maria; Vieira, Joseana; Guimarães Freire, Anna Cláudia; Bonatti, Vanessa; Bomtorin, Ana Durvalina; Barchuk, Angel Roberto; Simões, Zilá Luz Paulino

    2013-01-01

    The differential feeding regimes experienced by the queen and worker larvae of the honeybee Apis mellifera shape a complex endocrine response cascade that ultimately gives rise to differences in brain morphologies. Brain development analyzed at the morphological level from the third (L3) through fifth (L5) larval instars revealed an asynchrony between queens and workers. In the feeding phase of the last larval instar (L5F), two well-formed structures, pedunculi and calyces, are identifiable in the mushroom bodies of queens, both of which are not present in workers until a later phase (spinning phase, L5S). Genome-wide expression analyses and normalized transcript expression experiments monitoring specific genes revealed that this differential brain development starts earlier, during L3. Analyzing brains from L3 through L5S1 larvae, we identified 21 genes with caste-specific transcription patterns (e.g., APC-4, GlcAT-P, fax, kr-h1 and shot), which encode proteins that are potentially involved in the development of brain tissues through controlling the cell proliferation rate (APC4, kr-h1) and fasciculation (GlcAT-P, fax, and shot). Shot, whose expression is known to be required for axon extension and cell proliferation, was found to be transcribed at significantly higher levels in L4 queens compared with worker larvae. Moreover, the protein encoded by this gene was immunolocalized to the cytoplasm of cells near the antennal lobe neuropiles and proximal to the Kenyon cells in the brains of L4 queens. In conclusion, during the larval period, the brains of queens are larger and develop more rapidly than workers' brains, which represents a developmental heterochrony reflecting the effect of the differential feeding regime of the two castes on nervous system development. Furthermore, this differential development is characterized by caste-specific transcriptional profiles of a set of genes, thus pointing to a link between differential nutrition and differential

  7. Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer

    PubMed Central

    Yu, Jun; Wu, William K K; Li, Xiangchun; He, Jun; Li, Xiao-Xing; Ng, Simon S M; Yu, Chang; Gao, Zhibo; Yang, Jie; Li, Miao; Wang, Qiaoxiu; Liang, Qiaoyi; Pan, Yi; Tong, Joanna H; To, Ka F; Wong, Nathalie; Zhang, Ning; Chen, Jie; Lu, Youyong; Lai, Paul B S; Chan, Francis K L; Li, Yingrui; Kung, Hsiang-Fu; Yang, Huanming; Wang, Jun; Sung, Joseph J Y

    2015-01-01

    Background Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. Objective To identify recurrent somatic mutations with prognostic significance in patients with CRC. Method Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Results Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6–14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. Conclusions The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging. PMID:24951259

  8. Preliminary screening of differentially expressed genes involved in methyl-CpG-binding protein 2 gene-mediated proliferation in human osteosarcoma cells.

    PubMed

    Meng, Gang; Li, Yi; Lv, YangFan; Dai, Huanzi; Zhang, Xi; Guo, Qiao-Nan

    2015-04-01

    Methyl-CpG-binding protein 2 (MeCP2) is essential in human brain development and has been linked to several cancer types and neuro-developmental disorders. This study aims to screen the MeCP2 related differentially expressed genes and discover the therapeutic targets for osteosarcoma. CCK8 assay was used to detect the proliferation and SaOS2 and U2OS cells. Apoptosis of cells was detected by flow cytometry analysis that monitored Annexin V-APC/7-DD binding and 7-ADD uptake simultaneously. Denaturing formaldehyde agarose gel electrophoresis was employed to examine the quality of total RNA 18S and 28S units. Gene chip technique was utilized to discover the differentially expressed genes correlated with MeCP2 gene. Differential gene screening criteria were used to screen the changed genes. The gene up-regulation or down-regulation more than 1.5 times was regarded as significant differential expression genes. The CCK8 results indicated that the cell proliferation of MeCP2 silencing cells (LV-MeCP2-RNAi) was significantly decreased compared to non-silenced cells (LV-MeCP2-RNAi-CN) (P < 0.05). MeCP2 silencing could also induce significant apoptosis compared to non-silenced cells (P < 0.05); 107 expression changed genes were screened from a total of 49,395 transcripts. Among the total 107 transcripts, 34 transcripts were up-regulated and 73 transcripts were down-regulated. There were five significant differentially expressed genes, including IGFBP4, HOXC8, LMO4, MDK, and CTGF, which correlated with the MeCP2 gene. The methylation frequency of CpG in IGFBP4 gene could achieve 55%. In conclusion, the differentially expressed IGFBP4, HOXC8, LMO4, MDK, and CTGF genes may be involved in MeCP2 gene-mediated proliferation and apoptosis in osteosarcoma cells.

  9. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer

    SciTech Connect

    Goyette, M.C.; Fasching, C.L.; Stanbridge, E.J. ); Cho, K.; Levy, D.B.; Kinzler, K.W.; Vogelstein, B. ); Paraskeva, C. )

    1992-03-01

    Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, the authors have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 5 express the APC gene present on that chromosome as well as the endogenous mutant transcript. Expression of the putative tumor suppressor gene, DCC, was seen in the clones containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.

  10. Rehab center cuts LOS with interdisciplinary teams.

    PubMed

    1997-04-01

    Faced with an average length of stay (LOS) a full twenty days longer than the national average for spinal cord-injured patients, case managers at one hospital took advantage of their rehab center's existing spirit of interdisciplinary cooperation to foster a pathway that would bring LOS in line. The pathway team rejected a discipline-specific approach, choosing instead to break the pathway down by areas of outcome for the patient. By focusing on outcomes rather than specific responsibilities, members of the spinal cord team are encouraged to act collaboratively in helping patients achieve positive outcomes. Staff perform concurrent monitoring of patient compliance with the pathway in an effort to collect outcomes measures beyond LOS and cost per case.

  11. Acute Rheumatic Fever in Los Angeles County

    PubMed Central

    Propp, Richard P.

    1965-01-01

    There was a general downward trend in the reported incidence of acute rheumatic fever in Los Angeles County during the years 1954-1963. A survey of hospital records in five large hospitals in 1962 revealed 100 cases diagnosed, 39 of which were reported. Diagnoses in the charts reviewed conformed to the Modified Jones Criteria. Most of the patients were born in Los Angeles County. Mortality rates for acute rheumatic fever during the same period were greatly in excess of those expected from the reported morbidity. The mean crude mortality rate for the period concerned was higher than for New York City, although not as high as for Boston. Acute rheumatic fever appears to constitute a health problem in need of review in Los Angeles County. PMID:5834286

  12. Status of Monte Carlo at Los Alamos

    SciTech Connect

    Thompson, W.L.; Cashwell, E.D.

    1980-01-01

    At Los Alamos the early work of Fermi, von Neumann, and Ulam has been developed and supplemented by many followers, notably Cashwell and Everett, and the main product today is the continuous-energy, general-purpose, generalized-geometry, time-dependent, coupled neutron-photon transport code called MCNP. The Los Alamos Monte Carlo research and development effort is concentrated in Group X-6. MCNP treats an arbitrary three-dimensional configuration of arbitrary materials in geometric cells bounded by first- and second-degree surfaces and some fourth-degree surfaces (elliptical tori). Monte Carlo has evolved into perhaps the main method for radiation transport calculations at Los Alamos. MCNP is used in every technical division at the Laboratory by over 130 users about 600 times a month accounting for nearly 200 hours of CDC-7600 time.

  13. New Generation of Los Alamos Opacity Tables

    NASA Astrophysics Data System (ADS)

    Colgan, James; Kilcrease, D. P.; Magee, N. H.; Sherrill, M. E.; Abdallah, J.; Hakel, P.; Fontes, C. J.; Guzik, J. A.; Mussack, K. A.

    2016-05-01

    We present a new generation of Los Alamos OPLIB opacity tables that have been computed using the ATOMIC code. Our tables have been calculated for all 30 elements from hydrogen through zinc and are publicly available through our website. In this poster we discuss the details of the calculations that underpin the new opacity tables. We also show several recent applications of the use of our opacity tables to solar modeling and other astrophysical applications. In particular, we demonstrate that use of the new opacities improves the agreement between solar models and helioseismology, but does not fully resolve the long-standing `solar abundance' problem. The Los Alamos National Laboratory is operated by Los Alamos National Security, LLC for the National Nuclear Security Administration of the U.S. Department of Energy under Contract No. DE-AC5206NA25396.

  14. Publications of Los Alamos Research, 1983

    SciTech Connect

    Sheridan, C.J.; McClary, W.J.; Rich, J.A.; Rodriguez, L.L.

    1984-10-01

    This bibliography is a compilation of unclassified publications of work done at the Los Alamos National Laboratory for 1983. Papers published in 1982 are included regardless of when they were actually written. Publications received too late for inclusion in earlier compilations have also been listed. Declassification of previously classified reports is considered to constitute publication. All classified issuances are omitted - even those papers, themselves unclassified, which were published only as part of a classified document. If a paper was published more than once, all places of publication are included. The bibliography includes Los Alamos National Laboratory reports, papers released as non-Laboratory reports, journal articles, books, chapters of books, conference papers either published separately or as part of conference proceedings issued as books or reports, papers publishd in congressional hearings, theses, and US patents. Publications by Los Alamos authors that are not records of Laboratory-sponsored work are included when the Library becomes aware of them.

  15. Publications of Los Alamos research 1980

    SciTech Connect

    Salazar, C.A.; Willis, J.K.

    1981-09-01

    This bibliography is a compilation of unclassified publications of work done at the Los Alamos National Laboratory for 1980. Papers published in 1980 are included regardless of when they were actually written. Publications received too late for inclusion in earlier compilations have also been listed. Declassification of previously classified reports is considered to constitute publication. All classified issuances are omitted-even those papers, themselves unclassified, which were published only as part of a classified document. If a paper was pubished more than once, all places of publication are included. The bibliography includes Los Alamos National Laboratory reports, papers released as non-laboratory reports, journal articles, books, chapters of books, conference papers published either separately or as part of conference proceedings issued as books or reports, papers published in congressional hearings, theses, and US patents. Publications by Los Alamos authors that are not records of Laboratory-sponsored work are included when the Library becomes aware of them.

  16. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  17. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  18. SEDs at Los Alamos: A Personal Memoir

    NASA Astrophysics Data System (ADS)

    Bederson, Benjamin

    2001-03-01

    I have written this personal memoir approximately 55 years after the events I describe. It is based almost exclusively on memory, since apart from the diary I kept while on Tinian, I have few documents concerning it. It covers my service in the U.S. Army's Special Engineering Detachment (SED) in Oak Ridge and Los Alamos in 1944-45, on Tinian island, the launching pad for the bombing raids on Japan, in the summer and fall of 1945, and my return to Los Alamos until my discharge in January 1946.

  19. Bidirectional transcription of lipooligosaccharide synthesis genes from Campylobacter jejuni.

    PubMed

    Phongsisay, Vongsavanh; Fry, Benjamin N

    2007-10-01

    The lipooligosaccharide (LOS) molecules of Campylobacter jejuni are involved in virulence and induction of the Guillain-Barré syndrome (GBS). This study analysed the transcription of the LOS synthesis genes from the GBS-inducing C. jejuni strain HB 93-13 under microaerobic conditions. Fourteen consecutive genes Cj1132c, waaC, htrB, wlaNC, wlaND, cgtA, cgtB, cstII, neuB, neuC, neuA, wlaVA, wlaQA, and waaF were included. The results of rapid amplification of cDNA ends and single-stranded ligation of complementary ends showed initiation sites with potential promoter regions on both DNA strands in the Cj1132c/waaC, cgtB/cstII, and wlaQA/waaF strand-switch regions. Other termini without recognisable promoter region were also found throughout the LOS gene cluster, suggesting a low specificity of the polymerase during transcription. In addition, all gene junction regions were cloned into the shuttle vector pMW10 carrying the promoterless lacZ gene to identify functional promoter sites. Bidirectional active promoters were found in the strand-switch regions. The results of RT-PCR and cDNA blotting indicated that transcriptional linkage occurred between different operons, indicating a lack of transcription termination within the LOS gene cluster. Moreover, the results of semi-quantitative RT-PCR and real-time RT-PCR showed that both DNA strands were transcribed but transcription of the coding strand was at a higher rate. The results presented here provide an insight into transcription of the LOS synthesis gene cluster of C. jejuni.

  20. HER2/neu DNA vaccination by intradermal gene delivery in a mouse tumor model: Gene gun is superior to jet injector in inducing CTL responses and protective immunity.

    PubMed

    Nguyen-Hoai, Tam; Kobelt, Dennis; Hohn, Oliver; Vu, Minh D; Schlag, Peter M; Dörken, Bernd; Norley, Steven; Lipp, Martin; Walther, Wolfgang; Pezzutto, Antonio; Westermann, Jörg

    2012-12-01

    DNA vaccines are potential tools for the induction of immune responses against both infectious disease and cancer. The dermal application of DNA vaccines is of particular interest since the epidermal and dermal layers of the skin are characterized by an abundance of antigen-presenting cells (APCs). The aim of our study was to compare tumor protection as obtained by two different methods of intradermal DNA delivery (gene gun and jet injector) in a well-established HER2/neu mouse tumor model. BALB/c mice were immunized twice with a HER2/neu-coding plasmid by gene gun or jet injector. Mice were then subcutaneously challenged with HER2/neu(+) syngeneic D2F2/E2 tumor cells. Protection against subsequent challenges with tumor cells as well as humoral and T-cell immune responses induced by the vaccine were monitored. Gene gun immunization was far superior to jet injector both in terms of tumor protection and induction of HER2/neu-specific immune responses. After gene gun immunization, 60% of the mice remained tumor-free until day 140 as compared with 25% after jet injector immunization. Furthermore, gene gun vaccination was able to induce both a strong T(H)1-polarized T-cell response with detectable cytotoxic T-lymphocyte (CTL) activity and a humoral immune response against HER2/neu, whereas the jet injector was not. Although the disadvantages that were associated with the use of the jet injector in our model may be overcome with methodological modifications and/or in larger animals, which exhibit a thicker skin and/or subcutaneous muscle tissue, we conclude that gene gun delivery constitutes the method of choice for intradermal DNA delivery in preclinical mouse models and possibly also for the clinical development of DNA-based vaccines.