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Sample records for protein 1b tumor

  1. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.

    PubMed Central

    White, E; Sabbatini, P; Debbas, M; Wold, W S; Kusher, D I; Gooding, L R

    1992-01-01

    The adenovirus E1A and E1B proteins are required for transformation of primary rodent cells. When expressed in the absence of the 19,000-dalton (19K) E1B protein, however, the E1A proteins are acutely cytotoxic and induce host cell chromosomal DNA fragmentation and cytolysis, analogous to cells undergoing programmed cell death (apoptosis). E1A alone can efficiently initiate the formation of foci which subsequently undergo abortive transformation whereby stimulation of cell growth is counteracted by continual cell death. Cell lines with an immortalized growth potential eventually arise with low frequency. Coexpression of the E1B 19K protein with E1A is sufficient to overcome abortive transformation to produce high-frequency transformation. Like E1A, the tumoricidal cytokine tumor necrosis factor alpha (TNF-alpha) evokes a programmed cell death response in many tumor cell lines by inducing DNA fragmentation and cytolysis. Expression of the E1B 19K protein by viral infection, by transient expression, or in transformed cells completely and specifically blocks this TNF-alpha-induced DNA fragmentation and cell death. Cosegregation of 19K protein transforming activity with protection from TNF-alpha-mediated cytolysis demonstrates that both activities are likely the consequence of the same function of the protein. Therefore, we propose that by suppressing an intrinsic cell death mechanism activated by TNF-alpha or E1A, the E1B 19K protein enhances the transforming activity of E1A and enables adenovirus to evade TNF-alpha-dependent immune surveillance. Images PMID:1317006

  2. Human adenovirus 2 E1B-19K and E1B-53K tumor antigens: antipeptide antibodies targeted to the NH2 and COOH termini.

    PubMed Central

    Green, M; Brackmann, K H; Lucher, L A; Symington, J S; Kramer, T A

    1983-01-01

    The human adenovirus 2 (Ad2) transforming region is located in the left 11.1% of the viral genome and encodes two early transcription units, E1A and E1B. Based on the amino acid sequence deduced from the Ad2 E1B DNA sequence (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982), we have prepared antibodies against synthetic peptides, 8 to 16 amino acids in length, encoded at the NH2 and COOH termini of the major E1B-19K and E1B-53K tumor antigens. The antipeptide antibodies immunoprecipitated the targeted E1B-19K or E1B-53K tumor antigens from extracts of Ad2-infected cells. The specificity of the peptide competition studies. Antipeptide antibodies directed to the NH2 and COOH termini immunoprecipitated the E1B-19K and E1B-53K tumor antigens from two Ad2-transformed rat cell lines, F17 and F4, providing evidence that identical tumor antigens are synthesized in Ad2-infected and Ad2-transformed cells. These results show that the E1B-19K and E1B-53K T antigens are not processed proteolytically at either the NH2 or COOH terminus. Our data provide strong evidence at the protein level that the E1B-19K and E1B-53K tumor antigens partially overlap in DNA sequence, with the E1B-19K initiating translation at the first ATG at nucleotide 1711 in translation reading frame 1 and the E1B-53K tumor antigen initiating translation at the second ATG at nucleotide 2016 in reading frame 3. This confirms the results of others on the N-terminal amino acid sequence of E1B-19K and theoretical deductions based on the DNA sequence. Our findings prove that the large E1B-53K T antigen initiates translation at the second ATG at nucleotide 2016 and not at equally plausible initiation codons located farther downstream at nucleotides 2202 and 2235. Thus, the E1B-53K T antigen is another example of a protein which initiates translation at an internal ATG rather than at the 5'-proximal ATG. Images PMID:6632083

  3. Down-regulated expression of the protein-tyrosine phosphatase 1B (PTP1B) is associated with aggressive clinicopathologic features and poor prognosis in hepatocellular carcinoma

    SciTech Connect

    Zheng, Long-Yi; Zhou, Dong-Xun; Lu, Jin; Zhang, Wen-Jun; Zou, Da-Jin

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer PTP1B protein showed decreased expression in 67.79% of the HCC patients. Black-Right-Pointing-Pointer Low PTP1B expression predicts poor prognosis of HCC. Black-Right-Pointing-Pointer Low PTP1B expression is correlated with expansion of OV6{sup +} tumor-initiating cells. Black-Right-Pointing-Pointer Down-regulation of PTP1B is associated with activation of Wnt/{beta}-Catenin signaling. -- Abstract: The protein-tyrosine phosphatase 1B (PTP1B) is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and can exert both tumor suppressing and tumor promoting effects in different cancers depending on the substrate involved and the cellular context. However, the expression level and function of PTP1B in hepatocellular carcinoma (HCC) remain unclear. In this study, PTP1B expression was detected by immunohistochemistry in normal liver tissue (n = 16) and hepatocellular carcinoma (n = 169). The correlations between PTP1B expression level and clinicopathologic features and patient survival were also analyzed. One hundred and eleven of 169 HCC patients (65.7%) had negative or low PTP1B expression in tumorous tissues, whereas normal tissues always expressed strong PTP1B. Decreased PTP1B expression was significantly associated with aggressive clinicopathologic features and poor prognosis. Immunohistochemistry also showed that low PTP1B expression level was correlated with high percentage of OV6{sup +} tumor-initiating cells (T-ICs) and high frequency of nuclear {beta}-Catenin expression in HCC specimens. Our findings demonstrate for the first time that the loss of inhibitory effect of PTP1B may contribute to progression and invasion of HCC through activation of Wnt/{beta}-Catenin signaling and expansion of liver T-ICs. PTP1B may serve as a valuable prognostic biomarker and potential therapeutic target in HCC.

  4. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  5. Membrane associated cancer-oocyte neoantigen SAS1B/ovastacin is a candidate immunotherapeutic target for uterine tumors

    PubMed Central

    Pires, Eusebio S.; D'Souza, Ryan S.; Needham, Marisa A.; Herr, Austin K.; Jazaeri, Amir A.; Li, Hui; Stoler, Mark H.; Anderson-Knapp, Kiley L.; Thomas, Theodore; Mandal, Arabinda; Gougeon, Alain; Flickinger, Charles J.; Bruns, David E.; Pollok, Brian A.; Herr, John C.

    2015-01-01

    The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes. PMID:26327203

  6. Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

    PubMed

    Bettaieb, Ahmed; Koike, Shinichiro; Chahed, Samah; Bachaalany, Santana; Griffey, Stephen; Sastre, Juan; Haj, Fawaz G

    2016-08-01

    Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis. PMID:27461362

  7. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    PubMed

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

  8. Distinctive role of vasohibin-1A and its splicing variant vasohibin-1B in tumor angiogenesis.

    PubMed

    Horie, S; Suzuki, Y; Kobayashi, M; Kadonosono, T; Kondoh, S; Kodama, T; Sato, Y

    2016-05-01

    Vasohibin-1 (VASH1) was isolated as a negative-feedback regulator of angiogenesis expressed in endothelial cells (ECs). There are two transcripts of VASH1, that is, the full-length VASH1A consisting of seven exons and the splicing variant VASH1B consisting of four exons. Here, we compared the effects of VASH1A and VASH1B on tumor angiogenesis. When ECs were transfected with VASH1A or VASH1B cDNAs, VASH1B transfectants, but not VASH1A ones, induced autophagic cell death of ECs. With sonoporation, the VASH1A or VASH1B gene were transfected specifically in ECs of tumor vessels in mice. Both VASH1A and VASH1B decreased tumor vessel density and inhibited tumor growth. VASH1A normalized the remaining tumor vessels, increased their rate of perfusion, decreased tumor hypoxia and enhanced the efficacy of anticancer chemotherapy, whereas VASH1B pruned tumor vessels without causing normalization, increased tumor hypoxia and tumor necrosis and did not enhance the efficacy of anticancer chemotherapy. The alternate transfection of mice with the VASH1A and VASH1B gene showed the highest effects on antitumor activity and normalization of tumor vessels. Our present findings on VASH1A and VASH1B should provide an innovative approach that would improve the efficacy of antiangiogenic cancer therapy by balancing vascular normalization and pruning. PMID:27080222

  9. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades

    PubMed Central

    Jiang, Cheng-shi; Liang, Lin-fu; Guo, Yue-wei

    2012-01-01

    This article provides an overview of approximately 300 secondary metabolites with inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), which were isolated from various natural sources or derived from synthetic process in the last decades. The structure-activity relationship and the selectivity of some compounds against other protein phosphatases were also discussed. Potential pharmaceutical applications of several PTP1B inhibitors were presented. PMID:22941286

  10. AMYLOID-β PEPTIDE BINDS TO MICROTUBULE-ASSOCIATED PROTEIN 1B (MAP1B)

    PubMed Central

    Gevorkian, Goar; Gonzalez-Noriega, Alfonso; Acero, Gonzalo; Ordoñez, Jorge; Michalak, Colette; Munguia, Maria Elena; Govezensky, Tzipe; Cribbs, David H.; Manoutcharian, Karen

    2008-01-01

    Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer’s disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer’s disease. PMID:18079022

  11. Amyloid-beta peptide binds to microtubule-associated protein 1B (MAP1B).

    PubMed

    Gevorkian, Goar; Gonzalez-Noriega, Alfonso; Acero, Gonzalo; Ordoñez, Jorge; Michalak, Colette; Munguia, Maria Elena; Govezensky, Tzipe; Cribbs, David H; Manoutcharian, Karen

    2008-05-01

    Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease. PMID:18079022

  12. UBC9-dependent Association between Calnexin and Protein Tyrosine Phosphatase 1B (PTP1B) at the Endoplasmic Reticulum*

    PubMed Central

    Lee, Dukgyu; Kraus, Allison; Prins, Daniel; Groenendyk, Jody; Aubry, Isabelle; Liu, Wen-Xin; Li, Hao-Dong; Julien, Olivier; Touret, Nicolas; Sykes, Brian D.; Tremblay, Michel L.; Michalak, Marek

    2015-01-01

    Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein, molecular chaperone, and a component of the translocon. We discovered a novel interaction between the calnexin cytoplasmic domain and UBC9, a SUMOylation E2 ligase, which modified the calnexin cytoplasmic domain by the addition of SUMO. We demonstrated that calnexin interaction with the SUMOylation machinery modulates an interaction with protein tyrosine phosphatase 1B (PTP1B), an ER-associated protein tyrosine phosphatase involved in the negative regulation of insulin and leptin signaling. We showed that calnexin and PTP1B form UBC9-dependent complexes, revealing a previously unrecognized contribution of calnexin to the retention of PTP1B at the ER membrane. This work shows that the SUMOylation machinery links two ER proteins from divergent pathways to potentially affect cellular protein quality control and energy metabolism. PMID:25586181

  13. Pharmacophore modeling for protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Bharatham, Kavitha; Bharatham, Nagakumar; Lee, Keun Woo

    2007-05-01

    A three dimensional chemical feature based pharmacophore model was developed for the inhibitors of protein tyrosine phosphatase 1B (PTP1B) using the CATALYST software, which would provide useful knowledge for performing virtual screening to identify new inhibitors targeted toward type II diabetes and obesity. A dataset of 27 inhibitors, with diverse structural properties, and activities ranging from 0.026 to 600 microM, was selected as a training set. Hypol, the most reliable quantitative four featured pharmacophore hypothesis, was generated from a training set composed of compounds with two H-bond acceptors, one hydrophobic aromatic and one ring aromatic features. It has a correlation coefficient, RMSD and cost difference (null cost-total cost) of 0.946, 0.840 and 65.731, respectively. The best hypothesis (Hypol) was validated using four different methods. Firstly, a cross validation was performed by randomizing the data using the Cat-Scramble technique. The results confirmed that the pharmacophore models generated from the training set were valid. Secondly, a test set of 281 molecules was scored, with a correlation of 0.882 obtained between the experimental and predicted activities. Hypol performed well in correctly discriminating the active and inactive molecules. Thirdly, the model was investigated by mapping on two PTP1B inhibitors identified by different pharmaceutical companies. The Hypol model correctly predicted these compounds as being highly active. Finally, docking simulations were performed on few compounds to substantiate the role of the pharmacophore features at the binding site of the protein by analyzing their binding conformations. These multiple validation approaches provided confidence in the utility of this pharmacophore model as a 3D query for virtual screening to retrieve new chemical entities showing potential as potent PTP1B inhibitors. PMID:17615669

  14. Consequence of the tumor-associated conversion to cyclin D1b

    PubMed Central

    Augello, Michael A; Berman-Booty, Lisa D; Carr, Richard; Yoshida, Akihiro; Dean, Jeffry L; Schiewer, Matthew J; Feng, Felix Y; Tomlins, Scott A; Gao, Erhe; Koch, Walter J; Benovic, Jeffrey L; Diehl, John Alan; Knudsen, Karen E

    2015-01-01

    Clinical evidence suggests that cyclin D1b, a variant of cyclin D1, is associated with tumor progression and poor outcome. However, the underlying molecular basis was unknown. Here, novel models were created to generate a genetic switch from cyclin D1 to cyclin D1b. Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo. Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo. Further molecular interrogation uncovered unexpected links between cyclin D1b and the DNA damage/PARP1 regulatory networks, which could be exploited to suppress cyclin D1b-driven tumors. Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant. PMID:25787974

  15. Expression Patterns of Organic Anion Transporting Polypeptides 1B1 and 1B3 Protein in Human Pediatric Liver.

    PubMed

    Thomson, Margaret M S; Hines, Ronald N; Schuetz, Erin G; Meibohm, Bernd

    2016-07-01

    Determining appropriate pharmacotherapy in young children can be challenging due to uncertainties in the development of drug disposition pathways. With knowledge of the ontogeny of drug-metabolizing enzymes and an emerging focus on drug transporters, the developmental pattern of the uptake transporters organic anion transporting polypeptide (OATP) 1B1 and 1B3 was assessed by relative protein quantification using Western blotting in 80 human pediatric liver specimens covering an age range from 9 days to 12 years. OATP1B3 exhibited high expression at birth, which declined over the first months of life, and then increased again in the preadolescent period. In comparison with children 6-12 years of age, the relative protein expression of highly glycosylated (total) OATP1B3 was 235% (357%) in children <3 months of age, 33% (64%) in the age group from 3 months to 2 years, and 50% (59%) in children 2-6 years of age. The fraction of highly glycosylated to total OATP1B3 increased with age, indicating ontogenic processes not only at the transcriptional level but also at the post-translational level. Similar to OATP1B3, OATP1B1 showed high interindividual variability in relative protein expression but no statistically significant difference among the studied age groups. PMID:27098745

  16. RPRD1B promotes tumor growth by accelerating the cell cycle in endometrial cancer.

    PubMed

    Wang, Yuan; Qiu, Haifeng; Hu, Weixu; Li, Shaoru; Yu, Jinjin

    2014-03-01

    RPRD1B, the regulation of nuclear pre-mRNA domain containing 1B gene, functions as a cell cycle manipulator and has been found overexpressed in a small panel of endometrial cancer types. In the present study, we investigated the roles of RPRD1B in endometrial cancer using various in vitro and in vivo experiments. According to our results, RPRD1B mRNA was significantly upregulated in endometrial cancer tissues (P=0.0012). RPRD1B overexpression was correlated with tumor stage (P=0.0004), histology type (P=0.0146) and depth of myometrial invasion (P=0.024). In vitro, RPRD1B promoted cellular proliferation (P=0.032 for MTT assay and P=0.018 for colony formation assay), and accelerated the cell cycle (P=0.007) by upregulating cyclin D1, CDK4 and CDK6, while knockdown of RPRD1B suppressed cellular proliferation (P=0.02 for MTT assay and P=0.031 for colony formation assay), and led to G1 phase arrest (P=0.025) through downregulating cyclin D1, CDK4 and CDK6. Consistently, in the nude mice model, RPRD1B overexpression significantly accelerated the tumor xenograft growth (P=0.0012), accompanied by elevated Ki-67 and cyclin D1. In addition, we demonstrated that downregulating RPRD1B could sensitize Ishikawa cells to Raloxifene (P=0.01). In summary, we demonstrated that RPRD1B was frequently overexpressed in human endometrial cancer. Both in vitro and in vivo, over-abundant RPRD1B could promote tumor growth and accelerate cellular cell cycle. In addition, knockdown of RPRD1B also increased cell sensitivity to Raloxifene, making RPRD1B a potent therapeutic target for endometrial cancer, particularly in patients with resistance to the selective ER modulators. PMID:24452636

  17. Novel chromenedione derivatives displaying inhibition of protein tyrosine phosphatase 1B (PTP1B) from Flemingia philippinensis.

    PubMed

    Wang, Yan; Yuk, Heung Joo; Kim, Jeong Yoon; Kim, Dae Wook; Song, Yeong Hun; Tan, Xue Fei; Curtis-Long, Marcus J; Park, Ki Hun

    2016-01-15

    Protein tyrosine phosphatase 1B (PTP1B) is an important target to treat obesity and diabetes due to its key roles in insulin and leptin signaling. The MeOH extracts of the root bark of Flemingia philippinensis yielded eight inhibitory molecules (1-8) capable of targeting PTP1B. Three of them were identified to be novel compounds, philippin A (1), philippin B (2), and philippin C (3) which have a rare 3-phenylpropanoyl chromenedione skeleton. The other compounds (4-8) were known prenylated isoflavones. All compounds (1-8) inhibited PTP1B in a dose dependent manner with IC50s ranging between 2.4 and 29.4μM. The most potent compound emerged to be prenylated isoflavone 5 (IC50=2.4μM). In kinetic studies, chromenedione derivatives (1-3) emerged to be reversible, competitive inhibitors, whereas prenylated isoflavones (5-8) were noncompetitive inhibitors. PMID:26704263

  18. The ING1b tumor suppressor facilitates nucleotide excision repair by promoting chromatin accessibility to XPA

    SciTech Connect

    Kuo, Wei-Hung W.; Wang Yemin; Wong, Ronald P.C.; Campos, Eric I.; Li Gang . E-mail: gangli@interchange.ubc.ca

    2007-05-01

    ING1b is the most studied ING family protein and perhaps the most ubiquitously and abundantly expressed. This protein is involved in the regulation of various biological functions ranging from senescence, cell cycle arrest, apoptosis, to DNA repair. ING1b is upregulated by UV irradiation and enhances the removal of bulky nucleic acid photoproducts. In this study, we provide evidence that ING1b mediates nucleotide excision repair by facilitating the access to damaged nucleosomal DNA. We demonstrate that ING1b is not recruited to UV-induced DNA lesions but enhances nucleotide excision repair only in XPC-proficient cells, implying an essential role in early steps of the 'access, repair, restore' model. We also find that ING1b alters histone acetylation dynamics upon exposure to UV radiation and induces chromatin relaxation in microccocal nuclease digestion assay, revealing that ING1b may allow better access to nucleotide excision repair machinery. More importantly, ING1b associates with chromatin in a UV-inducible manner and facilitates DNA access to nucleotide excision repair factor XPA. Furthermore, depletion of the endogenous ING1b results to the sensitization of cells at S-phase to UV irradiation. Taken together, these observations establish a role of ING1b acting as a chromatin accessibility factor for DNA damage recognition proteins upon genotoxic injury.

  19. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    SciTech Connect

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-10-02

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  20. The Nuclear Envelope Protein, LAP1B, Is a Novel Protein Phosphatase 1 Substrate

    PubMed Central

    Santos, Mariana; Rebelo, Sandra; Van Kleeff, Paula J. M.; Kim, Connie E.; Dauer, William T.; Fardilha, Margarida; da Cruz e Silva, Odete A.; da Cruz e Silva, Edgar F.

    2013-01-01

    Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases. PMID:24116158

  1. HIV-1 Tat Protein Induces PD-L1 (B7-H1) Expression on Dendritic Cells through Tumor Necrosis Factor Alpha- and Toll-Like Receptor 4-Mediated Mechanisms

    PubMed Central

    Planès, Rémi; BenMohamed, Lbachir; Leghmari, Kaoutar; Delobel, Pierre; Izopet, Jacques

    2014-01-01

    ABSTRACT Chronic human immunodeficiency virus type 1 (HIV-1) infection is associated with induction of T-cell coinhibitory pathways. However, the mechanisms by which HIV-1 induces upregulation of coinhibitory molecules remain to be fully elucidated. The aim of the present study was to determine whether and how HIV-1 Tat protein, an immunosuppressive viral factor, induces the PD-1/PD-L1 coinhibitory pathway on human dendritic cells (DCs). We found that treatment of DCs with whole HIV-1 Tat protein significantly upregulated the level of expression of PD-L1. This PD-L1 upregulation was observed in monocyte-derived dendritic cells (MoDCs) obtained from either uninfected or HIV-1-infected patients as well as in primary myeloid DCs from HIV-negative donors. In contrast, no effect on the expression of PD-L2 or PD-1 molecules was detected. The induction of PD-L1 on MoDCs by HIV-1 Tat (i) occurred in dose- and time-dependent manners, (ii) was mediated by the N-terminal 1–45 fragment of Tat, (iii) did not require direct cell-cell contact but appeared rather to be mediated by soluble factor(s), (iv) was abrogated following neutralization of tumor necrosis factor alpha (TNF-α) or blocking of Toll-like receptor 4 (TLR4), (v) was absent in TLR4-knockoout (KO) mice but could be restored following incubation with Tat-conditioned medium from wild-type DCs, (vi) impaired the capacity of MoDCs to functionally stimulate T cells, and (vii) was not reversed functionally following PD-1/PD-L1 pathway blockade, suggesting the implication of other Tat-mediated coinhibitory pathways. Our results demonstrate that HIV-1 Tat protein upregulates PD-L1 expression on MoDCs through TNF-α- and TLR4-mediated mechanisms, functionally compromising the ability of DCs to stimulate T cells. The findings offer a novel potential molecular target for the development of an anti-HIV-1 treatment. IMPORTANCE The objective of this study was to investigate the effect of human immunodeficiency virus type 1 (HIV

  2. Integrative Bioinformatics Links HNF1B with Clear Cell Carcinoma and Tumor-Associated Thrombosis

    PubMed Central

    Cuff, Justin; Salari, Keyan; Clarke, Nicole; Esheba, Ghada E.; Forster, Andrew D.; Huang, Stephanie; West, Robert B.; Higgins, John P.; Longacre, Teri A.; Pollack, Jonathan R.

    2013-01-01

    Clear cell carcinoma (CCC) is a histologically distinct carcinoma subtype that arises in several organ systems and is marked by cytoplasmic clearing, attributed to abundant intracellular glycogen. Previously, transcription factor hepatocyte nuclear factor 1-beta (HNF1B) was identified as a biomarker of ovarian CCC. Here, we set out to explore more broadly the relation between HNF1B and carcinomas with clear cell histology. HNF1B expression, evaluated by immunohistochemistry, was significantly associated with clear cell histology across diverse gynecologic and renal carcinomas (P<0.001), as was hypomethylation of the HNF1B promoter (P<0.001). From microarray analysis, an empirically-derived HNF1B signature was significantly enriched for computationally-predicted targets (with HNF1 binding sites) (P<0.03), as well as genes associated with glycogen metabolism, including glucose-6-phophatase, and strikingly the blood clotting cascade, including fibrinogen, prothrombin and factor XIII. Enrichment of the clotting cascade was also evident in microarray data from ovarian CCC versus other histotypes (P<0.01), and HNF1B-associated prothrombin expression was verified by immunohistochemistry (P = 0.015). Finally, among gynecologic carcinomas with cytoplasmic clearing, HNF1B immunostaining was linked to a 3.0-fold increased risk of clinically-significant venous thrombosis (P = 0.043), and with a 2.3-fold increased risk (P = 0.011) in a combined gynecologic and renal carcinoma cohort. Our results define HNF1B as a broad marker of clear cell phenotype, and support a mechanistic link to glycogen accumulation and thrombosis, possibly reflecting (for gynecologic CCC) derivation from secretory endometrium. Our findings also implicate a novel mechanism of tumor-associated thrombosis (a major cause of cancer mortality), based on the direct production of clotting factors by cancer cells. PMID:24040285

  3. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance.

    PubMed

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  4. Oligouridylate Binding Protein 1b Plays an Integral Role in Plant Heat Stress Tolerance

    PubMed Central

    Nguyen, Cam Chau; Nakaminami, Kentaro; Matsui, Akihiro; Kobayashi, Shuhei; Kurihara, Yukio; Toyooka, Kiminori; Tanaka, Maho; Seki, Motoaki

    2016-01-01

    Stress granules (SGs), which are formed in the plant cytoplasm under stress conditions, are transient dynamic sites (particles) for mRNA storage. SGs are actively involved in protecting mRNAs from degradation. Oligouridylate binding protein 1b (UBP1b) is a component of SGs. The formation of microscopically visible cytoplasmic foci, referred to as UBP1b SG, was induced by heat treatment in UBP1b-overexpressing Arabidopsis plants (UBP1b-ox). A detailed understanding of the function of UBP1b, however, is still not clear. UBP1b-ox plants displayed increased heat tolerance, relative to control plants, while ubp1b mutants were more sensitive to heat stress than control plants. Microarray analysis identified 117 genes whose expression was heat-inducible and higher in the UBP1b-ox plants. RNA decay analysis was performed using cordycepin, a transcriptional inhibitor. In order to determine if those genes serve as targets of UBP1b, the rate of RNA degradation of a DnaJ heat shock protein and a stress-associated protein (AtSAP3) in UBP1b-ox plants was slower than in control plants; indicating that the mRNAs of these genes were protected within the UBP1b SG granule. Collectively, these data demonstrate that UBP1b plays an integral role in heat stress tolerance in plants. PMID:27379136

  5. Modulation of p53-mediated transcriptional repression and apoptosis by the adenovirus E1B 19K protein.

    PubMed Central

    Sabbatini, P; Chiou, S K; Rao, L; White, E

    1995-01-01

    BRK cell lines that stably express adenovirus E1A and a murine temperature-sensitive p53 undergo apoptosis when p53 assumes the wild-type conformation. Expression of the E1B 19,000-molecular-weight (19K) protein rescues cells from this p53-mediated apoptosis and diverts cells to a growth-arrested state. As p53 likely functions as a tumor suppressor by regulating transcription, the ability of the E1B 19K protein to regulate p53-mediated transactivation and transcriptional repression was investigated. In promoter-reporter assays the E1B 19K did not block p53-mediated transactivation but did alleviate p53-mediated transcriptional repression. E1B 19K expression permitted efficient transcriptional activation of the p21/WAF-1/cip-1 mRNA by p53, consistent with maintenance of the growth arrest function of p53. The E1B 19K protein is thereby unique among DNA virus-transforming proteins that target p53 for inactivation in that it selectively modulates the transcriptional properties of p53. The E1B 19K protein also rescued cells from apoptosis induced by inhibitors of transcription and protein synthesis. This suggests that cell death may result from the inhibition of expression of survival factors which function to maintain cell viability. p53 may induce apoptosis through generalized transcriptional repression. In turn, the E1B 19K protein may prevent p53-mediated apoptosis by alleviating p53-mediated transcriptional repression. PMID:7823921

  6. Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

    PubMed Central

    Smiley, J K; Young, M A; Flint, S J

    1990-01-01

    The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells. Images PMID:2143545

  7. Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

    PubMed

    Saifudin, Azis; Tanaka, Ken; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2012-08-01

    A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC₅₀ values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC₅₀, 5.51 ± 0.04 µM). PMID:22763740

  8. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins.

    PubMed Central

    White, E; Cipriani, R; Sabbatini, P; Denton, A

    1991-01-01

    Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these

  9. Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes

    PubMed Central

    Fatima, Tahira; Topuz, Muhamet; Bernadec, Anne; Sicher, Richard; Handa, Avtar K.; Mattoo, Autar K.

    2016-01-01

    Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines – putrescine (Put), spermidine (Spd), and spermine (Spm) – are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent

  10. Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes.

    PubMed

    Goyal, Ravinder K; Fatima, Tahira; Topuz, Muhamet; Bernadec, Anne; Sicher, Richard; Handa, Avtar K; Mattoo, Autar K

    2016-01-01

    Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines - putrescine (Put), spermidine (Spd), and spermine (Spm) - are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of

  11. Microtubule-associated protein 1B (MAP1B)-deficient neurons show structural presynaptic deficiencies in vitro and altered presynaptic physiology.

    PubMed

    Bodaleo, Felipe J; Montenegro-Venegas, Carolina; Henríquez, Daniel R; Court, Felipe A; Gonzalez-Billault, Christian

    2016-01-01

    Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro. PMID:27425640

  12. Microtubule-associated protein 1B (MAP1B)-deficient neurons show structural presynaptic deficiencies in vitro and altered presynaptic physiology

    PubMed Central

    Bodaleo, Felipe J.; Montenegro-Venegas, Carolina; Henríquez, Daniel R.; Court, Felipe A.; Gonzalez-Billault, Christian

    2016-01-01

    Microtubule-associated protein 1B (MAP1B) is expressed predominantly during the early stages of development of the nervous system, where it regulates processes such as axonal guidance and elongation. Nevertheless, MAP1B expression in the brain persists in adult stages, where it participates in the regulation of the structure and physiology of dendritic spines in glutamatergic synapses. Moreover, MAP1B expression is also found in presynaptic synaptosomal preparations. In this work, we describe a presynaptic phenotype in mature neurons derived from MAP1B knockout (MAP1B KO) mice. Mature neurons express MAP1B, and its deficiency does not alter the expression levels of a subgroup of other synaptic proteins. MAP1B KO neurons display a decrease in the density of presynaptic and postsynaptic terminals, which involves a reduction in the density of synaptic contacts, and an increased proportion of orphan presynaptic terminals. Accordingly, MAP1B KO neurons present altered synaptic vesicle fusion events, as shown by FM4-64 release assay, and a decrease in the density of both synaptic vesicles and dense core vesicles at presynaptic terminals. Finally, an increased proportion of excitatory immature symmetrical synaptic contacts in MAP1B KO neurons was detected. Altogether these results suggest a novel role for MAP1B in presynaptic structure and physiology regulation in vitro. PMID:27425640

  13. Myelin protein zero gene mutated in Charcot-Marie-Tooth type 1B patients

    SciTech Connect

    Su, Ying; Li, Lanying; Lepercq, J.; Lebo, R.V. ); Brooks, D.G.; Ravetch, J.V. ); Trofatter, J.A. )

    1993-11-15

    The autosomal dominant of Charcot-Marie-Tooth disease (CMT), whose gene is type 1B (CMT1B), has slow nerve conduction with demyelinated Schwann cells. In this study the abundant peripheral myelin protein zero (MPZ) gene, MPZ, was mapped 130 kb centromeric to the Fc receptor immunoglobulin gene cluster in band 1q22, and a major MPZ point mutation was found to cosegregate with CMT1B in one large CMT1B family. The MPZ point mutation in 18 of 18 related CMT1B pedigree 1 patients converts a positively charged lysine in codon 96 to a negatively charged glutamate. The same MPZ locus cosegregates with the CMT1B disease gene in a second CMT1B family [total multipoint logarithm of odds (lod) = 11.4 at [theta] = 0.00] with a splice junction mutation. Both mutations occur in MPZ protein regions otherwise conserved identically in human, rat, and cow since these species diverged 100 million years ago. MPZ protein, expressed exclusively in myelinated peripheral nerve Schwann cells, constitutes >50% of myelin protein. These mutations are anticipated to disrupt homophilic MPZ binding and result in CMT1B peripheral nerve demyelination.

  14. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    PubMed

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  15. Caveolin 1 (Cav-1) and actin-related protein 2/3 complex, subunit 1B (ARPC1B) expressions as prognostic indicators for oral squamous cell carcinoma (OSCC).

    PubMed

    Auzair, Lukman Bin Md; Vincent-Chong, Vui King; Ghani, Wan Maria Nabillah; Kallarakkal, Thomas George; Ramanathan, Anand; Lee, Chia Ee; Rahman, Zainal Ariff Abdul; Ismail, Siti Mazlipah; Abraham, Mannil Thomas; Zain, Rosnah Binti

    2016-07-01

    Caveolin-1 (Cav-1) and Actin-Related Protein 2/3 Complex, Subunit 1B (ARPC1B) have been implicated in various human cancers, yet its role in tumorigenesis remains controversial. Therefore, this study aims to determine the protein expression of these two genes in oral squamous cell carcinomas (OSCCs) and to evaluate the clinical and prognostic impact of these genes in OSCC. Protein expressions of these two genes were determined by immunohistochemistry technique. The association between Cav-1 and ARPC1B with clinico-pathological parameters was evaluated by Chi-square test (or Fisher exact test where appropriate). Correlation between the protein expressions of these 2 genes with survival was analyzed using Kaplan-Meier and Cox regression models. Cav-1 and ARPC1B were found to be significantly over-expressed in OSCC compared to normal oral mucosa (p = 0.002 and p = 0.033, respectively). Low level of ARPC1B protein expression showed a significant correlation with lymph node metastasis (LNM) (p = 0.010) and advanced tumor staging (p = 0.003). Kaplan-Meier survival analyses demonstrated that patients with over-expression of Cav-1 protein were associated with poor prognosis (p = 0.030). Adjusted multivariate Cox regression model revealed that over-expression of Cav-1 remained as an independent significant prognostic factor for OSCC (HRR = 2.700, 95 % CI 1.013-7.198, p = 0.047). This study demonstrated that low-expression of ARPC1B is significantly associated with LNM and advanced tumor staging whereas high expression of Cav-1 can be a prognostic indicator for poor prognosis in OSCC patients. PMID:26138391

  16. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    PubMed

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  17. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B

    PubMed Central

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  18. The Mechanism of Allosteric Inhibition of Protein Tyrosine Phosphatase 1B

    PubMed Central

    Lu, Shaoyong; Huang, Wenkang; Geng, Lv; Shen, Qiancheng; Zhang, Jian

    2014-01-01

    As the prototypical member of the PTP family, protein tyrosine phosphatase 1B (PTP1B) is an attractive target for therapeutic interventions in type 2 diabetes. The extremely conserved catalytic site of PTP1B renders the design of selective PTP1B inhibitors intractable. Although discovered allosteric inhibitors containing a benzofuran sulfonamide scaffold offer fascinating opportunities to overcome selectivity issues, the allosteric inhibitory mechanism of PTP1B has remained elusive. Here, molecular dynamics (MD) simulations, coupled with a dynamic weighted community analysis, were performed to unveil the potential allosteric signal propagation pathway from the allosteric site to the catalytic site in PTP1B. This result revealed that the allosteric inhibitor compound-3 induces a conformational rearrangement in helix α7, disrupting the triangular interaction among helix α7, helix α3, and loop11. Helix α7 then produces a force, pulling helix α3 outward, and promotes Ser190 to interact with Tyr176. As a result, the deviation of Tyr176 abrogates the hydrophobic interactions with Trp179 and leads to the downward movement of the WPD loop, which forms an H-bond between Asp181 and Glu115. The formation of this H-bond constrains the WPD loop to its open conformation and thus inactivates PTP1B. The discovery of this allosteric mechanism provides an overall view of the regulation of PTP1B, which is an important insight for the design of potent allosteric PTP1B inhibitors. PMID:24831294

  19. Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

    PubMed

    Schubert, Julian; Siekierska, Aleksandra; Langlois, Mélanie; May, Patrick; Huneau, Clément; Becker, Felicitas; Muhle, Hiltrud; Suls, Arvid; Lemke, Johannes R; de Kovel, Carolien G F; Thiele, Holger; Konrad, Kathryn; Kawalia, Amit; Toliat, Mohammad R; Sander, Thomas; Rüschendorf, Franz; Caliebe, Almuth; Nagel, Inga; Kohl, Bernard; Kecskés, Angela; Jacmin, Maxime; Hardies, Katia; Weckhuysen, Sarah; Riesch, Erik; Dorn, Thomas; Brilstra, Eva H; Baulac, Stephanie; Møller, Rikke S; Hjalgrim, Helle; Koeleman, Bobby P C; Jurkat-Rott, Karin; Lehman-Horn, Frank; Roach, Jared C; Glusman, Gustavo; Hood, Leroy; Galas, David J; Martin, Benoit; de Witte, Peter A M; Biskup, Saskia; De Jonghe, Peter; Helbig, Ingo; Balling, Rudi; Nürnberg, Peter; Crawford, Alexander D; Esguerra, Camila V; Weber, Yvonne G; Lerche, Holger

    2014-12-01

    Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes. PMID:25362483

  20. Prediction and verification of novel peptide targets of protein tyrosine phosphatase 1B.

    PubMed

    Li, Xun; Köhn, Maja

    2016-08-01

    Phosphotyrosine peptides are useful starting points for inhibitor design and for the search for protein tyrosine phosphatase (PTP) phosphoprotein substrates. To identify novel phosphopeptide substrates of PTP1B, we developed a computational prediction protocol based on a virtual library of protein sequences with known phosphotyrosine sites. To these we applied sequence-based methods, biologically meaningful filters and molecular docking. Five peptides were selected for biochemical testing of their potential as PTP1B substrates. All five peptides were equally good substrates for PTP1B compared to a known peptide substrate whereas appropriate control peptides were not recognized, showing that our protocol can be used to identify novel peptide substrates of PTP1B. PMID:27025565

  1. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    PubMed

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  2. SMC1B is present in mammalian somatic cells and interacts with mitotic cohesin proteins

    PubMed Central

    Mannini, Linda; Cucco, Francesco; Quarantotti, Valentina; Amato, Clelia; Tinti, Mara; Tana, Luigi; Frattini, Annalisa; Delia, Domenico; Krantz, Ian D.; Jessberger, Rolf; Musio, Antonio

    2015-01-01

    Cohesin is an evolutionarily conserved protein complex that plays a role in many biological processes: it ensures faithful chromosome segregation, regulates gene expression and preserves genome stability. In mammalian cells, the mitotic cohesin complex consists of two structural maintenance of chromosome proteins, SMC1A and SMC3, the kleisin protein RAD21 and a fourth subunit either STAG1 or STAG2. Meiotic paralogs in mammals were reported for SMC1A, RAD21 and STAG1/STAG2 and are called SMC1B, REC8 and STAG3 respectively. It is believed that SMC1B is only a meiotic-specific cohesin member, required for sister chromatid pairing and for preventing telomere shortening. Here we show that SMC1B is also expressed in somatic mammalian cells and is a member of a mitotic cohesin complex. In addition, SMC1B safeguards genome stability following irradiation whereas its ablation has no effect on chromosome segregation. Finally, unexpectedly SMC1B depletion impairs gene transcription, particularly at genes mapping to clusters such as HOX and PCDHB. Genome-wide analyses show that cluster genes changing in expression are enriched for cohesin-SMC1B binding. PMID:26673124

  3. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    PubMed

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

  4. Distinct Features of Cap Binding by eIF4E1b Proteins

    PubMed Central

    Kubacka, Dorota; Miguel, Ricardo Núñez; Minshall, Nicola; Darzynkiewicz, Edward; Standart, Nancy; Zuberek, Joanna

    2015-01-01

    eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m7GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α + β fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N7 of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N7 position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding. PMID:25463438

  5. Protein 4.1B Contributes to the Organization of Peripheral Myelinated Axons

    PubMed Central

    Devaux, Jérôme; Carnaud, Michèle; Levasseur, Grégoire; Niwa-Kawakita, Michiko; Harroch, Sheila; Girault, Jean-Antoine; Giovannini, Marco; Goutebroze, Laurence

    2011-01-01

    Neurons are characterized by extremely long axons. This exceptional cell shape is likely to depend on multiple factors including interactions between the cytoskeleton and membrane proteins. In many cell types, members of the protein 4.1 family play an important role in tethering the cortical actin-spectrin cytoskeleton to the plasma membrane. Protein 4.1B is localized in myelinated axons, enriched in paranodal and juxtaparanodal regions, and also all along the internodes, but not at nodes of Ranvier where are localized the voltage-dependent sodium channels responsible for action potential propagation. To shed light on the role of protein 4.1B in the general organization of myelinated peripheral axons, we studied 4.1B knockout mice. These mice displayed a mildly impaired gait and motility. Whereas nodes were unaffected, the distribution of Caspr/paranodin, which anchors 4.1B to the membrane, was disorganized in paranodal regions and its levels were decreased. In juxtaparanodes, the enrichment of Caspr2, which also interacts with 4.1B, and of the associated TAG-1 and Kv1.1, was absent in mutant mice, whereas their levels were unaltered. Ultrastructural abnormalities were observed both at paranodes and juxtaparanodes. Axon calibers were slightly diminished in phrenic nerves and preterminal motor axons were dysmorphic in skeletal muscle. βII spectrin enrichment was decreased along the axolemma. Electrophysiological recordings at 3 post-natal weeks showed the occurrence of spontaneous and evoked repetitive activity indicating neuronal hyperexcitability, without change in conduction velocity. Thus, our results show that in myelinated axons 4.1B contributes to the stabilization of membrane proteins at paranodes, to the clustering of juxtaparanodal proteins, and to the regulation of the internodal axon caliber. PMID:21966409

  6. Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells.

    PubMed

    Henriques, Andreia F A; Barros, Patrícia; Moyer, Mary Pat; Matos, Paulo; Jordan, Peter

    2015-12-28

    Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated β-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS. PMID:26341689

  7. Myelin protein zero gene sequencing diagnoses Charcot-Marie-Tooth Type 1B disease

    SciTech Connect

    Su, Y.; Zhang, H.; Madrid, R.

    1994-09-01

    Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, affects about 1 in 2600 people in Norway and is found worldwide. CMT Type 1 (CMT1) has slow nerve conduction with demyelinated Schwann cells. Autosomal dominant CMT Type 1B (CMT1B) results from mutations in the myelin protein zero gene which directs the synthesis of more than half of all Schwann cell protein. This gene was mapped to the chromosome 1q22-1q23.1 borderline by fluorescence in situ hybridization. The first 7 of 7 reported CMT1B mutations are unique. Thus the most effective means to identify CMT1B mutations in at-risk family members and fetuses is to sequence the entire coding sequence in dominant or sporadic CMT patients without the CMT1A duplication. Of the 19 primers used in 16 pars to uniquely amplify the entire MPZ coding sequence, 6 primer pairs were used to amplify and sequence the 6 exons. The DyeDeoxy Terminator cycle sequencing method used with four different color fluorescent lables was superior to manual sequencing because it sequences more bases unambiguously from extracted genomic DNA samples within 24 hours. This protocol was used to test 28 CMT and Dejerine-Sottas patients without CMT1A gene duplication. Sequencing MPZ gene-specific amplified fragments identified 9 polymorphic sites within the 6 exons that encode the 248 amino acid MPZ protein. The large number of major CMT1B mutations identified by single strand sequencing are being verified by reverse strand sequencing and when possible, by restriction enzyme analysis. This protocol can be used to distringuish CMT1B patients from othre CMT phenotypes and to determine the CMT1B status of relatives both presymptomatically and prenatally.

  8. Early region 1B of adenovirus 2 encodes two coterminal proteins of 495 and 155 amino acid residues.

    PubMed Central

    Anderson, C W; Schmitt, R C; Smart, J E; Lewis, J B

    1984-01-01

    Partial sequence analysis of tryptic peptides has identified the E1B-495R (E1b-57K) (early transcription region 1B of 495 amino acid residues, with an approximate molecular weight of 57,000) protein of adenovirus 2 as encoded by the 495 amino acid open reading frame located in the adenovirus 2 DNA sequence between nucleotides 2016 and 3500. Additional proteins of 16,000 Mr and 18,000 Mr that are related to the E1B-495R protein were identified by cell-free translation of hybridization-selected mRNA. Analysis of [35S]methionine-containing amino terminal tryptic peptides by thin-layer chromatography showed that the E1B-495R, E1B-18K, and E1B-16K proteins all begin at the same initiation codon. The E1B-495R protein from 293 cells also has the same initial tryptic peptide, acetyl-methionyl-glutamyl-arginine. Sequence analysis of E1B-18K tryptic peptides indicated that this protein also has the same carboxy terminus as the E1B-495R protein and that it is derived from an mRNA that is spliced to remove sequences between nucleotides 2250 and 3269, resulting in a protein product of 155 amino acid residues. Analysis of E1B-16K tryptic peptides has not yet revealed the carboxy terminal structure of this protein. Both the E1B-495R and the E1B-155R (E1B-18K) proteins, as well as the E1B-16K protein, were precipitated from cell-free translations and from extracts of infected cells by antiserum against an amino terminal nonapeptide common to these proteins. Images PMID:6323739

  9. A 130-kDa Protein 4.1B Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization*

    PubMed Central

    Wang, Jie; Song, Jinlei; An, Chao; Dong, Wenji; Zhang, Jingxin; Yin, Changcheng; Hale, John; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2014-01-01

    Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer. PMID:24381168

  10. α1B-adrenergic receptors differentially associate with Rab proteins during homologous and heterologous desensitization.

    PubMed

    Castillo-Badillo, Jean A; Sánchez-Reyes, Omar B; Alfonzo-Méndez, Marco A; Romero-Ávila, M Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  11. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    PubMed Central

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  12. Protein Tyrosine Phosphatase 1B Inhibitors from the Stems of Akebia quinata.

    PubMed

    An, Jin-Pyo; Ha, Thi Kim Quy; Kim, Jinwoong; Cho, Tae Oh; Oh, Won Keun

    2016-01-01

    PTP1B deficiency in mouse mammary tumor virus (MMTV)-NeuNT transgenic mice inhibited the onset of MMTV-NeuNT-evoked breast cancer, while its overexpression was observed in breast cancer. Thus, PTP1B inhibitors are considered chemopreventative agents for breast cancer. As part of our program to find PTP1B inhibitors, one new diterpene glycoside (1) and 13 known compounds (2-14) were isolated from the methanol extract of the stems of Akebia quinata. All isolates were identified based on extensive spectroscopic data analysis, including UV, IR, NMR and MS. Compounds 2, 3, 6, 8 and 11 showed significant inhibitory effects on the PTP1B enzyme, with IC50 values ranging from 4.08 ± 1.09 to 21.80 ± 4.74 μM. PTP1B inhibitors also had concentration-dependent cytotoxic effects on breast cancer cell lines, such as MCF7, MDA-MB-231 and tamoxifen-resistant MCF7 (MCF7/TAMR) (IC50 values ranging from 0.84 ± 0.04 to 7.91 ± 0.39 μM). These results indicate that compounds 6 and 8 from Akebia quinata may be lead compounds acting as anti-breast cancer agents. PMID:27548130

  13. Impaired Intestinal Calcium Absorption in Protein 4.1R-deficient Mice Due to Altered Expression of Plasma Membrane Calcium ATPase 1b (PMCA1b)*

    PubMed Central

    Liu, Congrong; Weng, Haibao; Chen, Lixiang; Yang, Shaomin; Wang, Hua; Debnath, Gargi; Guo, Xinhua; Wu, Liancheng; Mohandas, Narla; An, Xiuli

    2013-01-01

    Protein 4.1R was first identified in the erythrocyte membrane skeleton. It is now known that the protein is expressed in a variety of epithelial cell lines and in the epithelia of many tissues, including the small intestine. However, the physiological function of 4.1R in the epithelial cells of the small intestine has not so far been explored. Here, we show that 4.1R knock-out mice exhibited a significantly impaired small intestinal calcium absorption that resulted in secondary hyperparathyroidism as evidenced by increased serum 1,25-(OH)2-vitamin D3 and parathyroid hormone levels, decreased serum calcium levels, hyperplasia of the parathyroid, and demineralization of the bones. 4.1R is located on the basolateral membrane of enterocytes, where it co-localizes with PMCA1b (plasma membrane calcium ATPase 1b). Expression of PMCA1b in enterocytes was decreased in 4.1−/− mice. 4.1R directly associated with PMCA1b, and the association involved the membrane-binding domain of 4.1R and the second intracellular loop and C terminus of PMCA1b. Our findings have enabled us to define a functional role for 4.1R in small intestinal calcium absorption through regulation of membrane expression of PMCA1b. PMID:23460639

  14. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    SciTech Connect

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-09-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells.

  15. SARS Coronavirus 3b Accessory Protein Modulates Transcriptional Activity of RUNX1b

    PubMed Central

    Varshney, Bhavna; Agnihotram, Sudhakar; Tan, Yee-Joo; Baric, Ralph; Lal, Sunil K.

    2012-01-01

    Background The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein. Methodology/Principal Findings In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells. Conclusions/Significance These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection. PMID:22253733

  16. SynCAM1 recruits NMDA receptors via protein 4.1B.

    PubMed

    Hoy, Jennifer L; Constable, John R; Vicini, Stefano; Fu, Zhanyan; Washbourne, Philip

    2009-12-01

    Cell adhesion molecules have been implicated as key organizers of synaptic structures, but there is still a need to determine how these molecules facilitate neurotransmitter receptor recruitment to developing synapses. Here, we identify erythrocyte protein band 4.1-like 3 (protein 4.1B) as an intracellular effector molecule of Synaptic Cell Adhesion Molecule 1 (SynCAM1) that is sufficient to recruit NMDA-type receptors (NMDARs) to SynCAM1 adhesion sites in COS7 cells. Protein 4.1B in conjunction with SynCAM1 also increased the frequency of NMDAR-mediated mEPSCs and area of presynaptic contact in an HEK293 cell/ neuron co-culture assay. Studies in cultured hippocampal neurons reveal that manipulation of protein 4.1B expression levels specifically affects NMDAR-mediated activity and localization. Finally, further experimentation in COS7 cells show that SynCAM1 may also interact with protein 4.1N to specifically effect AMPA type receptor (AMPAR) recruitment. Thus, SynCAM1 may recruit both AMPARs and NMDARs by independent mechanisms during synapse formation. PMID:19796685

  17. The Potent Inhibitors of Protein Tyrosine Phosphatase 1B from the Fruits of Melaleuca leucadendron

    PubMed Central

    Saifudin, Azis; Lallo, Subehan Ab; Tezuka, Yasuhiro

    2016-01-01

    Background: Melaleuca leucadendron (Myrtaceae) is a kind of fruit used as Indonesian medicinal component and recorded in Jamu (tonic made of medical herbs) prescription records for the diabetes treatment. Its methanol extract exhibited a strong inhibitory activity with the half maximal inhibitory concentration (IC50) value of 2.05 μg/mL, while it is the same value with positive control RK-682. Objective: To isolate the chemical constituents of M. leucadendron and to evaluate their activity against protein tyrosine phosphatase 1B (PTP1B). Further, determine their toxicity potential against T-cell protein tyrosine phosphatase (TCPTP). Materials and Methods: Methanol extract was fractionated using silica column chromatography, and the obtained fraction was purified using Sephadex 20-LH. The structure of isolated compounds was identified based on 1H and 13Nuclear Magnetic Resonance Spectrometry. Furthermore, the compounds were examined against PTP1B and TCPTP. Results: Methanol extract of M. leucadendron (Myrtaceae) afforded two triterpenes: Betulinic acid and ursolic acid in high quantities. Both compounds exhibited a strong inhibitory activity against PTP1B inhibition with IC50 value of 1.5 and 2.3 μg/mL, respectively (positive control RK-682, IC50 = 2.05 μg/mL). Their activity toward TCPTP, on the other hand, were at 2.4 and 3.1 μg/mL, respectively. Based on this purification work, betulinic acid and ursolic acid presented 7.6% and 2.4%, respectively, as markedly M. leucadendron most potential for betulinic acid source among Indonesian plants. The result should have demonstrated that the antidiabetes of M. dendron could be through the inhibition of PTP1B. SUMMARY Melaleuca leucadendron is a good source for ursolic acid.Confirming traditional use for type II diabetes via PTP1B inhibition. PMID:27114690

  18. Protein tyrosine phosphatase 1B (PTP1B) inhibitors from Morinda citrifolia (Noni) and their insulin mimetic activity.

    PubMed

    Nguyen, Phi-Hung; Yang, Jun-Li; Uddin, Mohammad N; Park, So-Lim; Lim, Seong-Il; Jung, Da-Woon; Williams, Darren R; Oh, Won-Keun

    2013-11-22

    As part of our ongoing search for new antidiabetic agents from medicinal plants, we found that a methanol extract of Morinda citrifolia showed potential stimulatory effects on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3-8, 11-13, and 15). The absolute configurations of compounds 9, 10, and 14 were determined by ECD spectra analysis. Compounds 3, 6, 7, and 15 showed inhibitory effects on PTP1B enzyme with IC50 values of 21.86 ± 0.48, 15.01 ± 0.20, 16.82 ± 0.42, and 4.12 ± 0.09 μM, respectively. Furthermore, compounds 3, 6, 7, and 15 showed strong stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. This study indicated the potential of compounds 3, 6, 7, and 15 as lead molecules for antidiabetic agents. PMID:24224843

  19. Co-expression of protein tyrosine kinases EGFR-2 and PDGFRβ with protein tyrosine phosphatase 1B in Pichia pastoris.

    PubMed

    Tu, Pham Ngoc; Wang, Yamin; Cai, Menghao; Zhou, Xiangshan; Zhang, Yuanxing

    2014-02-28

    The regulation of protein tyrosine phosphorylation is mediated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) and is essential for cellular homeostasis. Coexpression of PTKs with PTPs in Pichia pastoris was used to facilitate the expression of active PTKs by neutralizing their apparent toxicity to cells. In this study, the gene encoding phosphatase PTP1B with or without a blue fluorescent protein or peroxisomal targeting signal 1 was cloned into the expression vector pAG32 to produce four vectors. These vectors were subsequently transformed into P. pastoris GS115. The tyrosine kinases EGFR-2 and PDGFRβ were expressed from vector pPIC3.5K and were fused with a His-tag and green fluorescent protein at the N-terminus. The two plasmids were transformed into P. pastoris with or without PTP1B, resulting in 10 strains. The EGFR-2 and PDGFRβ fusion proteins were purified by Ni(2+) affinity chromatography. In the recombinant P. pastoris, the PTKs co-expressed with PTP1B exhibited higher kinase catalytic activity than did those expressing the PTKs alone. The highest activities were achieved by targeting the PTKs and PTP1B into peroxisomes. Therefore, the EGFR-2 and PDGFRβ fusion proteins expressed in P. pastoris may be attractive drug screening targets for anticancer therapeutics. PMID:24248091

  20. Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b

    PubMed Central

    Elhaddad, Nagat S.; Hunt, Lee; Sloan, Jennifer; Gray, Julie E.

    2014-01-01

    Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure. PMID:24828466

  1. Light-induced stomatal opening is affected by the guard cell protein kinase APK1b.

    PubMed

    Elhaddad, Nagat S; Hunt, Lee; Sloan, Jennifer; Gray, Julie E

    2014-01-01

    Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure. PMID:24828466

  2. The PCNA-associated factor KIAA0101/p15{sup PAF} binds the potential tumor suppressor product p33ING1b

    SciTech Connect

    Simpson, Fiona; Lammerts van Bueren, Kelly; Butterfield, Natalie; Bennetts, Jennifer S.; Bowles, Josephine; Adolphe, Christelle; Simms, Lisa A.; Young, Joanne; Walsh, Michael D.; Leggett, Barbara; Fowles, Lindsay F.; Wicking, Carol . E-mail: C.Wicking@imb.uq.edu.au

    2006-01-01

    The KIAA0101/p15{sup PAF}/OEATC-1 protein was initially isolated in a yeast two-hybrid screen for proliferating cell nuclear antigen (PCNA) binding partners, and was shown to bind PCNA competitively with the cell cycle regulator p21{sup WAF}. PCNA is involved in DNA replication and damage repair. Using polyclonal antisera raised against a p15{sup PAF} fusion protein, we have shown that in a range of mammalian tumor and non-tumor cell lines the endogenous p15{sup PAF} protein localises to the nucleus and the mitochondria. Under normal conditions no co-localisation with PCNA could be detected, however following exposure to UV it was possible to co-immunoprecipitate p15{sup PAF} and PCNA from a number of cell lines, suggesting a UV-enhanced association of the two proteins. Overexpression of p15{sup PAF} in mammalian cells was also found to protect cells from UV-induced cell death. Based on similarities between the behaviour of p15{sup PAF} and the potential tumor suppressor product p33ING1b, we have further shown that these two proteins interact in the same complex in cell cultures. This suggests that p15{sup PAF} forms part of a larger protein complex potentially involved in the regulation of DNA repair, apoptosis and cell cycle progression.

  3. Phosphorylation at the carboxy terminus of the 55-kilodalton adenovirus type 5 E1B protein regulates transforming activity.

    PubMed Central

    Teodoro, J G; Halliday, T; Whalen, S G; Takayesu, D; Graham, F L; Branton, P E

    1994-01-01

    The 55-kDa product of early region 1B (E1B) of human adenoviruses is required for viral replication and participates in cell transformation through complex formation with and inactivation of the cellular tumor suppressor p53. We have used both biochemical and genetic approaches to show that this 496-residue (496R) protein of adenovirus type 5 is phosphorylated at serine and threonine residues near the carboxy terminus within sequences characteristic of substrates of casein kinase II. Mutations which converted serines 490 and 491 to alanine residues decreased viral replication and greatly reduced the efficiency of transformation of primary baby rat kidney cells. Such mutant 496R proteins interacted with p53 at efficiencies similar to those of wild-type 496R but only partially inhibited p53 transactivation activity. These results indicated that phosphorylation at these carboxy-terminal sites either regulates the inhibition of p53 or regulates some other 496R function required for cell transformation. Images PMID:8289381

  4. INHIBITORY POTENTIAL OF POLYHYDROXYLATED FULLERENES AGAINST PROTEIN TYROSINE PHOSPHATASE 1B.

    PubMed

    Kobzar, O L; Trush, V V; Tanchuk, V Yu; Vovk, A I

    2015-01-01

    Inhibition of PTP1B by polyhydroxylated fullerenes was studied in silico and in vitro. The enzyme kinetics in the presence of polyhydroxy small gap fullerenes showed that reciprocal value of maximum velocity non-linearly increases with increasing the inhibitor concentration. Analysis of the dose-dependent curve of PTP1B inhibition suggests an apparent positive cooperativity with involvement of at least two binding sites for the hydroxylated fullerene cages. Molecular docking calculations indicated that highly hydroxylated fullerene C60 may occupy the active site and additional allosteric binding site with similar affinity. In silico analysis of a number of fullerenols with 6, 12, 18, 24, 30, and 36 hydroxyl groups showed that the inhibitory activity may depend on the degree of hydroxylation of the nanoparticles surface. These data provide some understanding of the mechanisms of inhibitory action of fullerenols on activity of protein tyrosine phosphatases. PMID:26547960

  5. Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B).

    PubMed

    Parsons, Zachary D; Ruddraraju, Kasi Viswanatharaju; Santo, Nicholas; Gates, Kent S

    2016-06-15

    Redox regulation of protein tyrosine phosphatase 1B (PTP1B) involves oxidative conversion of the active site cysteine thiolate into an electrophilic sulfenyl amide residue. Reduction of the sulfenyl amide by biological thiols regenerates the native cysteine residue. Here we explored fundamental chemical reactions that may enable covalent capture of the sulfenyl amide residue in oxidized PTP1B. Various sulfone-containing carbon acids were found to react readily with a model peptide sulfenyl amide via attack of the sulfonyl carbanion on the electrophilic sulfur center in the sulfenyl amide. Both the products and the rates of these reactions were characterized. The results suggest that capture of a peptide sulfenyl amide residue by sulfone-stabilized carbanions can slow, but not completely prevent, thiol-mediated generation of the corresponding cysteine-containing peptide. Sulfone-containing carbon acids may be useful components in the construction of agents that knock down PTP1B activity in cells via transient covalent capture of the sulfenyl amide oxoform generated during insulin signaling processes. PMID:27132865

  6. Long-Range Communication Network in the Type 1B Bone Morphogenetic Protein Receptor.

    PubMed

    Evangelista, Wilfredo; Yeh, Lee-Chuan C; Gmyrek, Aleksandra; Lee, J Ching; Lee, John C

    2015-12-01

    Protein-protein interactions are recognized as a fundamental phenomenon that is intimately associated with biological functions and thus are ideal targets for developing modulators for regulating biological functions. A challenge is to identify a site that is situated away from but functionally connected to the protein-protein interface. We employed bone morphogenetic proteins (BMPs) and their receptors as a model system to develop a strategy for identifying such a network of communication. Accordingly, using computational analyses with the COREX/BEST algorithm, we uncovered an overall pattern connecting various regions of BMPR-1B ectodomain, including the four conserved residues in the protein-protein interface. In preparation for testing the long-range effects of mutations of distal residues for future studies, we examined the extent of measurable perturbation of the four conserved residues by determination of the conformation and relative affinities of these BMPR-1B mutants for ligands BMP-2, -6, and -7 and GDF-5. Results suggest no significant structural changes in the receptor but do suggest that the four residues play different roles in defining ligand affinity and both intra- and intermolecular interactions play a role in defining ligand affinity. Thus, these results established two primary but necessary goals: (1) the baseline knowledge of perturbation of conserved interfacial residues for future reference and (2) the ability of the computational approach to identify the distal residues connecting to the interfacial residues. The data presented here provide the foundation for future experiments to identify the effects of distal residues that affect the specificity and affinity of BMP recognition. Protein-protein interactions are integral reactions in essentially all biological activities such as gene regulation and age-related development. Often, diseases are consequences of the alteration of these intermacromolecular interactions, which are thus recognized

  7. ING1b negatively regulates HIF1α protein levels in adipose-derived stromal cells by a SUMOylation-dependent mechanism.

    PubMed

    Bigot, N; Guérillon, C; Loisel, S; Bertheuil, N; Sensebé, L; Tarte, K; Pedeux, R

    2015-01-01

    Hypoxic niches help maintain mesenchymal stromal cell properties, and their amplification under hypoxia sustains their immature state. However, how MSCs maintain their genomic integrity in this context remains elusive, since hypoxia may prevent proper DNA repair by downregulating expression of BRCA1 and RAD51. Here, we find that the ING1b tumor suppressor accumulates in adipose-derived stromal cells (ADSCs) upon genotoxic stress, owing to SUMOylation on K193 that is mediated by the E3 small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT protein γ (PIAS4). We demonstrate that ING1b finely regulates the hypoxic response by triggering HIF1α proteasomal degradation. On the contrary, when mutated on its SUMOylation site, ING1b failed to efficiently decrease HIF1α levels. Consistently, we observed that the adipocyte differentiation, generally described to be downregulated by hypoxia, was highly dependent on ING1b expression, during the early days of this process. Accordingly, contrary to what was observed with HIF1α, the absence of ING1b impeded the adipogenic induction under hypoxic conditions. These data indicate that ING1b contributes to adipogenic induction in adipose-derived stromal cells, and thus hinders the phenotype maintenance of ADSCs. PMID:25611387

  8. Microtubule-associated protein 1B interaction with tubulin tyrosine ligase contributes to the control of microtubule tyrosination.

    PubMed

    Utreras, Elías; Jiménez-Mateos, Eva Maria; Contreras-Vallejos, Erick; Tortosa, Elena; Pérez, Mar; Rojas, Sebastián; Saragoni, Lorena; Maccioni, Ricardo B; Avila, Jesús; González-Billault, Christian

    2008-01-01

    Microtubule-associated protein 1B (MAP1B) is the first microtubule-associated protein to be expressed during nervous system development. MAP1B belongs to a large family of proteins that contribute to the stabilization and/or enhancement of microtubule polymerization. These functions are related to the control of the dynamic properties of microtubules. The C-terminal domain of the neuronal alpha-tubulin isotype is characterized by the presence of an acidic polypeptide, with the last amino acid being tyrosine. This tyrosine residue may be enzymatically removed from the protein by an unknown carboxypeptidase activity. Subsequently, the tyrosine residue is again incorporated into this tubulin by another enzyme, tubulin tyrosine ligase, to yield tyrosinated tubulin. Because neurons lacking MAP1B have a reduced proportion of tyrosinated microtubules, we analyzed the possible interaction between MAP1B and tubulin tyrosine ligase. Our results show that these proteins indeed interact and that the interaction is not affected by MAP1B phosphorylation. Additionally, neurons lacking MAP1B, when exposed to drugs that reversibly depolymerize microtubules, do not fully recover tyrosinated microtubules upon drug removal. These results suggest that MAP1B regulates tyrosination of alpha-tubulin in neuronal microtubules. This regulation may be important for general processes involved in nervous system development such as axonal guidance and neuronal migration. PMID:18075266

  9. Substrate Specificity of Protein Tyrosine Phosphatases 1B, RPTPα, SHP-1, and SHP-2†

    PubMed Central

    Ren, Lige; Chen, Xianwen; Luechapanichkul, Rinrada; Selner, Nicholas G.; Meyer, Tiffany M.; Wavreille, Anne-Sophie; Chan, Richard; Iorio, Caterina; Zhou, Xiang; Neel, Benjamin G.; Pei, Dehua

    2011-01-01

    We determined the substrate specificities of the protein tyrosine phosphatases (PTPs) PTP1B, RPTPα, SHP-1, and SHP-2 by on-bead screening of combinatorial peptide libraries and solution-phase kinetic analysis of individually synthesized phosphotyrosyl (pY) peptides. These PTPs exhibit different levels of sequence specificity and catalytic efficiency. The catalytic domain of RPTPα has very weak sequence specificity and is approximately two orders of magnitude less active than the other three PTPs. The PTP1B catalytic domain has modest preference for acidic residues on both sides of pY, is highly active towards multiply phosphorylated peptides, but disfavors basic residues at any position, a Gly at the pY−1 position, or a Pro at the pY+1 position. By contrast, SHP-1 and SHP-2 share similar but much narrower substrate specificities, with a strong preference for acidic and aromatic hydrophobic amino acids on both sides of the pY residue. An efficient SHP-1/2 substrate generally contains two or more acidic residues on the N-terminal side and one or more acidic residues on the C-terminal side of pY, but no basic residues. Subtle differences exist between SHP-1 and SHP-2 in that SHP-1 has a stronger preference for acidic residues at the pY−1 and pY+1 positions and the two SHPs prefer acidic residues at different positions N-terminal to pY. A survey of the known protein substrates of PTP1B, SHP-1, and SHP-2 shows an excellent agreement between the in vivo dephosphorylation pattern and the in vitro specificity profiles derived from library screening. These results suggest that different PTPs have distinct sequence specificity profiles and the intrinsic activity/specificity of the PTP domain is an important determinant of the enzyme’s in vivo substrate specificity. PMID:21291263

  10. Ring1B Contains a Ubiquitin-Like Docking Module for Interaction with Cbx Proteins

    SciTech Connect

    Bezsonova, Irina; Walker, John R.; Bacik, John P.; Duan, Shili; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.

    2010-04-19

    Polycomb group (PcG) proteins are a special set of repressive transcription factors involved in epigenetic modifications of chromatin. They form two functionally distinct groups of catalytically active complexes: Polycomb repressive complex 1 (PRC1) and 2 (PRC2). The PRC1 complex is an important yet poorly characterized multiprotein histone ubiquitylation machine responsible for maintaining transcriptionally silent states of genes through histone H2A K119 modification. The Ring domain containing subunits of PRC1 also have substrate-targeting domains that interact with Cbx proteins, which have been implicated in chromatin and RNA binding. In this work, we present a high resolution structure of the C-terminal domain of Ring1B, revealing a variant ubiquitin-like fold with a distinct conserved surface region. On the basis of crystal structure and mutational analysis of this domain we show that the conserved surface is responsible for interaction with Cbx members of the PRC1 and homodimer formation. These data suggest a mechanism by which Ring1B serves as an adaptor that mediates binding between the members of the PRC1 complex and the nucleosome.

  11. Structure and Configuration of Phosphoeleganin, a Protein Tyrosine Phosphatase 1B Inhibitor from the Mediterranean Ascidian Sidnyum elegans.

    PubMed

    Imperatore, Concetta; Luciano, Paolo; Aiello, Anna; Vitalone, Rocco; Irace, Carlo; Santamaria, Rita; Li, Jia; Guo, Yue-W; Menna, Marialuisa

    2016-04-22

    A new phosphorylated polyketide, phosphoeleganin (1), has been isolated from the Mediterranean ascidian Sidnyum elegans. Its structure and configuration have been determined by extensive use of 2D NMR and microscale chemical degradation and/or derivatization. Phosphoeleganin (1) inhibited the protein tyrosine phosphatase 1B (PTP1B) activity. PMID:27064611

  12. Phosphoinositide-specific Phospholipase C β 1b (PI-PLCβ1b) Interactome: Affinity Purification-Mass Spectrometry Analysis of PI-PLCβ1b with Nuclear Protein*

    PubMed Central

    Piazzi, Manuela; Blalock, William L.; Bavelloni, Alberto; Faenza, Irene; D'Angelo, Antonietta; Maraldi, Nadir M.; Cocco, Lucio

    2013-01-01

    Two isoforms of inositide-dependent phospholipase C β1 (PI-PLCβ1) are generated by alternative splicing (PLCβ1a and PLCβ1b). Both isoforms are present within the nucleus, but in contrast to PLCβ1a, the vast majority of PLCβ1b is nuclear. In mouse erythroid leukemia cells, PI-PLCβ1 is involved in the regulation of cell division and the balance between cell proliferation and differentiation. It has been demonstrated that nuclear localization is crucial for the enzymatic function of PI-PLCβ1, although the mechanism by which this nuclear import occurs has never been fully characterized. The aim of this study was to characterize both the mechanism of nuclear localization and the molecular function of nuclear PI-PLCβ1 by identifying its interactome in Friend's erythroleukemia isolated nuclei, utilizing a procedure that coupled immuno-affinity purification with tandem mass spectrometry analysis. Using this procedure, 160 proteins were demonstrated to be in association with PI-PLCβ1b, some of which have been previously characterized, such as the splicing factor SRp20 (Srsf3) and Lamin B (Lmnb1). Co-immunoprecipitation analysis of selected proteins confirmed the data obtained via mass spectrometry. Of particular interest was the identification of the nuclear import proteins Kpna2, Kpna4, Kpnb1, Ran, and Rangap1, as well as factors involved in hematological malignancies and several anti-apoptotic proteins. These data give new insight into possible mechanisms of nuclear trafficking and functioning of this critical signaling molecule. PMID:23665500

  13. Structural analysis of anti-tumor heteropolysaccharide GFPS1b from the cultured mycelia of Grifola frondosa GF9801.

    PubMed

    Cui, F J; Tao, W Y; Xu, Z H; Guo, W J; Xu, H Y; Ao, Z H; Jin, J; Wei, Y Q

    2007-01-01

    A 21-kDa heteropolysaccharide, coded as GFPS1b, was obtained from the cultured mycelia of Grifola frondosa GF9801 by hot-water extraction, ethanol precipitation, and fractioned by DEAE Sepharose Fast-flow, followed by the purification with Sephadex G-100 column chromatography using an AKTA purifier. It exhibited more potent anti-proliferative activity on MCF-7 cells than other polysaccharide fractions. GFPS1b was an acidic polysaccharide with approximately 16.60% protein and 4.3% uronic acid. Gas chromatography of absolute acid hydrolysate of GFPS1b suggested that it was composed of D-glucose, D-galactose, and L-arabinose with a molar ratio of 4:2:1. Periodate oxidation, Smith degradation, partial acid hydrolyzation, methylation analysis, FT-IR, and (1)H, (13)C NMR spectroscopy analysis revealed that GFPS1b had a backbone consisting of alpha-(1-->4)-linked D-galacopyranosyl and alpha-(1-->3)-linked D-glucopyranosyl residues substituted at O-6 with glycosyl residues composed of alpha-L-arabinose-(1-->4)-alpha-D-glucose (1--> linked residues. PMID:16459075

  14. Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli.

    PubMed Central

    Spindler, K R; Rosser, D S; Berk, A J

    1984-01-01

    Plasmid vectors were constructed which expressed three adenovirus tumor antigens fused to a portion of the trpE protein of Escherichia coli. Insertion of adenovirus type 2 DNA from early region 1A (E1A) into such a plasmid led to a fusion protein which contained the C-terminal 266 amino acids of the 289-amino acid protein encoded by the viral 13S mRNA. Similarly, insertion of adenovirus type 5 DNA corresponding to the E1B 55- and 21-kilodalton proteins led to production of fusion proteins containing amino acid sequences from these proteins. After induction with indoleacrylic acid, fusion proteins accumulated stably in the E. coli cells. By using a simple extraction of insoluble protein, 1 to 10 mg of fusion protein per liter of culture was obtained. The fusion proteins were purified on preparative polyacrylamide gels and used to immunize rabbits. Specific antisera for the E1A 289- and closely related 243-amino acid proteins and the E1B 55- and 21-kilodalton proteins were obtained. These sera were used to immunoprecipitate the tumor antigens in cells infected with wild-type and various mutants of adenovirus or to analyze them by an immunoblotting procedure. Mutant E1A proteins in which the C-terminal 70 amino acids are deleted were phosphorylated to much lower extents than the wild-type E1A proteins. This indicates that the deleted region is important for the process of phosphorylation. The E1A proteins were extracted, sedimented in glycerol gradients, analyzed by immunoprecipitation, and found to sediment primarily as monomers. Images PMID:6361277

  15. Phosphonate monoesters on a thiacalix[4]arene framework as potential inhibitors of protein tyrosine phosphatase 1B.

    PubMed

    Trush, Viacheslav V; Kharchenko, Sergiy G; Tanchuk, Vsevolod Yu; Kalchenko, Vitaly I; Vovk, Andriy I

    2015-09-01

    Monoester derivatives of thiacalix[4]arene tetrakis(methylphosphonic) acid were found to be capable of inhibiting protein tyrosine phosphatase 1B. In addition, these compounds can strongly bind to human serum albumin. PMID:26205135

  16. Structural insights into the lipoprotein outer membrane regulator of penicillin-binding protein 1B.

    PubMed

    King, Dustin T; Lameignere, Emilie; Strynadka, Natalie C J

    2014-07-01

    In bacteria, the synthesis of the protective peptidoglycan sacculus is a dynamic process that is tightly regulated at multiple levels. Recently, the lipoprotein co-factor LpoB has been found essential for the in vivo function of the major peptidoglycan synthase PBP1b in Enterobacteriaceae. Here, we reveal the crystal structures of Salmonella enterica and Escherichia coli LpoB. The LpoB protein can be modeled as a ball and tether, consisting of a disordered N-terminal region followed by a compact globular C-terminal domain. Taken together, our structural data allow us to propose new insights into LpoB-mediated regulation of peptidoglycan synthesis. PMID:24808177

  17. Microtubule Associated Protein 1b (MAP1B) Is a Marker of the Microtubular Cytoskeleton in Podocytes but Is Not Essential for the Function of the Kidney Filtration Barrier in Mice

    PubMed Central

    Gödel, Markus; Temerinac, Dunja; Grahammer, Florian; Hartleben, Björn; Kretz, Oliver; Riederer, Beat M.; Propst, Friedrich

    2015-01-01

    Podocytes are essential for the function of the kidney glomerular filter. A highly differentiated cytoskeleton is requisite for their integrity. Although much knowledge has been gained on the organization of cortical actin networks in podocyte’s foot processes, less is known about the molecular organization of the microtubular cytoskeleton in primary processes and the cell body. To gain an insight into the organization of the microtubular cytoskeleton of the podocyte, we systematically analyzed the expression of microtubule associated proteins (Maps), a family of microtubules interacting proteins with known functions as regulator, scaffold and guidance proteins. We identified microtubule associated protein 1b (MAP1B) to be specifically enriched in podocytes in human and rodent kidney. Using immunogold labeling in electron microscopy, we were able to demonstrate an enrichment of MAP1B in primary processes. A similar association of MAP1B with the microtubule cytoskeleton was detected in cultured podocytes. Subcellular distribution of MAP1B HC and LC1 was analyzed using a double fluorescent reporter MAP1B fusion protein. Subsequently we analyzed mice constitutively depleted of MAP1B. Interestingly, MAP1B KO was not associated with any functional or structural alterations pointing towards a redundancy of MAP proteins in podocytes. In summary, we established MAP1B as a specific marker protein of the podocyte microtubular cytoskeleton. PMID:26448484

  18. Tomato Protein Kinase 1b Mediates Signaling of Plant Responses to Necrotrophic Fungi and Insect Herbivory[W

    PubMed Central

    AbuQamar, Synan; Chai, Mao-Feng; Luo, Hongli; Song, Fengming; Mengiste, Tesfaye

    2008-01-01

    The tomato protein kinase 1 (TPK1b) gene encodes a receptor-like cytoplasmic kinase localized to the plasma membrane. Pathogen infection, mechanical wounding, and oxidative stress induce expression of TPK1b, and reducing TPK1b gene expression through RNA interference (RNAi) increases tomato susceptibility to the necrotrophic fungus Botrytis cinerea and to feeding by larvae of tobacco hornworm (Manduca sexta) but not to the bacterial pathogen Pseudomonas syringae. TPK1b RNAi seedlings are also impaired in ethylene (ET) responses. Notably, susceptibility to Botrytis and insect feeding is correlated with reduced expression of the proteinase inhibitor II gene in response to Botrytis and 1-aminocyclopropane-1-carboxylic acid, the natural precursor of ET, but wild-type expression in response to mechanical wounding and methyl-jasmonate. TPK1b functions independent of JA biosynthesis and response genes required for resistance to Botrytis. TPK1b is a functional kinase with autophosphorylation and Myelin Basis Protein phosphorylation activities. Three residues in the activation segment play a critical role in the kinase activity and in vivo signaling function of TPK1b. In sum, our findings establish a signaling role for TPK1b in an ET-mediated shared defense mechanism for resistance to necrotrophic fungi and herbivorous insects. PMID:18599583

  19. Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy.

    PubMed

    Roosing, Susanne; Lamers, Ideke J C; de Vrieze, Erik; van den Born, L Ingeborgh; Lambertus, Stanley; Arts, Heleen H; Peters, Theo A; Hoyng, Carel B; Kremer, Hannie; Hetterschijt, Lisette; Letteboer, Stef J F; van Wijk, Erwin; Roepman, Ronald; den Hollander, Anneke I; Cremers, Frans P M

    2014-08-01

    Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors. PMID:25018096

  20. ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis

    PubMed Central

    Moll, Iris; Yang, Wan-Jen; Wüstehube-Lausch, Joycelyn; Fischer, Andreas

    2015-01-01

    Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis. PMID:26698571

  1. Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons

    PubMed Central

    Bolte, Petra; Bleibaum, Florian; Einwich, Angelika; Günther, Anja; Liedvogel, Miriam; Heyers, Dominik; Depping, Anne; Wöhlbrand, Lars; Rabus, Ralf; Janssen‐Bienhold, Ulrike; Mouritsen, Henrik

    2016-01-01

    Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism. PMID:26953791

  2. Localisation of the Putative Magnetoreceptive Protein Cryptochrome 1b in the Retinae of Migratory Birds and Homing Pigeons.

    PubMed

    Bolte, Petra; Bleibaum, Florian; Einwich, Angelika; Günther, Anja; Liedvogel, Miriam; Heyers, Dominik; Depping, Anne; Wöhlbrand, Lars; Rabus, Ralf; Janssen-Bienhold, Ulrike; Mouritsen, Henrik

    2016-01-01

    Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism. PMID:26953791

  3. Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes*

    PubMed Central

    Mobasher, Maysa Ahmed; de Toro-Martín, Juan; González-Rodríguez, Águeda; Ramos, Sonia; Letzig, Lynda G.; James, Laura P.; Muntané, Jordi; Álvarez, Carmen; Valverde, Ángela M.

    2014-01-01

    Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B−/− mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver. PMID:25204659

  4. Protein tyrosine phosphatase 1B negatively regulates S100A9-mediated lung damage during respiratory syncytial virus exacerbations.

    PubMed

    Foronjy, R F; Ochieng, P O; Salathe, M A; Dabo, A J; Eden, E; Baumlin, N; Cummins, N; Barik, S; Campos, M; Thorp, E B; Geraghty, P

    2016-09-01

    Protein tyrosine phosphatase 1B (PTP1B) has anti-inflammatory potential but PTP1B responses are desensitized in the lung by prolonged cigarette smoke exposure. Here we investigate whether PTP1B expression affects lung disease severity during respiratory syncytial viral (RSV) exacerbations of chronic obstructive pulmonary disease (COPD). Ptp1b(-/-) mice infected with RSV exhibit exaggerated immune cell infiltration, damaged epithelial cell barriers, cytokine production, and increased apoptosis. Elevated expression of S100A9, a damage-associated molecular pattern molecule, was observed in the lungs of Ptp1b(-/-) mice during RSV infection. Utilizing a neutralizing anti-S100A9 IgG antibody, it was determined that extracellular S100A9 signaling significantly affects lung damage during RSV infection. Preexposure to cigarette smoke desensitized PTP1B activity that coincided with enhanced S100A9 secretion and inflammation in wild-type animals during RSV infection. S100A9 levels in human bronchoalveolar lavage fluid had an inverse relationship with lung function in healthy subjects, smokers, and COPD subjects. Fully differentiated human bronchial epithelial cells isolated from COPD donors cultured at the air liquid interface secreted more S100A9 than cells from healthy donors or smokers following RSV infection. Together, these findings show that reduced PTP1B responses contribute to disease symptoms in part by enhancing S100A9 expression during viral-associated COPD exacerbations. PMID:26813343

  5. Adenoviral E4orf3 and E4orf6 Proteins, But Not E1B55K, Increase Killing of Cancer Cells by Radiotherapy in vivo

    SciTech Connect

    Liikanen, Ilkka; Dias, Joao D.; Nokisalmi, Petri; Sloniecka, Marta; Kangasniemi, Lotta; Rajecki, Mari; Dobner, Thomas; Tenhunen, Mikko; Kanerva, Anna; Pesonen, Sari; Ahtiainen, Laura Ph.D.; Hemminki, Akseli

    2010-11-15

    Purpose: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. Methods and Materials: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. Results: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. Conclusions: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.

  6. Identification of adenovirus type 2 early region 1B proteins that share the same amino terminus as do the 495R and 155R proteins.

    PubMed Central

    Lewis, J B; Anderson, C W

    1987-01-01

    Adenovirus type 2 early region 1B (E1B) proteins synthesized in vitro were fractionated chromatographically and characterized by peptide and sequence analysis and by reaction with peptide-specific antisera targeted to either the N or C terminus of either of two overlapping E1B reading frames (175 or 495 codons). In addition to the previously identified E1B-495R, E1B-175R, and E1B-155R species, two other E1B proteins of similar electrophoretic mobility to the 175R protein were identified. E1B-82R is an abundant product in vitro and in vivo that has the same N terminus as that of the 495R and 155R proteins but a different C terminus. The structure of 82R is predicted by the structure of the abundant 13S (1.02-kilobase) E1B mRNA. E1B-168R is a novel minor species consisting of the 24 amino-terminal residues of the 495R protein fused to the entire polypeptide IX sequence. An additional, minor 16,000-molecular-weight polypeptide was detected that may correspond to a predicted 92R E1B protein, but definitive identification was not possible. These observations establish that the leftmost portion (78 codons) of the 495-codon reading frame, which overlaps the right half of the 175-codon reading frame, is expressed as an abundant protein that does not contain other 495R sequences. This region, which may participate in the regulation of region E1A expression, may thus constitute a functional domain distinct from the rightward portion of the 495R protein. Images PMID:2960832

  7. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    PubMed

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. PMID:26337385

  8. The 4.1B cytoskeletal protein regulates the domain organization and sheath thickness of myelinated axons

    PubMed Central

    Einheber, Steven; Maurel, Patrice; Meng, Xiaosong; Rubin, Marina; Lam, Isabel; Mohandas, Narla; An, Xiuli; Shrager, Peter; Kissil, Joseph; Salzer, James L.

    2012-01-01

    Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e. nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains and, unexpectedly, that it regulates myelin sheath thickness. PMID:23109359

  9. The 4.1B cytoskeletal protein regulates the domain organization and sheath thickness of myelinated axons.

    PubMed

    Einheber, Steven; Meng, Xiaosong; Rubin, Marina; Lam, Isabel; Mohandas, Narla; An, Xiuli; Shrager, Peter; Kissil, Joseph; Maurel, Patrice; Salzer, James L

    2013-02-01

    Myelinated axons are organized into specialized domains critical to their function in saltatory conduction, i.e., nodes, paranodes, juxtaparanodes, and internodes. Here, we describe the distribution and role of the 4.1B protein in this organization. 4.1B is expressed by neurons, and at lower levels by Schwann cells, which also robustly express 4.1G. Immunofluorescence and immuno-EM demonstrates 4.1B is expressed subjacent to the axon membrane in all domains except the nodes. Mice deficient in 4.1B have preserved paranodes, based on marker staining and EM in contrast to the juxtaparanodes, which are substantially affected in both the PNS and CNS. The juxtaparanodal defect is evident in developing and adult nerves and is neuron-autonomous based on myelinating cocultures in which wt Schwann cells were grown with 4.1B-deficient neurons. Despite the juxtaparanodal defect, nerve conduction velocity is unaffected. Preservation of paranodal markers in 4.1B deficient mice is associated with, but not dependent on an increase of 4.1R at the axonal paranodes. Loss of 4.1B in the axon is also associated with reduced levels of the internodal proteins, Necl-1 and Necl-2, and of alpha-2 spectrin. Mutant nerves are modestly hypermyelinated and have increased numbers of Schmidt-Lanterman incisures, increased expression of 4.1G, and express a residual, truncated isoform of 4.1B. These results demonstrate that 4.1B is a key cytoskeletal scaffold for axonal adhesion molecules expressed in the juxtaparanodal and internodal domains that unexpectedly regulates myelin sheath thickness. PMID:23109359

  10. Development of Novel Triazolo-Thiadiazoles from Heterogeneous “Green” Catalysis as Protein Tyrosine Phosphatase 1B Inhibitors

    PubMed Central

    Baburajeev, C. P.; Dhananjaya Mohan, Chakrabhavi; Ananda, Hanumappa; Rangappa, Shobith; Fuchs, Julian E.; Jagadish, Swamy; Sivaraman Siveen, Kodappully; Chinnathambi, Arunachalam; Ali Alharbi, Sulaiman; Zayed, M. E.; Zhang, Jingwen; Li, Feng; Sethi, Gautam; Girish, Kesturu S.; Bender, Andreas; Basappa; Rangappa, Kanchugarakoppal S.

    2015-01-01

    Condensed-bicyclic triazolo-thiadiazoles were synthesized via an efficient “green” catalyst strategy and identified as effective inhibitors of PTP1B in vitro. The lead compound, 6-(2-benzylphenyl)-3-phenyl-[1,2,4]triazolo[3][1,3,4]thiadiazole (BPTT) was most effective against human hepatoma cells, inhibits cell invasion, and decreases neovasculature in HUVEC and also tumor volume in EAT mouse models. This report describes an experimentally unidentified class of condensed-bicyclic triazolo-thiadiazoles targeting PTP1B and its analogs could be the therapeutic drug-seeds. PMID:26388336

  11. Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

    SciTech Connect

    Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

    2003-06-11

    Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

  12. Mitochondrial localization, ELK-1 transcriptional regulation and Growth inhibitory functions of BRCA1, BRCA1a and BRCA1b proteins

    PubMed Central

    Maniccia, Anna W; Lewis, Catherine; Begum, Nurjahan; Xu, Jingyao; Cui, Jianqi; Chipitsyna, Galina; Aysola, Kartik; Reddy, Vaishali; Bhat, Ganapathy; Fujimura, Yasuo; Henderson, Beric; Reddy, E. Shyam P.; Rao, Veena N.

    2009-01-01

    BRCA1 is a tumor suppressor gene that is mutated in families with breast and ovarian cancer. Several BRCA1 splice variants are found in different tissues, but their subcellular localization and functions are poorly understood at the moment. We previously described BRCA1 splice variant BRCA1a to induce apoptosis and function as a tumor suppressor of triple negative breast, ovarian and prostate cancers. In this study we have analyzed the function of BRCA1 isoforms (BRCA1a and BRCA1b) and compared them to the wild type BRCA1 protein using several criteria like studying expression in normal and tumor cells by RNase protection assays, sub cellular localization/fractionation by immunofluorescence microscopy and western blot analysis, transcription regulation of biological relevant proteins and growth suppression in breast cancer cells. We are demonstrating for the first time that ectopically expressed GFP-tagged BRCA1, BRCA1a, and BRCA1b proteins are localized to the mitochondria, repress ELK-1 transcriptional activity and possess antiproliferative activity on breast cancer cells. These results suggest that the exon 9,10 and 11 sequences (aa 263 – 1365) which contain two nuclear localization signals, p53, Rb, c-Myc, γ- tubulin, Stat, Rad 51, Rad 50 binding domains, angiopoietin-1 repression domain are not absolutely required for mitochondrial localization and growth suppressor function of these proteins. Since mitochondrial dysfunction is a hallmark of cancer, we can speculate that the mitochondrial localization of BRCA1 proteins may be functionally significant in regulating both the mitochondrial DNA damage as well as apoptotic activity of BRCA1 proteins and mislocalization causes cancer. PMID:19170108

  13. Pathogenesis-related protein 1b1 (PR1b1) is a major tomato fruit protein responsive to chilling temperature and upregulated in high polyamine transgenic genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. A group of commonly found metabolites implicated in protection against stresses such as chilling stress constitute ubiquitous biogenic amines calle...

  14. The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

    PubMed

    Pandey, Nihar R; Zhou, Xun; Qin, Zhaohong; Zaman, Tariq; Gomez-Smith, Mariana; Keyhanian, Kianoosh; Anisman, Hymie; Brunel, Jean Michel; Stewart, Alexandre F R; Chen, Hsiao-Huei

    2013-07-31

    Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat. PMID:23904601

  15. Light-switched inhibitors of protein tyrosine phosphatase PTP1B based on phosphonocarbonyl phenylalanine as photoactive phosphotyrosine mimetic.

    PubMed

    Wagner, Stefan; Schütz, Anja; Rademann, Jörg

    2015-06-15

    Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT). PMID:25907367

  16. Acute exercise decreases PTP-1B protein level and improves insulin signaling in the liver of old rats

    PubMed Central

    2013-01-01

    It is now commonly accepted that chronic inflammation associated with obesity during aging induces insulin resistance in the liver. In the present study, we investigated whether the improvement in insulin sensitivity and insulin signaling, mediated by acute exercise, could be associated with modulation of protein-tyrosine phosphatase 1B (PTP-1B) in the liver of old rats. Aging rats were subjected to swimming for two 1.5-h long bouts, separated by a 45 min rest period. Sixteen hours after the exercise, the rats were sacrificed and proteins from the insulin signaling pathway were analyzed by immunoblotting. Our results show that the fat mass was increased in old rats. The reduction in glucose disappearance rate (Kitt) observed in aged rats was restored 16 h after exercise. Aging increased the content of PTP-1B and attenuated insulin signaling in the liver of rats, a phenomenon that was reversed by exercise. Aging rats also increased the IRβ/PTP-1B and IRS-1/PTP-1B association in the liver when compared with young rats. Conversely, in the liver of exercised old rats, IRβ/PTP-1B and IRS-1/PTP-1B association was markedly decreased. Moreover, in the hepatic tissue of old rats, the insulin signalling was decreased and PEPCK and G6Pase levels were increased when compared with young rats. Interestingly, 16 h after acute exercise, the PEPCK and G6Pase protein level were decreased in the old exercised group. These results provide new insights into the mechanisms by which exercise restores insulin signalling in liver during aging. PMID:23442260

  17. Novel Mechanism of Impaired Function of Organic Anion-Transporting Polypeptide 1B3 in Human Hepatocytes: Post-Translational Regulation of OATP1B3 by Protein Kinase C Activation

    PubMed Central

    Powell, John; Farasyn, Taleah; Köck, Kathleen; Meng, Xiaojie; Pahwa, Sonia; Brouwer, Kim L. R.

    2014-01-01

    The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [3H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [3H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti–phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds. PMID:25200870

  18. Protein tyrosine phosphatase 1B inhibitors: a molecular level legitimate approach for the management of diabetes mellitus.

    PubMed

    Thareja, Suresh; Aggarwal, Saurabh; Bhardwaj, T R; Kumar, Manoj

    2012-05-01

    Diabetes mellitus is a systemic disease responsible for morbidity in the western world and is gradually becoming prevalent in developing countries too. The prevalence of diabetes is rapidly increasing in industrialized countries and type 2 diabetes accounts for 90% of the disease. Insulin resistance is a major pathophysiological factor in the development of type 2 diabetes, occurring mainly in muscle, adipose tissues, and liver leading to reduced glucose uptake and utilization and increased glucose production. The prevalence and rising incidence of diabetes emphasized the need to explore new molecular targets and strategies to develop novel antihyperglycemic agents. Protein Tyrosine Phosphatase 1B (PTP 1B) has recently emerged as a promising molecular level legitimate therapeutic target in the effective management of type 2 diabetes. PTP 1B, a cytosolic nonreceptor PTPase, has been implicated as a negative regulator of insulin signal transduction. Therefore, PTP 1B inhibitors would increase insulin sensitivity by blocking the PTP 1B-mediated negative insulin signaling pathway and might be an attractive target for type 2 diabetes mellitus and obesity. With X-ray crystallography and NMR-based fragment screening, the binding interactions of several classes of inhibitors have been elucidated, which could help the design of future PTP 1B inhibitors. The drug discovery research in PTP 1B is a challenging area to work with and many pharmaceutical organizations and academic research laboratories are focusing their research toward the development of potential PTP 1B inhibitors which would prove to be a milestone for the management of diabetes. PMID:20814956

  19. Actin related protein complex subunit 1b controls sperm release, barrier integrity and cell division during adult rat spermatogenesis.

    PubMed

    Kumar, Anita; Dumasia, Kushaan; Deshpande, Sharvari; Gaonkar, Reshma; Balasinor, N H

    2016-08-01

    Actin remodeling is a vital process for signaling, movement and survival in all cells. In the testes, extensive actin reorganization occurs at spermatid-Sertoli cell junctions during sperm release (spermiation) and at inter Sertoli cell junctions during restructuring of the blood testis barrier (BTB). During spermiation, tubulobulbar complexes (TBCs), rich in branched actin networks, ensure recycling of spermatid-Sertoli cell junctional molecules. Similar recycling occurs during BTB restructuring around the same time as spermiation occurs. Actin related protein 2/3 complex is an essential actin nucleation and branching protein. One of its subunits, Arpc1b, was earlier found to be down-regulated in an estrogen-induced rat model of spermiation failure. Also, Arpc1b was found to be estrogen responsive through estrogen receptor beta in seminiferous tubule culture. Here, knockdown of Arpc1b by siRNA in adult rat testis led to defects in spermiation caused by failure in TBC formation. Knockdown also compromised BTB integrity and caused polarity defects of mature spermatids. Apart from these effects pertaining to Sertoli cells, Arpc1b reduction perturbed ability of germ cells to enter G2/M phase thus hindering cell division. In summary, Arpc1b, an estrogen responsive gene, is a regulator of spermiation, mature spermatid polarity, BTB integrity and cell division during adult spermatogenesis. PMID:27113856

  20. New Hippolide Derivatives with Protein Tyrosine Phosphatase 1B Inhibitory Activity from the Marine Sponge Hippospongia lachne

    PubMed Central

    Piao, Shu-Juan; Jiao, Wei-Hua; Yang, Fan; Yi, Yang-Hua; Di, Ying-Tong; Han, Bing-Nan; Lin, Hou-Wen

    2014-01-01

    Five new sesterterpenoids, compounds 1–5, have been isolated from the sponge Hippospongia lachne off Yongxing Island in the South China Sea. The structures of compounds 1–5 were elucidated through extensive spectroscopic analysis, including HRMS, 1D, and 2D NMR experiments. The stereochemistry, including absolute configurations of these compounds, was determined by spectroscopic, chemical, and computational methods. Compounds 1 and 5 showed moderate protein tyrosine phosphatase 1B (PTP1B) inhibitory activities with IC50 values of 5.2 μM and 8.7 μM, respectively, more potent than previously reported hippolides. PMID:25007159

  1. Gi-protein-coupled 5-HT1B/D receptor agonist sumatriptan induces type I hyperalgesic priming.

    PubMed

    Araldi, Dioneia; Ferrari, Luiz F; Levine, Jon D

    2016-08-01

    We have recently described a novel form of hyperalgesic priming (type II) induced by agonists at two clinically important Gi-protein-coupled receptors (Gi-GPCRs), mu-opioid and A1-adenosine. Like mu-opioids, the antimigraine triptans, which act at 5-HT1B/D Gi-GPCRs, have been implicated in pain chronification. We determined whether sumatriptan, a prototypical 5-HT1B/D agonist, produces type II priming. Characteristic of hyperalgesic priming, intradermal injection of sumatriptan (10 ng) induced a change in nociceptor function such that a subsequent injection of prostaglandin-E2 (PGE2) induces prolonged mechanical hyperalgesia. However, onset to priming was delayed 3 days, characteristic of type I priming. Also characteristic of type I priming, a protein kinase Cε, but not a protein kinase A inhibitor attenuated the prolongation phase of PGE2 hyperalgesia. The prolongation of PGE2 hyperalgesia was also permanently reversed by intradermal injection of cordycepin, a protein translation inhibitor. Also, hyperalgesic priming did not occur in animals pretreated with pertussis toxin or isolectin B4-positive nociceptor toxin, IB4-saporin. Finally, as observed for other agonists that induce type I priming, sumatriptan did not induce priming in female rats. The prolongation of PGE2 hyperalgesia induced by sumatriptan was partially prevented by coinjection of antagonists for the 5-HT1B and 5-HT1D, but not 5-HT7, serotonin receptors and completely prevented by coadministration of a combination of the 5-HT1B and 5-HT1D antagonists. Moreover, the injection of selective agonists, for 5-HT1B and 5-HT1D receptors, also induced hyperalgesic priming. Our results suggest that sumatriptan, which signals through Gi-GPCRs, induces type I hyperalgesic priming, unlike agonists at other Gi-GPCRs, which induce type II priming. PMID:27075428

  2. Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

    SciTech Connect

    Nicholas, R.A.; Suzuki, H.; Hirota, Y.; Strominger, J.L.

    1985-07-02

    This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with (/sup 14/C)penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the (/sup 14/C)penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.

  3. Artifactual processing of penicillin-binding proteins 7 and 1b by the OmpT protease of Escherichia coli.

    PubMed Central

    Henderson, T A; Dombrosky, P M; Young, K D

    1994-01-01

    Penicillin-binding proteins (PBPs) were visualized in strains of Escherichia coli that carried mutations in one or more of the following protease genes: tsp, degP, ptr, and ompT. In the absence of a functional ompT gene, PBPs 1b alpha and 7 were not processed to the shortened forms 1b beta and 8, respectively. Cleavage of PBPs 1b alpha and 7 could be restored by introduction of a plasmid carrying the wild-type ompT gene. These PBPs were processed only after cell lysis or after membrane perturbation of whole cells by freeze-thaw, suggesting that the cleavage was a nonspecific artifact due to contact with OmpT, an outer membrane protease, and that such processing was not biologically significant in vivo. The degradation of other PBPs during purification or storage may also be effected by OmpT. Images PMID:8282705

  4. Characterisation of iron regulatory protein 1A and 1B in the blood-feeding copepod Lepeophtheirus salmonis.

    PubMed

    Tröße, Christiane; Kongshaug, Heidi; Dondrup, Michael; Nilsen, Frank

    2015-10-01

    During its parasitic life stages, the marine ectoparasitic copepod Lepeophtheirus salmonis ingests large amounts of host blood, which contains high amounts of iron. Iron is an essential micronutrient, but also toxic in high dosages, and blood-feeding parasites like the salmon louse must thus possess an efficient system to handle the excess iron. Iron regulatory protein 1 and 2 (IRP1 and IRP2) are known to play crucial roles in this process, by regulating several proteins involved in iron transport and storage, depending on the cellular iron concentration. To gain knowledge about the regulation of the iron metabolism in salmon lice, two IRP homologues (LsIRP1A and LsIRP1B) were identified by sequence and predicted structure similarity to known IRPs in other species. In situ hybridisation revealed that LsIRP1A and LsIRP1B mRNAs were expressed in the ovaries, oviducts and vitellogenic oocytes of adult females. Transcription levels of LsIRP1A and LsIRP1B mRNAs did not differ significantly between the different developmental stages of the salmon louse. Adults in the absence of blood as a feed source had decreased levels of LsIRP1A, but not LsIRP1B mRNA. RNA binding experiments indicated the presence of functioning IRP in salmon lice. In order to explore the biological functions of LsIRP1A and LsIRP1B, the mRNAs of both proteins were knocked down by RNA interference (RNAi) in preadult females. The knockdown was confirmed by qRT-PCR. LsIRP1B knockdown lice produced less offspring than control lice due to slightly shorter egg strings and had decreased levels of transcripts involved in egg development. Knockdown of both LsIRP1A and LsIRP1B caused increased expression of a salmon louse Ferritin (LsFer). These results confirm that salmon lice have two IRP1 homologues, LsIRP1A and LsIRP1B, and might suggest a function in cellular iron regulation in the reproductive organs and eggs. PMID:26115940

  5. The Adaptor Protein-1 μ1B Subunit Expands the Repertoire of Basolateral Sorting Signal Recognition in Epithelial Cells

    PubMed Central

    Guo, Xiaoli; Mattera, Rafael; Ren, Xuefeng; Chen, Yu; Retamal, Claudio; González, Alfonso; Bonifacino, Juan S.

    2014-01-01

    SUMMARY An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the μ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous μ1A and the epithelial-specific μ1B. Previous studies led to the notion that μ1A and μ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that μ1A and μ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, μ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a μ1B-dependent manner. We conclude that expression of distinct μ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface. PMID:24229647

  6. Synthesis of protein tyrosine phosphatase 1B inhibitors: model validation and docking studies.

    PubMed

    Saxena, Anil K; Pandey, Gyanendra; Gupta, Swati; Singh, Amar Bahadur; Srivastava, Arvind K

    2009-04-15

    The designed and synthesized 2-(4-methoxyphenyl) ethyl] acetamide derivatives (3a, 3b and 3c) were evaluated for their PTP1B inhibitory activity where they showed IC(50) values 69 microM, 87 microM and 71 microM, respectively. These results correlated well with the docking studies and in vivo screening of the compounds for their antidiabetic activity in SLM and STZ models. PMID:19282172

  7. The Polycomb group protein RING1B is overexpressed in ductal breast carcinoma and is required to sustain FAK steady state levels in breast cancer epithelial cells

    PubMed Central

    Bosch, Almudena; Panoutsopoulou, Konstantina; Corominas, Josep Maria; Gimeno, Ramón; Moreno-Bueno, Gema; Martín-Caballero, Juan; Morales, Saleta; Lobato, Tania; Martínez-Romero, Carles; Farias, Eduardo F.; Mayol, Xavier; Cano, Amparo; Hernández-Muáoz, Inmaculada

    2014-01-01

    In early stages of metastasis malignant cells must acquire phenotypic changes to enhance their migratory behavior and their ability to breach the matrix surrounding tumors and blood vessel walls. Epigenetic regulation of gene expression allows the acquisition of these features that, once tumoral cells have escape from the primary tumor, can be reverted. Here we report that the expression of the Polycomb epigenetic repressor Ring1B is enhanced in tumoral cells that invade the stroma in human ductal breast carcinoma and its expression is coincident with that of Fak in these tumors. Ring1B knockdown in breast cancer cell lines revealed that Ring1B is required to sustain Fak expression in basal conditions as well as in Tgfβ-treated cells. Functionally, endogenous Ring1B is required for cell migration and invasion in vitro and for in vivo invasion of the mammary fat pad by tumoral cells. Finally we identify p63 as a target of Ring1B to regulate Fak expression: Ring1B depletion results in enhanced p63 expression, which in turns represses Fak expression. Importantly, Fak downregulation upon Ring1B depletion is dependent on p63 expression. Our findings provide new insights in the biology of the breast carcinoma and open new avenues for breast cancer prognosis and therapy. PMID:24742605

  8. FK506-binding protein 1b/12.6: a key to aging-related hippocampal Ca2+ dysregulation?

    PubMed Central

    Gant, JC; Blalock, EM; K-C, Chen; Kadish, I; Porter, NM; Norris, CM; Thibault, O; Landfield, PW

    2014-01-01

    It has been recognized for some time that the Ca2+-dependent slow afterhyperpolarization (sAHP) is larger in hippocampal neurons of aged compared with young animals. In addition, extensive studies since have shown that other Ca2+-mediated electrophysiological responses are increased in hippocampus with aging, including Ca2+ transients, L-type voltage-gated Ca2+ channel activity, Ca2+ spike duration and action potential accommodation. Elevated Ca2+-induced Ca2+ release from ryanodine receptors (RyRs) appears to drive amplification of the Ca2+ responses. Components of this Ca2+ dysregulation phenotype correlate with deficits in cognitive function and plasticity, indicating they may play critical roles in aging-related impairment of brain function. However, the molecular mechanisms underlying aging-related Ca2+ dysregulation are not well understood. FK506-binding proteins 1a and 1b (FKBP1a/1b, also known as FKBP12/12.6) are immunophilin proteins that bind the immunosuppressant drugs FK506 and rapamycin. In muscle cells, FKBP1a/1b also bind RyRs and inhibits Ca2+-induced Ca2+ release, but it is not clear whether FKBPs act similarly in brain cells. Recently, we found that selectively disrupting hippocampal FKBP1b function in young rats, either by microinjecting adeno-associated viral vectors containing siRNA, or by treatment with rapamycin, increases the sAHP and recapitulates much of the hippocampal Ca2+ dysregulation phenotype. Moreover, in microarray studies, we found FKBP1b gene expression was downregulated in hippocampus of aging rats and early-stage Alzheimer’s disease subjects. These results suggest the novel hypothesis that declining FKBP function is a key factor in aging-related Ca2+ dysregulation in the brain and point to potential new therapeutic targets for counteracting unhealthy brain aging. PMID:24291098

  9. Mutational analysis of the myelin protein zero (MPZ) gene associated with Charcot-Marie-Tooth neuropathy type 1B

    SciTech Connect

    Roa, B.B.; Warner, L.E.; Lupski, J.R.

    1994-09-01

    The MPZ gene that maps to chromosome 1q22q23 encodes myelin protein zero, which is the most abundant peripheral nerve myelin protein that functions as a homophilic adhesion molecule in myelin compaction. Association of the MPZ gene with the dysmyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B) and the more severe Dejerine-Sottas syndrome (DSS) was previously demonstrated by MPZ mutations identified in CMT1B and in rare DSS patients. In this study, the coding region of the MPZ gene was screened for mutations in a cohort of 74 unrelated patients with either CMT type 1 or DSS who do not carry the most common CMT1-associated molecular lesion of a 1.5 Mb DNA duplication on 17p11.2-p12. Heteroduplex analysis detected base mismatches in ten patients that were distributed over three exons of MPZ. Direct sequencing of PCR-amplified genomic DNA identified a de novo MPZ mutation associated with CMT1B that predicts an Ile(135)Thr substitution. This finding further confirms the role of MPZ in the CMT1B disease process. In addition, two polymorphisms were identified within the Gly(200) and Ser(228) codons that do not alter the respective amino acid residues. A fourth base mismatch in MPZ exon 3 detected by heteroduplex analysis is currently being characterized by direct sequence determination. Previously, four unrelated patients in this same cohort were found to have unique point mutations in the coding region of the PMP22 gene. The collective findings on CMT1 point mutations could suggest that regulatory region mutations, and possibly mutations in CMT gene(s) apart from the MPZ, PMP22 and Cx32 genes identified thus far, may prove to be significant for a number of CMT1 cases that do not involve DNA duplication.

  10. SAS1B Protein [Ovastacin] Shows Temporal and Spatial Restriction to Oocytes in Several Eutherian Orders and Initiates Translation at the Primary to Secondary Follicle Transition

    PubMed Central

    Pires, Eusebio S; Hlavin, Callie; Macnamara, Ellen; Ishola-Gbenla, Khadijat; Doerwaldt, Christa; Chamberlain, Catherine; Klotz, Kenneth; Herr, Austin K.; Khole, Aalok; Chertihin, Olga; Curnow, Eliza; Feldman, Sandford H; Mandal, Arabinda; Shetty, Jagathpala; Flickinger, Charles; Herr, John C

    2014-01-01

    Background Sperm Acrosomal SLLP1 Binding (SAS1B) protein (ovastacin) is an oolemmal binding partner for the intra-acrosomal sperm protein SLLP1. Results Immunohistochemical localization revealed that SAS1B translation is restricted among adult tissues to the ovary and oocytes, SAS1B appearing first in follicles at the primary-secondary transition. Quiescent oocytes within primordial follicles and primary follicles did not stain for SAS1B. Examination of neonatal rat ovaries revealed SAS1B expression first as faint signals in postnatal day 3 oocytes, with SAS1B protein staining intensifying with oocyte growth. Irrespective of animal age or estrus stage, SAS1B was seen only in oocytes of follicles that initiated a second granulosa cell layer. The precise temporal and spatial onset of SAS1B expression was conserved in adult ovaries in 7 eutherian species, including non-human primates. Immunoelectron micrographs localized SAS1B within cortical granules in MII oocytes. A population of SAS1B localized on the oolemma predominantly in the microvillar region anti-podal to the nucleus in ovulated MII rat oocytes and on the oolemma in macaque GV oocytes. Conclusions The restricted expression of SAS1B protein in growing oocytes, absence in the ovarian reserve, and localization on the oolemma suggest this zinc metalloprotease deserves consideration as a candidate target for reversible female contraceptive strategies. PMID:24038607

  11. Synthesis and protein tyrosine phosphatase 1B inhibition activities of two new synthetic bromophenols and their methoxy derivatives

    NASA Astrophysics Data System (ADS)

    Cui, Yongchao; Shi, Dayong; Hu, Zhiqiang

    2011-11-01

    3-bromo-4,5-bis(2,3-dibromo-4,5-dihydroxybenzyl)-1,2-benzenediol ( 1) is a natural bromophenol isolated from the red algae Rhodomela confervoides that exhibits significant inhibition against protein tyrosine phosphatase 1B (PTP1B). Based on its activity, we synthesized two new synthetic bromophenols and their methoxy derivatives from vanillin using the structure of natural bromophenol 1 as a scaffold. The structures of these bromophenols were elucidated from 1H NMR, 13C NMR, and high resolution electron ionization mass spectrometry as 2,3-dibromo-1-(2'-bromo-6'-(3″,4″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 2), 2,3-dibromo-1-(2'-bromo-6'-(2″-bromo-4″,5″-dimethoxybenzyl)-3',4'-dimethoxybenzyl)-4,5-dimethoxybenzene ( 3), 3,4-dibromo-5-(2'-bromo-6'-(2″-bromo-4″,5″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 4) and 3,4-dibromo-5-(2'-bromo-6'-(3″,4″-dihydroxybenzyl)-3',4'-dihydroxybenzyl)pyrocatechol ( 5). PTP1B inhibition activities of these compounds were evaluated using a colorimetric assay, and compounds 3 and 4 demonstrated interesting activity against PTP1B.

  12. Isopetrosynol, a New Protein Tyrosine Phosphatase 1B Inhibitor, from the Marine Sponge Halichondria cf. panicea Collected at Iriomote Island.

    PubMed

    Abdjul, Delfly Booby; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2016-01-01

    A new polyacetylene compound, isopetrosynol (1), was isolated from the Okinawan marine sponge Halichondria cf. panicea together with petrosynol (2), adociacetylene D (3), (5R)-3,15,27-triacontatriene-1,29-diyn-5-ol (4), and petrosterol (5). The structure of 1 was assigned on the basis of spectroscopic data for 1 and 2. Compound 1 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 8.2±0.3 µM, while compound 2, a diastereomer of 1, showed only 28.9±4.5% inhibition at 21.6 µM. The IC50 values of compounds 3 and 4 were 7.8±0.5 and 12.2±0.5 µM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7±0.1 µM (IC50) in the same experiment. The inhibitory activity of 1 was stronger than that of its diastereomer (2). This is the first study to show the inhibitory effects of polyacetylene compounds on PTP1B. PMID:27373628

  13. Strongylophorines, new protein tyrosine phosphatase 1B inhibitors, from the marine sponge Strongylophora strongilata collected at Iriomote Island.

    PubMed

    Lee, Jong-Soo; Abdjul, Delfly B; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2015-09-15

    A new meroditerpene, 26-O-ethylstrongylophorine-14 (1), was isolated from the Okinawan marine sponge Strongylophora strongilata together with six known strongylophorines: 26-O-methylstrongylophorine-16 (2) and strongylophorines-2 (3), -3 (4), -8 (5), -15 (6), and -17 (7). The structure of 1 was assigned on the basis of its spectroscopic data. Compound 1 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with an IC50 value of 8.7 μM, while known compounds 2-8 gave IC50 values of 8.5, >24.4, 9.0, 21.2, 11.9, and 14.8 μM, respectively. Oleanolic acid, a positive control, inhibited PTP1B activity at 0.7 μM (IC50). The inhibitory activities of strongylophorines possessing the acetal moiety at C-26 (1, 2, and 6) were stronger than those of the lactone derivatives (3 and 5). This is the first study to demonstrate that meroditerpenes inhibit PTP1B activity. PMID:26253631

  14. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs via a novel mechanism involving a Cullin-containing complex

    PubMed Central

    Querido, Emmanuelle; Blanchette, Paola; Yan, Qin; Kamura, Takumi; Morrison, Megan; Boivin, Dominique; Kaelin, William G.; Conaway, Ronald C.; Conaway, Joan Weliky; Branton, Philip E.

    2001-01-01

    Although MDM2 plays a major role in regulating the stability of the p53 tumor suppressor protein, other poorly understood MDM2-independent pathways also exist. Human adenoviruses have evolved strategies to regulate p53 function and stability to permit efficient viral replication. One mechanism involves adenovirus E1B55K and E4orf6 proteins, which collaborate to target p53 for degradation. To determine the mechanism of this process, a multiprotein E4orf6-associated complex was purified and shown to contain a novel Cullin-containing E3 ubiquitin ligase that is (1) composed of Cullin family member Cul5, Elongins B and C, and the RING-H2 finger protein Rbx1(ROC1); (2) remarkably similar to the von Hippel-Lindau tumor suppressor and SCF (Skp1–Cul1/Cdc53–F-box) E3 ubiquitin ligase complexes; and (3) capable of stimulating ubiquitination of p53 in vitro in the presence of E1/E2 ubiquitin-activating and -conjugating enzymes. Cullins are activated by NEDD8 modification; therefore, to determine whether Cullin complexes are required for adenovirus-induced p53 degradation, studies were conducted in ts41 Chinese hamster ovary cells that are temperature sensitive for the NEDD8 pathway. E4orf6/E1B55K failed to induce the degradation of p53 at the nonpermissive temperature. Thus, our results identify a novel role for the Cullin-based machinery in regulation of p53. PMID:11731475

  15. A Functional Complex of Adenovirus Proteins E1B-55kDa and E4orf6 Is Necessary To Modulate the Expression Level of p53 but Not Its Transcriptional Activity

    PubMed Central

    Cathomen, Toni; Weitzman, Matthew D.

    2000-01-01

    In adenovirus-infected cells, binding of E1B-55kDa and E4orf6 to the tumor suppressor protein p53 inhibits its transcriptional activity and causes rapid turnover of the protein. To investigate the requirements of the E1B-E4orf6 complex to modulate p53 function, we generated an E4orf6 mutant that failed to associate functionally and physically with E1B-55kDa but still interacted with p53. We confirm that E4orf6 and E1B-55kDa reduce p53 transactivation individually and show that their combined inhibition is additive rather than synergistic. Furthermore, we found that downregulation of p53's expression level, but not transcriptional inhibition of p53, depends on a functional E1B-E4 complex. A functional interaction of E1B-55kDa with p53, on the other hand, is a prerequisite for both transcriptional repression and downregulation of p53. The separation of these two functions will enable further dissection of the requirements for oncogenicity by the E4orf6 protein. PMID:11070042

  16. Direct interaction and functional coupling between human 5-HT6 receptor and the light chain 1 subunit of the microtubule-associated protein 1B (MAP1B-LC1).

    PubMed

    Kim, Soon-Hee; Kim, Dong Hyuk; Lee, Kang Ho; Im, Sun-Kyoung; Hur, Eun-Mi; Chung, Kwang Chul; Rhim, Hyewhon

    2014-01-01

    Serotonin (5-HT) receptors of type 6 (5-HT6R) play important roles in mood, psychosis, and eating disorders. Recently, a growing number of studies support the use of 5-HT6R-targeting compounds as promising drug candidates for treating cognitive dysfunction associated with Alzheimer's disease. However, the mechanistic linkage between 5-HT6R and such functions remains poorly understood. By using yeast two-hybrid, GST pull-down, and co-immunoprecipitation assays, here we show that human 5-HT6R interacts with the light chain 1 (LC1) subunit of MAP1B protein (MAP1B-LC1), a classical microtubule-associated protein highly expressed in the brain. Functionally, we have found that expression of MAP1B-LC1 regulates serotonin signaling in a receptor subtype-specific manner, specifically controlling the activities of 5-HT6R, but not those of 5-HT4R and 5-HT7R. In addition, we have demonstrated that MAP1B-LC1 increases the surface expression of 5-HT6R and decreases its endocytosis, suggesting that MAP1B-LC1 is involved in the desensitization and trafficking of 5-HT6R via a direct interaction. Together, we suggest that signal transduction pathways downstream of 5-HT6R are regulated by MAP1B, which might play a role in 5-HT6R-mediated signaling in the brain. PMID:24614691

  17. Direct Interaction and Functional Coupling between Human 5-HT6 Receptor and the Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B-LC1)

    PubMed Central

    Kim, Soon-Hee; Kim, Dong Hyuk; Lee, Kang Ho; Im, Sun-Kyoung; Hur, Eun-Mi; Chung, Kwang Chul; Rhim, Hyewhon

    2014-01-01

    Serotonin (5-HT) receptors of type 6 (5-HT6R) play important roles in mood, psychosis, and eating disorders. Recently, a growing number of studies support the use of 5-HT6R-targeting compounds as promising drug candidates for treating cognitive dysfunction associated with Alzheimer’s disease. However, the mechanistic linkage between 5-HT6R and such functions remains poorly understood. By using yeast two-hybrid, GST pull-down, and co-immunoprecipitation assays, here we show that human 5-HT6R interacts with the light chain 1 (LC1) subunit of MAP1B protein (MAP1B-LC1), a classical microtubule-associated protein highly expressed in the brain. Functionally, we have found that expression of MAP1B-LC1 regulates serotonin signaling in a receptor subtype-specific manner, specifically controlling the activities of 5-HT6R, but not those of 5-HT4R and 5-HT7R. In addition, we have demonstrated that MAP1B-LC1 increases the surface expression of 5-HT6R and decreases its endocytosis, suggesting that MAP1B-LC1 is involved in the desensitization and trafficking of 5-HT6R via a direct interaction. Together, we suggest that signal transduction pathways downstream of 5-HT6R are regulated by MAP1B, which might play a role in 5-HT6R-mediated signaling in the brain. PMID:24614691

  18. Chemical constituents of Blumea balsamifera of Indonesia and their protein tyrosine phosphatase 1B inhibitory activity.

    PubMed

    Saifudin, Azis; Tanaka, Ken; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2012-07-01

    A methanol extract of the leaves of Blumea balsamifera (L.) DC. (Asteraceae) afforded a new guaian-type sesquiterpene, epiblumeaene K (1), together with four known guaian-type sesquiterpenes (2-5), three known sesquiterpenes (6-8), and nine known flavonoids (9-17) by a combination of chromatography and preparative TLC techniques. Their structures were elucidated by extensive spectroscopic methods and comparison with the literature data. Among the isolated compounds, a known sesquiterpene, beta-caryophyllene 8R,9R-oxide (6), exhibited a significant PTP1B inhibitory activity in a dose-dependent manner, with an IC50 value of 25.8 microM (5.62 microg/mL). PMID:22908553

  19. IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses

    PubMed Central

    Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

    2012-01-01

    Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in

  20. Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

    PubMed Central

    Sahyoun, N; McDonald, O B; Farrell, F; Lapetina, E G

    1991-01-01

    A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release. Images PMID:1901412

  1. Verruculides A and B, two new protein tyrosine phosphatase 1B inhibitors from an Indonesian ascidian-derived Penicillium verruculosum.

    PubMed

    Yamazaki, Hiroyuki; Nakayama, Wataru; Takahashi, Ohgi; Kirikoshi, Ryota; Izumikawa, Yuta; Iwasaki, Kohei; Toraiwa, Kengo; Ukai, Kazuyo; Rotinsulu, Henki; Wewengkang, Defny S; Sumilat, Deiske A; Mangindaan, Remy E P; Namikoshi, Michio

    2015-08-15

    Two new merosesquiterpenes, verruculides A (1) and B (2), were isolated from a culture broth of the Indonesian ascidian-derived Penicillium verruculosum TPU1311, together with three known congeners, chrodrimanins A (3), B (4), and H (5). The structures of 1 and 2 were assigned on the basis of their spectroscopic data (1D and 2D NMR, HRMS, UV, CD, and IR). Compound 2 had a linear sesquiterpene moiety and was considered to be the derivative of the biosynthetic precursor for 1 and 3-5. Compounds 1, 3, and 5 inhibited the activity of protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 8.4, 8.5, and 14.9 μM, respectively. Compound 2 showed 40% inhibition at 23.1 μM, while 4 was not active at 20.7 μM. PMID:26115570

  2. HTLV-1 bZIP factor protein targets the Rb/E2F-1 pathway to promote proliferation and apoptosis of primary CD4(+) T cells.

    PubMed

    Kawatsuki, A; Yasunaga, J-I; Mitobe, Y; Green, P L; Matsuoka, M

    2016-08-25

    Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4(+) T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3(+) T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4(+) T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4(+) T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4(+) T cells infected with HTLV-1. PMID:26804169

  3. Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

    PubMed

    Radke, Jay R; Yong, Sherri L; Cook, James L

    2016-01-01

    Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased

  4. Identification of a third Protein 4.1 tumor suppressor, Protein 4.1R, in meningioma pathogenesis.

    PubMed

    Robb, Victoria A; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H

    2003-08-01

    Meningiomas are common central nervous system tumors; however, the mechanisms underlying their pathogenesis are largely undefined. In this report, we demonstrate that a third Protein 4.1 family member, Protein 4.1R, functions as a meningioma tumor suppressor. We observed loss of Protein 4.1R expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningiomas by immunohistochemistry and fluorescence in situ hybridization. In support of a meningioma tumor suppressor function, Protein 4.1R overexpression resulted in reduced IOMM-Lee and CH157-MN cell proliferation. Similar to the Protein 4.1B and merlin tumor suppressors, Protein 4.1R membrane localization increased significantly under conditions of growth arrest in vitro. Lastly, we show that Protein 4.1R interacted with a subset of merlin/Protein 4.1B interactors including CD44 and betaII-spectrin. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor in the molecular pathogenesis of meningioma. PMID:12901833

  5. PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas.

    PubMed

    Cimino-Mathews, Ashley; Thompson, Elizabeth; Taube, Janis M; Ye, Xiaobu; Lu, Yao; Meeker, Alan; Xu, Haiying; Sharma, Rajni; Lecksell, Kristen; Cornish, Toby C; Cuka, Nathan; Argani, Pedram; Emens, Leisha A

    2016-01-01

    Programmed death ligand 1 (PD-L1) expression by tumor-infiltrating lymphocytes (TILs) and tumor cells in breast cancer has been reported, but the relationships between PD-L1 expression by TIL, carcinoma cells, and other immunologic features of the breast tumor microenvironment remain unclear. We therefore evaluated the interrelationships between tumor cell surface and TIL PD-L1 expression, lymphocyte subpopulations, and patterns of immune cell infiltration in cohorts of treatment-naive, primary breast cancers (PBCs) (n = 45) and matched PBC and metastatic breast cancers (MBC) (n = 26). Seventy-eight percent of untreated PBCs contained PD-L1(+) TILs, but only 21% had PD-L1(+) carcinoma cells. Carcinoma PD-L1 expression localized to the tumor invasive front and was associated with high tumor grade (P = .04). Eighty-nine percent of PD-L1(+) carcinomas contained brisk TIL infiltrates, compared to only 24% of PD-L1(-) carcinomas; this included CD3(+) (P = .02), CD4(+) (P = .04), CD8(+) (P = .002), and FoxP3(+) T cells (P = .02). PD-L1(+) PBCs were more likely to contain PD-L1(+) TIL than PD-L1(-) PBCs (P = .04). Peripheral lymphoid aggregates were present in 100% of PD-L1(+) compared to 41% of PD-L1(-) PBC (P < .001). No patient with PD-L1(+) PBC developed distant recurrence, compared to 15% of patients with PD-L1(-) PBC. For the matched PBC and MBC cohort, 2 patients (8%) had PD-L1(+) tumors, with 1 case concordant and 1 case discordant for carcinoma PD-L1 expression in the PBC and MBC. Our data support PD-L1 expression by tumor cells as a biomarker of active breast tumor immunity and programmed death 1 blockade as a therapeutic strategy for breast cancer. PMID:26527522

  6. Interferonβ-1b Induces the Expression of RGS1 a Negative Regulator of G-Protein Signaling

    PubMed Central

    Tran, Tiffany; Paz, Pedro; Velichko, Sharlene; Cifrese, Jill; Belur, Praveen; Yamaguchi, Ken D.; Ku, Karin; Mirshahpanah, Parham; Reder, Anthony T.; Croze, Ed

    2010-01-01

    We present evidence of a link between interferonβ-1b (IFN-β) and G-protein signaling by demonstrating that IFN-β can induce the expression of the negative regulator of G-protein signaling 1 (RGS1). RGS1 reduces G-protein activation and immune cell migration by interacting with heterotrimeric G-proteins and enhancing their intrinsic GTPase activity. In this study, IFN-β treatment resulted in the induction of RGS1 in peripheral blood mononuclear cells (PBMCs), monocytes, T cells, and B cells. Induction of RGS1 by IFN-β was concentration dependent and observed at both the RNA and protein level. Other members of the RGS family were not induced by IFN-β, and induction of RGS1 required the activation of the IFN receptor. In addition, RGS1 induction was observed in PBMCs obtained from IFN-β-treated multiple sclerosis patients suggesting a possible, as yet unexplored, involvement of G-protein regulation in disease treatment. The upregulation of RGS1 by IFN-β has not been previously reported. PMID:21274427

  7. Regulation of Signaling at Regions of Cell-Cell Contact by Endoplasmic Reticulum-Bound Protein-Tyrosine Phosphatase 1B

    PubMed Central

    Haj, Fawaz G.; Sabet, Ola; Kinkhabwala, Ali; Wimmer-Kleikamp, Sabine; Roukos, Vassilis; Han, Hong-Mei; Grabenbauer, Markus; Bierbaum, Martin; Antony, Claude; Neel, Benjamin G.; Bastiaens, Philippe I.

    2012-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B. PMID:22655028

  8. The tumor necrosis factor receptor superfamily member 1B polymorphisms predict response to anti-TNF therapy in patients with autoimmune disease: A meta-analysis.

    PubMed

    Chen, Wenjuan; Xu, Hui; Wang, Xiuxiu; Gu, Junying; Xiong, Huizi; Shi, Yuling

    2015-09-01

    Numerous published data on the tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) gene polymorphisms are shown to be associated with response or non-response to anti-TNF therapy in autoimmune diseases such as rheumatoid arthritis (RA), psoriasis and Crohn's Disease (CD). The aim of this study is to investigate whether the TNFRSF1B rs1061622 T/G or TNFRSF1A A/G rs767455 polymorphisms can predict the response to anti-TNF-based therapy in patients with autoimmune diseases. We conducted a meta-analysis of studies on the association between TNFRSF1B rs1061622 T/G polymorphism or TNFRSF1A A/G rs767455 polymorphism and non-responsiveness to anti-TNF therapy in autoimmune diseases. A total of 8 studies involving 929 subjects for TNFRSF1B rs1061622 and 564 subjects for TNFRSF1A rs767455 were finally considered. These studies consisted of seven studies on the TNFRSF1B polymorphism and four studies on the TNFRSF1A polymorphism. Meta-analysis showed significant association between the TNFRSF1B rs1061622 allele and non-responders to anti-TNF therapy [T/G odds ratio (OR) 0.72, 95% confidence interval (CI) 0.57-0.93, p=0.01]. Stratification by disease type indicated an association between the TNFRSF1B rs1061622 allele and non-responders to TNF antagonist in RA (T/G OR 0.69, 95% CI 0.48-0.99, p<0.05) and psoriasis (T/G OR 0.39, 95% CI 0.23-0.67, p<0.001), but not in CD (T/G OR 1.14, 95% CI 0.57-0.93, p=0.57). And there was no association between TNFRSF1A rs767455 genotype and non-responders to the anti-TNF therapy (A/G OR 0.93, 95% CI 0.70-1.23, p=0.59). This meta-analysis demonstrates that TNFRSF1B T allele carriers show a better response to anti-TNF therapy, and individuals carrying TNFRSF1A A allele have no relationship with the response to anti-TNF therapy for autoimmune diseases. The genotyping of this polymorphism could help to optimize the treatment by identifying patients with a likely poor response to biological drugs. PMID:26071216

  9. Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis.

    PubMed Central

    Babiss, L E; Ginsberg, H S

    1984-01-01

    To determine the role adenovirus 5 early region 1b-encoded 21- and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (0 to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region 1b functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dl118, H5dl110, H5in127, and H5dl163) indicates that all of the viruses contain mutations which affect the 55-kilodalton protein, whereas dl118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the mutants which is phenotypically identical to wild-type adenovirus 5). When compared with characteristics of sub309, the early and late transcription and DNA replication of the mutants were similar, but synthesis of late polypeptides and late cytoplasmic mRNAs was greatly reduced. Quantitation of mutant virus-specific late mRNAs associated with polysomes revealed a threefold reduction when compared with that of sub309. Analysis of infected cell extracts further revealed that these mutants were incapable of efficiently shutting off host cell protein synthesis, suggesting that the 55-kilodalton protein plays a role in this process. These data suggest that early region 1b products may function by interacting with additional viral or host cell macromolecules to modulate host cell shutoff or that some late viral mRNA or

  10. Pharmacological analysis of G-protein activation mediated by guinea-pig recombinant 5-HT1B receptors in C6-glial cells: similarities with the human 5-HT1B receptor.

    PubMed

    Pauwels, P J; Wurch, T; Palmier, C; Colpaert, F C

    1998-01-01

    1. The guinea-pig recombinant 5-hydroxytryptamine1B (gp 5-HT1B) receptor stably transfected in rat C6-glial cells was characterized by monitoring G-protein activation in a membrane preparation with agonist-stimulated [35S]-GTPgammaS binding. The intrinsic activity of 5-HT receptor ligands was compared with that determined previously at the human recombinant 5-HT1B (h 5-HT1B) receptor under similar experimental conditions. 2. Membrane preparations of C6-glial/gp 5-HT1B cells exhibited [3H]-5-carboxamidotryptamine (5-CT) and [3H]-N-[4-methoxy-3,4-methylpiperazin-1-yl) phenyl]-3-methyl-4-(4-pyridinyl)benzamide (GR 125743) binding sites with a pKd of 9.62 to 9.85 and a Bmax between 2.1 to 6.4 fmol mg(-1) protein. The binding affinities of a series of 5-HT receptor ligands determined with [3H]-5-CT and [3H]-GR 125743 were similar. Ligand affinities were comparable to and correlated (r2: 0.74, P<0.001) with those determined at the recombinant h 5-HT1B receptor. 3. [35S]-GTPgammaS binding to membrane preparations of C6-glial/gp 5-HT1B cells was stimulated by the 5-HT receptor agonists that were being investigated. The maximal responses of naratriptan, zolmitriptan, sumatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulphonamide (CP 122638), rizatriptan and dihydroergotamine were between 0.76 and 0.85 compared to 5-HT. The potency of these agonists showed a positive correlation (r2: 0.72, P=0.015) with their potency at the recombinant h 5-HT1B receptor. 1-naphthylpiperazine, (+/-)-cyanopindolol and (2'-methyl-4'-(5-methyl[1,2,4] oxadiazole-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR 127935) elicited an even smaller response (Emax: 0.32 to 0.63). 4. The ligands 1'-methyl-5-(2'-methyl-4'-(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-carbonyl)-2,3,6,7tetrahydrospiro [furo[2,3-f]indole-3-spiro-4'-piperidine] (SB224289), methiothepin and ritanserin displayed inhibition of basal [35S]-GTPgammaS binding at concentrations

  11. 1B50-1, a mAb raised against recurrent tumor cells, targets liver tumor-initiating cells by binding to the calcium channel α2δ1 subunit.

    PubMed

    Zhao, Wei; Wang, Limin; Han, Haibo; Jin, Kemin; Lin, Na; Guo, Ting; Chen, Yangde; Cheng, Heping; Lu, Fengmin; Fang, Weigang; Wang, Yu; Xing, Baocai; Zhang, Zhiqian

    2013-04-15

    The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here, we generated a monoclonal antibody against recurrent HCC, 1B50-1, that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore, 1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally, α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs, whereas its overexpression led to enhanced sphere formation, which is regulated by calcium influx. Thus, α2δ1 is a functional liver TIC marker, and its inhibitors may serve as potential anti-HCC drugs. PMID:23597567

  12. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins.

    PubMed Central

    Rao, L; Debbas, M; Sabbatini, P; Hockenbery, D; Korsmeyer, S; White, E

    1992-01-01

    Cooperation between the adenovirus E1A and E1B oncogenes is required for transformation of primary quiescent rodent cells. Although expression of E1A alone will stimulate cell proliferation sufficient to initiate transformed focus formation, proliferation fails to be sustained and foci degenerate. Coexpression of either the 19-kDa or 55-kDa E1B oncoproteins with E1A permits high-frequency transformation by overcoming this cytotoxic response. Without E1B 19-kDa protein expression, however, transformants remain susceptible to induction of cell death. Rapid loss of viability is coincident with nucleolytic cleavage of DNA in intranucleosomal regions and chromatin condensation, hallmarks of programmed cell death (apoptosis). Furthermore, overexpression of a known suppressor of apoptosis, the Bcl-2 protooncogene, can rescue E1A-induced focus degeneration. Thus E1A-dependent stimulation of cell proliferation is accompanied by apoptosis and thereby insufficient to singly induce transformation. High-frequency transformation requires a second function encoded by the E1B 19-kDa protein to block apoptosis. Images PMID:1457005

  13. Matrix Gla protein in tumoral pathology.

    PubMed

    Gheorghe, Simona Roxana; Crăciun, Alexandra Mărioara

    2016-01-01

    Matrix Gla protein is a vitamin K-dependent protein secreted by chondrocytes and vascular smooth muscle cells. The presence of matrix Gla protein was reported in arterial and venous walls, lungs, kidney, uterus, heart, tooth cementum and eyes. Several studies identified matrix Gla protein in tumoral pathology. Until recently, it was thought to only have an inhibitory role of physiological and ectopic calcification. New studies demonstrated that it also has a role in physiological and pathological angiogenesis, as well as in tumorigenesis. The aim of this review is to report the latest findings related to the expression and clinical implications of matrix Gla protein in different types of cancer with an emphasis on cerebral tumors. PMID:27547048

  14. Matrix Gla protein in tumoral pathology

    PubMed Central

    GHEORGHE, SIMONA ROXANA; CRĂCIUN, ALEXANDRA MĂRIOARA

    2016-01-01

    Matrix Gla protein is a vitamin K-dependent protein secreted by chondrocytes and vascular smooth muscle cells. The presence of matrix Gla protein was reported in arterial and venous walls, lungs, kidney, uterus, heart, tooth cementum and eyes. Several studies identified matrix Gla protein in tumoral pathology. Until recently, it was thought to only have an inhibitory role of physiological and ectopic calcification. New studies demonstrated that it also has a role in physiological and pathological angiogenesis, as well as in tumorigenesis. The aim of this review is to report the latest findings related to the expression and clinical implications of matrix Gla protein in different types of cancer with an emphasis on cerebral tumors. PMID:27547048

  15. Protein tyrosine phosphatase 1B inhibitory activity of Indonesian herbal medicines and constituents of Cinnamomum burmannii and Zingiber aromaticum.

    PubMed

    Saifudin, Azis; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2013-04-01

    We screened water and methanol extracts of 28 Indonesian medicinal plants for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activities. Nine water extracts, i.e., Alstonia scholaris leaf, Blumea balsamifera, Cinnamomum burmannii, Cymbopogon nardus, Melaleuca leucadendra, Phyllanthus niruri, Piper nigrum, Syzygium aromaticum, and Sy. polyanthum, exhibited ≥70 % inhibition at 25 μg/mL, whereas 11 methanol extracts, i.e., Als. scholaris, Andrographis paniculata, B. balsamifera, Ci. burmannii, Curcuma heyneana, Glycyrrhiza glabra, M. leucadendra, Punica granatum, Rheum palmatum, Sy. polyanthum, and Z. aromaticum, exhibited ≥70 % inhibition at 25 μg/mL. Water extracts of B. balsamifera (IC50, 2.26 μg/mL) and M. leucadendra (IC50, 2.05 μg/mL), and methanol extracts of Ci. burmannii (IC50, 2.47 μg/mL), Pu. granatum (IC50, 2.40 μg/mL), and Sy. polyanthum (IC50, 1.03 μg/mL) exhibited strong inhibitory activity, which was comparable with that of the positive control, RK-682 (IC50, 2.05 μg/mL). The PTP1B inhibitory activity of the constituents of Ci. burmannii and Z. aromaticum was then evaluated. 5'-Hydroxy-5-hydroxymethyl-4″,5″-methylenedioxy-1,2,3,4-dibenzo-1,3,5-cycloheptatriene (2; IC50, 29.7 μM) and trans-cinnamaldehyde (5; IC50, 57.6 μM) were the active constituents of Ci. burmannii, while humulatrien-5-ol-8-one (21; IC50, 27.7 μM), kaempferol-3,4'-di-O-methyl ether (32; IC50, 17.5 μM), and (S)-6-gingerol (33; IC50, 28.1 μM) were those of Z. aromaticum. These results suggest that these medicinal plants may contribute to the treatment and/or prevention of type II diabetes and/or obesity through PTP1B inhibition. PMID:22645080

  16. HTLV-1 bZIP Factor RNA and Protein Impart Distinct Functions on T-cell Proliferation and Survival.

    PubMed

    Mitobe, Yuichi; Yasunaga, Jun-ichirou; Furuta, Rie; Matsuoka, Masao

    2015-10-01

    Infection of T cells with human T-cell leukemia virus type-1 (HTLV-1) induces clonal proliferation and is closely associated with the onset of adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. Although Tax expression is frequently suppressed in HTLV-1-infected cells, the accessory gene, HTLV-1 bZIP factor (HBZ), is continuously expressed and has been implicated in HTLV-1 pathogenesis. Here, we report that transduction of mouse T cells with specific mutants of HBZ that distinguish between its RNA and protein activity results in differential effects on T-cell proliferation and survival. HBZ RNA increased cell number by attenuating apoptosis, whereas HBZ protein induced apoptosis. However, both HBZ RNA and protein promoted S-phase entry of T cells. We further identified that the first 50 bp of the HBZ coding sequence are required for RNA-mediated cell survival. Transcriptional profiling of T cells expressing wild-type HBZ, RNA, or protein revealed that HBZ RNA is associated with genes involved in cell cycle, proliferation, and survival, while HBZ protein is more closely related to immunological properties of T cells. Specifically, HBZ RNA enhances the promoter activity of survivin, an inhibitor of apoptosis, to upregulate its expression. Inhibition of survivin using YM155 resulted in impaired proliferation of several ATL cell lines as well as a T-cell line expressing HBZ RNA. The distinct functions of HBZ RNA and protein may have several implications for the development of strategies to control the proliferation and survival mechanisms associated with HTLV-1 infection and ATL. PMID:26383166

  17. Trichoketides A and B, two new protein tyrosine phosphatase 1B inhibitors from the marine-derived fungus Trichoderma sp.

    PubMed

    Yamazaki, Hiroyuki; Saito, Ryo; Takahashi, Ohgi; Kirikoshi, Ryota; Toraiwa, Kengo; Iwasaki, Kohei; Izumikawa, Yuta; Nakayama, Wataru; Namikoshi, Michio

    2015-10-01

    Two new octaketides, trichoketides A (1) and B (2), were isolated from a culture broth of the seawater-derived fungus Trichoderma sp. TPU1237 together with two known analogs, trichodermaketones C (3) and D (4), by ODS column chromatography followed by preparative ODS and chiral HPLC. The structures of 1 and 2 were elucidated on the basis of their spectroscopic data, and absolute configurations were assigned by comparing their experimental electronic circular dichroism (ECD) spectra with the calculated ECD spectra. Compounds 1 and 2 were epimers at the C-8 position (α-position of dihydrofuran ring). The IC50 values of compounds 1-4 against protein tyrosine phosphatase 1B were 53.1, 65.1, 68.0 and 55.9 μM, respectively. PMID:25899128

  18. Sesquiterpene Hydroquinones with Protein Tyrosine Phosphatase 1B Inhibitory Activities from a Dysidea sp. Marine Sponge Collected in Okinawa.

    PubMed

    Abdjul, Delfly B; Yamazaki, Hiroyuki; Takahashi, Ohgi; Kirikoshi, Ryota; Ukai, Kazuyo; Namikoshi, Michio

    2016-07-22

    Three new sesquiterpene hydroquinones, avapyran (1), 17-O-acetylavarol (2), and 17-O-acetylneoavarol (3), were isolated from a Dysidea sp. marine sponge collected in Okinawa together with five known congeners: avarol (4), neoavarol (5), 20-O-acetylavarol (6), 20-O-acetylneoavarol (7), and 3'-aminoavarone (8). The structures of 1-3 were assigned on the basis of their spectroscopic data. Compounds 1-3 inhibited the activity of protein tyrosine phosphatase 1B with IC50 values of 11, 9.5, and 6.5 μM, respectively, while known compounds 4-8 gave IC50 values of 12, >32, 10, 8.6, and 18 μM, respectively. In a preliminary investigation on structure-activity relationships, six ester and methoxy derivatives (9-14) were prepared from 4 and 5. PMID:27336796

  19. Acylation of the 176R (19-kilodalton) early region 1B protein of human adenovirus type 5.

    PubMed Central

    McGlade, C J; Tremblay, M L; Yee, S P; Ross, R; Branton, P E

    1987-01-01

    Antipeptide sera were prepared in rabbits against synthetic peptides corresponding to the predicted amino and carboxy termini of the early region 1B 176R (19-kilodalton [kDa]) protein of human adenovirus type 5. Both antisera specifically immunoprecipitated the 19- and 18.5-kDa forms of the 176R protein observed previously with antitumor sera. These data suggested that both species are full-length molecules of 176 residues. To identify posttranslational modifications that could explain the formation of these multiple species and possibly their known association with membranes, studies were carried out to determine whether they are glycosylated or acylated. Neither the 19- nor the 18.5-kDa species appeared to be a glycoprotein, however, they were labeled with [3H]palmitate and [3H]myristate, indicating that both species are acylated. Thus, whereas acylation does not appear to be the cause of the multiple species, it could play a role in the membrane association of these viral proteins. The acylation of 176R was found to be unusual. The fatty acid linkage was resistant to treatment with hydroxylamine or methanol-KOH, suggesting that acylation was through an amide bond. In addition, both palmitate and myristate were present in 176R, suggesting either a lack of specificity in the acylation reaction or the existence of more than one acylation site. Images PMID:2957509

  20. Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

    PubMed Central

    Han, J; Sabbatini, P; White, E

    1996-01-01

    The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein. PMID:8816500

  1. Protein kinase A alterations in adrenocortical tumors.

    PubMed

    Espiard, S; Ragazzon, B; Bertherat, J

    2014-11-01

    Stimulation of the cAMP pathway by adrenocorticotropin (ACTH) is essential for adrenal cortex maintenance, glucocorticoid and adrenal androgens synthesis, and secretion. Various molecular and cellular alterations of the cAMP pathway have been observed in endocrine tumors. Protein kinase A (PKA) is a central key component of the cAMP pathway. Molecular alterations of PKA subunits have been observed in adrenocortical tumors. PKA molecular defects can be germline in hereditary disorders or somatic in sporadic tumors. Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) can be observed in patients with ACTH-independent Cushing's syndrome (CS) due to primary pigmented nodular adrenocortical disease (PPNAD). PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic cortisol-secreting adrenocortical adenomas. Germline gene duplication of the catalytic subunits Cα (PRKACA) has been observed in patients with PPNAD. Furthermore, exome sequencing revealed recently activating somatic mutations of PRKACA in about 40% of cortisol-secreting adrenocortical adenomas. In vitro and in vivo functional studies help in the progress to understand the mechanisms of adrenocortical tumors development due to PKA regulatory subunits alterations. All these alterations are observed in benign oversecreting tumors and are mimicking in some way cAMP pathway constitutive activation. On the long term, unraveling these alterations will open new strategies of pharmacological treatment targeting the cAMP pathway in adrenal tumors and cortisol-secretion disorders. PMID:25105543

  2. Bcl-2/adenovirus E1B 19 kDa interacting protein-3 knockdown enables growth of breast cancer metastases in the lung, liver, and bone.

    PubMed

    Manka, David; Spicer, Zachary; Millhorn, David E

    2005-12-15

    The mouse breast cancer cell lines 4T1, 4T07, and 67NR are highly tumorigenic but vary in metastatic potential: 4T1 widely disseminates, resulting in secondary tumors in the lung, liver, bone, and brain; 4T07 spreads to the lung and liver but is unable to establish metastatic nodules; 67NR is unable to metastasize. The Bcl-2/adenovirus E1B 19 kDa interacting protein-3 (Bnip-3) was recently shown to be absent after hypoxia in pancreatic cancer cell lines whereas its overexpression restored hypoxia-induced cell death. We found that Bnip-3 expression increased after 6 hours of hypoxia in all cell lines tested but was highest in the nonmetastatic 67NR cells and lowest in the highly metastatic 4T1 cells. Hypoxia-induced expression of Bnip-3 in the disseminating but nonmetastatic 4T07 cells was intermediate compared with 4T1 and 67NR cells. Cleaved caspase-3, a key downstream effector of cell death, increased after 6 hours of hypoxia in the 67NR and 4T07 cells by 1.9- and 2.5-fold, respectively. Conversely, cleaved caspase-3 decreased by 45% in the highly metastatic 4T1 cells after hypoxia. Small interfering RNA oligonucleotides targeting endogenous Bnip-3 blocked cell death and increased clonigenic survival after hypoxic challenge in vitro and increased primary tumor size and enabled metastasis to the lung, liver, and sternum of mice inoculated with 4T07 cells in vivo. These data inversely correlate the hypoxia-induced expression of the cell death protein Bnip-3 to metastatic potential and suggest that loss of Bnip-3 expression is critical for malignant and metastatic evasion of hypoxia-induced cell death. PMID:16357180

  3. Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

    NASA Astrophysics Data System (ADS)

    Sarma, Bani Kanta

    2013-09-01

    The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by nO → σ*S-OH orbital interactions, which force the -OH group to adopt a position trans to the S⋯O interaction, leading to an almost linear arrangement of the O⋯S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S⋯N or S⋯O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

  4. Protein 4.1B associates with both Caspr/paranodin and Caspr2 at paranodes and juxtaparanodes of myelinated fibres.

    PubMed

    Denisenko-Nehrbass, Natalia; Oguievetskaia, Ksénia; Goutebroze, Laurence; Galvez, Thierry; Yamakawa, Hisashi; Ohara, Osamu; Carnaud, Michèle; Girault, Jean-Antoine

    2003-01-01

    Caspr/paranodin, a neuronal transmembrane glycoprotein, is essential for the structure and function of septate-like paranodal axoglial junctions at nodes of Ranvier. A closely related protein, Caspr2, is concentrated in juxtaparanodal regions where it associates indirectly with the shaker-type potassium channels. Although ultrastructural studies indicate that paranodal complexes are linked to the cytoskeleton, the intracellular partners of Caspr/paranodin, as well as those of Caspr2, are poorly characterized. We show that the conserved intracellular juxtamembrane regions (GNP motif) of Caspr/paranodin and Caspr2 bind proteins 4.1R and 4.1B. 4.1B is known to be enriched in paranodal and juxtaparanodal regions. 4.1B immunoreactivity accumulates progressively at paranodes and juxtaparanodes during postnatal development, following the concentration of Caspr/paranodin and Caspr2, respectively, in central and peripheral myelinated axons. These two proteins coimmunoprecipitated with 4.1B in brain homogenates. Our results provide strong evidence for the association of 4.1B with Caspr/paranodin at paranodes and with Caspr2 at juxtaparanodes. We propose that 4.1B anchors these axonal proteins to the actin-based cytoskeleton in these two regions. PMID:12542678

  5. Optimised purification and characterisation of lipid transfer protein 1 (LTP1) and its lipid-bound isoform LTP1b from barley malt.

    PubMed

    Nieuwoudt, Melanie; Lombard, Nicolaas; Rautenbach, Marina

    2014-08-15

    In beer brewing, brewers worldwide strive to obtain product consistency in terms of flavour, colour and foam. Important proteins contributing to beer foam are lipid transfer proteins (LTPs), in particular LTP1 and its lipid-bound isoform LTP1b, which are known to transport lipids in vivo and prevent lipids from destabilising the beer foam. LTP1 and LTP1b were successfully purified using only five purification steps with a high purified protein yield (160 mg LTP1 and LTP1b from 200 g barley). Circular dichroism of LTP1 and LTP1b confirmed that both proteins are highly tolerant to high temperatures (>90 °C) and are pH stable, particularly at a neutral to a more basic pH. Only LTP1 exhibited antiyeast and thermo-stable lytic activity, while LTP1b was inactive, indicating that the fatty acid moiety compromised the antimicrobial activity of LTP1. This lack in antiyeast activity and the positive foam properties of LTP1b would benefit beer fermentation and quality. PMID:24679818

  6. Protein-based tumor molecular imaging probes

    PubMed Central

    Lin, Xin; Xie, Jin

    2013-01-01

    Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging. PMID:20232092

  7. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    PubMed

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  8. The Putative “Switch 2” Domain of the Ras-related GTPase, Rab1B, Plays an Essential Role in the Interaction with Rab Escort Protein

    PubMed Central

    Overmeyer, Jean H.; Wilson, Amy L.; Erdman, Robert A.; Maltese, William A.

    1998-01-01

    Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the α2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative α2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the α3 and α4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B α2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B α2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the α2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate. PMID:9437002

  9. Extracellular surface residues of the α1B-adrenoceptor critical for G protein-coupled receptor function.

    PubMed

    Ragnarsson, Lotten; Andersson, Åsa; Thomas, Walter G; Lewis, Richard J

    2015-01-01

    Ligand binding and conformational changes that accompany signaling from G protein-coupled receptors (GPCRs) have mostly focused on the role of transmembrane helices and intracellular loop regions. However, recent studies, including several GPCRs cocrystallized with bound ligands, suggest that the extracellular surface (ECS) of GPCRs plays an important role in ligand recognition, selectivity, and binding, as well as potentially contributing to receptor activation and signaling. This study applied alanine-scanning mutagenesis to investigate the role of the complete ECS of the α1B-adrenoreceptor on norepinephrine (NE) potency, affinity, and efficacy. Half (24 of 48) of the ECS mutations significantly decreased NE potency in an inositol 1-phosphate assay. Most mutations reduced NE affinity (17) determined from [(3)H]prazosin displacement studies, whereas four mutations at the entrance to the NE binding pocket enhanced NE affinity. Removing the influence of NE affinity and receptor expression levels on NE potency gave a measure of NE efficacy, which was significantly decreased for 11 of 48 ECS mutants. These different effects tended to cluster to different regions of the ECS, which is consistent with different regions of the ECS playing discrete functional roles. Exposed ECS residues at the entrance to the NE binding pocket mostly affected NE affinity, whereas buried or structurally significant residues mostly affected NE efficacy. The broad potential for ECS mutations to affect GPCR function has relevance for the increasing number of nonsynonymous single nucleotide polymorphisms now being identified in GPCRs. PMID:25352041

  10. Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

    PubMed

    Tan, Jeff S; Mohandas, Narla; Conboy, John G

    2005-12-01

    Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5'-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2'. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2'. These correlations are independent of cell type or species of origin. Since exon 2' contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. PMID:16242908

  11. The Cytoskeletal Adaptor Protein Band 4.1B is Required for the Maintenance of Paranodal Axo-Glial Septate Junctions in Myelinated Axons

    PubMed Central

    Buttermore, Elizabeth D.; Dupree, Jeffrey L.; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A.

    2011-01-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here we report the generation and characterization of 4.1B null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axo-glial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after one year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at about one year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  12. The cytoskeletal adaptor protein band 4.1B is required for the maintenance of paranodal axoglial septate junctions in myelinated axons.

    PubMed

    Buttermore, Elizabeth D; Dupree, Jeffrey L; Cheng, JrGang; An, Xiuli; Tessarollo, Lino; Bhat, Manzoor A

    2011-06-01

    Precise targeting and maintenance of axonal domains in myelinated axons is essential for saltatory conduction. Caspr and Caspr2, which localize at paranodal and juxtaparanodal domains, contain binding sites for the cytoskeletal adaptor protein 4.1B. The exact role of 4.1B in the organization and maintenance of axonal domains is still not clear. Here, we report the generation and characterization of 4.1B-null mice. We show that loss of 4.1B in the PNS results in mislocalization of Caspr at paranodes and destabilization of paranodal axoglial septate junctions (AGSJs) as early as postnatal day 30. In the CNS, Caspr localization is progressively disrupted and ultrastructural analysis showed paranodal regions that were completely devoid of AGSJs, with axolemma separated from the myelin loops, and loops coming off the axolemma. Most importantly, our phenotypic analysis of previously generated 4.1B mutants, used in the study by Horresh et al. (2010), showed that Caspr localization was not affected in the PNS, even after 1 year; and 4.1R was neither expressed, nor enriched at the paranodes. Furthermore, ultrastructural analysis of these 4.1B mutants showed destabilization of CNS AGSJs at ∼ 1 year. We also discovered that the 4.1B locus is differentially expressed in the PNS and CNS, and generates multiple splice isoforms in the PNS, suggesting 4.1B may function differently in the PNS versus CNS. Together, our studies provide direct evidence that 4.1B plays a pivotal role in interactions between the paranodal AGSJs and axonal cytoskeleton, and that 4.1B is critically required for long-term maintenance of axonal domains in myelinated axons. PMID:21632923

  13. Protein kinase A alterations in endocrine tumors.

    PubMed

    Yu, B; Ragazzon, B; Rizk-Rabin, M; Bertherat, J

    2012-09-01

    Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis. PMID:22752956

  14. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    SciTech Connect

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; Kobe, Bostjan

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex. We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.

  15. Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

    DOE PAGESBeta

    Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; Kobe, Bostjan

    2015-03-16

    Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

  16. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  17. Reactions of 1,3-Diketones with a Dipeptide Isothiazolidin-3-one: Toward Agents That Covalently Capture Oxidized Protein Tyrosine Phosphatase 1B.

    PubMed

    Ruddraraju, Kasi Viswanatharaju; Parsons, Zachary D; Llufrio, Elizabeth M; Frost, Natasha L; Gates, Kent S

    2015-12-18

    Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of type 2 diabetes; however, the enzyme has been classified by some as an "undruggable target". Here we describe studies directed toward the development of agents that covalently capture the sulfenyl amide "oxoform" of PTP1B generated during insulin signaling events. The sulfenyl amide residue found in oxidized PTP1B presents a unique electrophilic sulfur center that may be exploited in drug and probe design. Covalent capture of oxidized PTP1B could permanently disable the intracellular pool of enzyme involved in regulation of insulin signaling. Here, we employed a dipeptide model of oxidized PTP1B to investigate the nucleophilic capture of the sulfenyl amide residue by structurally diverse 1,3-diketones. All of the 1,3-diketones examined here reacted readily with the electrophilic sulfur center in the sulfenyl amide residue to generate stable covalent attachments. Several different types of products were observed, depending upon the substituents present on the 1,3-diketone. The results provide a chemical foundation for the development of agents that covalently capture the oxidized form of PTP1B generated in cells during insulin signaling events. PMID:26517018

  18. The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity

    PubMed Central

    Hobeika, Elias; Biesen, Robert; Kollert, Florian; Taddeo, Adriano; Voll, Reinhard E.; Hiepe, Falk

    2014-01-01

    Tyrosine phosphorylation of signaling molecules that mediate B cell activation in response to various stimuli is tightly regulated by protein tyrosine phosphatases (PTPs). PTP1B is a ubiquitously expressed tyrosine phosphatase with well-characterized functions in metabolic signaling pathways. We show here that PTP1B negatively regulates CD40, B cell activating factor receptor (BAFF-R), and TLR4 signaling in B cells. Specifically, PTP1B counteracts p38 mitogen-activated protein kinase (MAPK) activation by directly dephosphorylating Tyr182 of this kinase. Mice with a B cell–specific PTP1B deficiency show increased T cell–dependent immune responses and elevated total serum IgG. Furthermore, aged animals develop systemic autoimmunity with elevated serum anti-dsDNA, spontaneous germinal centers in the spleen, and deposition of IgG immune complexes and C3 in the kidney. In a clinical setting, we observed that B cells of rheumatoid arthritis patients have significantly reduced PTP1B expression. Our data suggest that PTP1B plays an important role in the control of B cell activation and the maintenance of immunological tolerance. PMID:24590766

  19. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription

    PubMed Central

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song

    2015-01-01

    ABSTRACT Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. IMPORTANCE Influenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we

  20. A simple and visible colorimetric method through Zr(4+)-phosphate coordination for the assay of protein tyrosine phosphatase 1B and screening of its inhibitors.

    PubMed

    Zhang, Juan; Lv, Jun; Wang, Xiaonan; Li, Defeng; Wang, Zhaoxia; Li, Genxi

    2015-08-21

    Inhibitors of protein tyrosine phosphatase 1B (PTP1B) are promising agents for the treatment of type 2 diabetes and obesity, so a colorimetric method has been developed in this work for PTP1B assay and screening of its inhibitors. The method is based on the chelation effect of zirconium (Zr(4+)) ions on the phosphate group, which may induce aggregation of 4-aminophenylphosphate-functionalized gold nanoparticles (APP/AuNPs) and the corresponding color change of the testing solution. Owing to the dephosphorylation of PTP1B, the aggregation of AuNPs will be influenced by PTP1B since there is no coordination reactivity between Zr(4+) ions and 4-aminophenol, the hydrolyzed product of APP catalyzed by the enzyme. Therefore, a simple colorimetric method for the assay of PTP1B activity can be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 650 nm to that at 522 nm vary linearly with the PTP1B activity in the range from 0.005 to 0.18 U mL(-1) with the lowest detection limit of 0.0017 U mL(-1). Moreover, using this proposed method, the inhibition effect of 6-chloro-3-formyl-7-methylchromone, betulinic acid, ursolic acid, and sodium orthovanadate on PTP1B activity can be tested with IC50 values of 10, 13, 9, and 1.1 μM, respectively. Therefore, this new method has great potential not only for the detection of PTP1B activity but also for the screening of the inhibitors. PMID:26153652

  1. Aldo-keto reductase family 1 B10 protein detoxifies dietary and lipid-derived alpha, beta-unsaturated carbonyls at physiological levels

    SciTech Connect

    Zhong, Linlin; Liu, Ziwen; Yan, Ruilan; Johnson, Stephen; Zhao, Yupei; Fang, Xiubin; Cao, Deliang

    2009-09-18

    Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 {mu}M, 4-hydroxynonenal (HNE) at 0.10 {mu}M, trans-2-hexanal at 0.10 {mu}M, and trans-2,4-hexadienal at 0.05 {mu}M, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 {mu}M (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.

  2. A Golgi-localized Mannosidase (MAN1B1) Plays a Non-enzymatic Gatekeeper Role in Protein Biosynthetic Quality Control*

    PubMed Central

    Iannotti, Michael J.; Figard, Lauren; Sokac, Anna M.; Sifers, Richard N.

    2014-01-01

    Conformation-based disorders are manifested at the level of protein structure, necessitating an accurate understanding of how misfolded proteins are processed by the cellular proteostasis network. Asparagine-linked glycosylation plays important roles for protein quality control within the secretory pathway. The suspected role for the MAN1B1 gene product MAN1B1, also known as ER mannosidase I, is to function within the ER similar to the yeast ortholog Mns1p, which removes a terminal mannose unit to initiate a glycan-based ER-associated degradation (ERAD) signal. However, we recently discovered that MAN1B1 localizes to the Golgi complex in human cells and uncovered its participation in ERAD substrate retention, retrieval to the ER, and subsequent degradation from this organelle. The objective of the current study was to further characterize the contribution of MAN1B1 as part of a Golgi-based quality control network. Multiple lines of experimental evidence support a model in which neither the mannosidase activity nor catalytic domain is essential for the retention or degradation of the misfolded ERAD substrate Null Hong Kong. Instead, a highly conserved, vertebrate-specific non-enzymatic decapeptide sequence in the luminal stem domain plays a significant role in controlling the fate of overexpressed Null Hong Kong. Together, these findings define a new functional paradigm in which Golgi-localized MAN1B1 can play a mannosidase-independent gatekeeper role in the proteostasis network of higher eukaryotes. PMID:24627495

  3. Assembly of juxtaparanodes in myelinating DRG culture: Differential clustering of the Kv1/Caspr2 complex and scaffolding protein 4.1B.

    PubMed

    Hivert, Bruno; Pinatel, Delphine; Labasque, Marilyne; Tricaud, Nicolas; Goutebroze, Laurence; Faivre-Sarrailh, Catherine

    2016-05-01

    The precise distribution of ion channels at the nodes of Ranvier is essential for the efficient propagation of action potentials along myelinated axons. The voltage-gated potassium channels Kv1.1/1.2 are clustered at the juxtaparanodes in association with the cell adhesion molecules, Caspr2 and TAG-1 and the scaffolding protein 4.1B. In the present study, we set up myelinating cultures of DRG neurons and Schwann cells to look through the formation of juxtaparanodes in vitro. We showed that the Kv1.1/Kv1.2 channels were first enriched at paranodes before being restricted to distal paranodes and juxtaparanodes. In addition, the Kv1 channels displayed an asymmetric expression enriched at the distal juxtaparanodes. Caspr2 was strongly co-localized with Kv1.2 whereas the scaffolding protein 4.1B was preferentially recruited at paranodes while being present at juxtaparanodes too. Kv1.2/Caspr2 but not 4.1B, also transiently accumulated within the nodal region both in myelinated cultures and developing sciatic nerves. Studying cultures and sciatic nerves from 4.1B KO mice, we further showed that 4.1B is required for the proper targeting of Caspr2 early during myelination. Moreover, using adenoviral-mediated expression of Caspr-GFP and photobleaching experiments, we analyzed the stability of paranodal junctions and showed that the lateral stability of paranodal Caspr was not altered in 4.1B KO mice indicating that 4.1B is not required for the assembly and stability of the paranodal junctions. Thus, developing an adapted culture paradigm, we provide new insights into the dynamic and differential distribution of Kv1 channels and associated proteins during myelination. GLIA 2016;64:840-852. PMID:26840208

  4. Validation of formylchromane derivatives as protein tyrosine phosphatase 1B inhibitors by pharmacophore modeling, atom-based 3D-QSAR and docking studies.

    PubMed

    Malla, Priyanka; Kumar, Rajnish; Kumar, Manoj

    2013-07-01

    Formylchromane derivatives were reported to possess irreversible and selective inhibition on human protein tyrosine phosphatase 1B (PTP 1B). Inhibition of PTP 1B is a molecular level legitimate approach for the management of type 2 diabetes mellitus (T2DM). 3D-QSAR studies were performed on a series of formylchromane derivatives using partial least square (PLS) analysis for correlating molecular architecture of the analogs with their PTP 1B inhibitory activity. A five-point pharmacophore hypothesis with three hydrogen bond acceptors (A) and two aromatic rings (R) as pharmacophoric features was developed using phase module of Schrödinger suite. The hypothesis AAARR.160 was considered as best hypothesis in this study characterized by survival score (3.483), the best cross-validated r² (Q²) (0.774), regression coefficient (0.960), Pearson-R (0.891), and F value (100.3). The results of hypothesis AAARR.160 complimented very closely to interactions witnessed in active site of the ligand-bound complex. The molecular docking simulations into PTP 1B active site also highlighted that biphenyl moiety favorably interacted with amino acid residues lining the lipophilic pocket, and provided hydrophobic interactions with receptor active site. These observations might be useful for further development and optimization of new chemical entities as potential PTP 1B inhibitors prior to its synthesis. PMID:23506477

  5. Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

    PubMed Central

    Fueller, Julia; Egorov, Mikhail V.; Walther, Kirstin A.; Sabet, Ola; Mallah, Jana; Grabenbauer, Markus; Kinkhabwala, Ali

    2015-01-01

    The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states. PMID:26431424

  6. Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat

    PubMed Central

    Guo, Yusong; Linstedt, Adam D

    2014-01-01

    Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it’s binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity. PMID:25332841

  7. Dephosphorylation of Tyrosine 393 in Argonaute 2 by Protein Tyrosine Phosphatase 1B Regulates Gene Silencing in Oncogenic RAS-Induced Senescence

    PubMed Central

    Yang, Ming; Haase, Astrid D.; Huang, Fang-Ke; Coulis, Gérald; Rivera, Keith D.; Dickinson, Bryan C.; Chang, Christopher J.; Pappin, Darryl J.; Neubert, Thomas A.; Hannon, Gregory J.; Boivin, Benoit; Tonks, Nicholas K.

    2014-01-01

    SUMMARY Oncogenic RAS (H-RASV12) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RASV12-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RASV12-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RASV12-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNA) and thus miRNA-mediated gene silencing, which counteracted the function of H-RASV12-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RASV12-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing. PMID:25175024

  8. BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis[W

    PubMed Central

    Feraru, Elena; Feraru, Mugurel I.; Asaoka, Rin; Paciorek, Tomasz; De Rycke, Riet; Tanaka, Hirokazu; Nakano, Akihiko; Friml, Jiří

    2012-01-01

    Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging–based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane. PMID:22773752

  9. Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

    PubMed Central

    Mohan, Vijay; Pendyala, Srikanth; He, Donghong; Ebenezer, David L.; Harijith, Anantha; Fu, Panfeng; Huang, Long Shuang; Bear, James E.; Garcia, Joe G. N.; Natarajan, Viswanathan

    2013-01-01

    Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis. PMID:23667561

  10. The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

    PubMed Central

    Schmitz, M L; Indorf, A; Limbourg, F P; Städtler, H; Traenckner, E B; Baeuerle, P A

    1996-01-01

    The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB. PMID:8754803

  11. Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle–Associated Replication of Peroxisomes[W

    PubMed Central

    Lingard, Matthew J.; Gidda, Satinder K.; Bingham, Scott; Rothstein, Steven J.; Mullen, Robert T.; Trelease, Richard N.

    2008-01-01

    Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ∼40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells. PMID:18539750

  12. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  13. Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation.

    PubMed

    Sun, Chun-Xiao; Robb, Victoria A; Gutmann, David H

    2002-11-01

    Members of the Protein 4.1 superfamily have highly conserved FERM domains that link cell surface glycoproteins to the actin cytoskeleton. Within this large and constantly expanding superfamily, at least five subgroups have been proposed. Two of these subgroups, the ERM and prototypic Protein 4.1 molecules, include proteins that function as tumor suppressors. The ERM subgroup member merlin/schwannomin is inactivated in the tumor-predisposition syndrome neurofibromatosis 2 (NF2), and the prototypic 4.1 subgroup member, Protein 4.1B, has been implicated in the molecular pathogenesis of breast, lung and brain cancers. This review focuses on what is known of mechanisms of action and critical protein interactions that may mediate the unique growth inhibitory signals of these two Protein 4.1 tumor suppressors. On the basis of insights derived from studying the NF2 tumor suppressor, we propose a model for merlin growth regulation in which CD44 links growth signals from plasma membrane to the nucleus by interacting with ERM proteins and merlin. PMID:12356905

  14. Interaction of 5-HT1B/D ligands with recombinant h 5-HT1A receptors: intrinsic activity and modulation by G-protein activation state.

    PubMed

    Pauwels, P J; Palmier, C; Dupuis, D S; Colpaert, F C

    1998-05-01

    Many 5-HT1B/D receptor ligands have affinity for 5-HT1A receptors. In the present study, the intrinsic activity of a series of 5-HT1B/D ligands was investigated at human 5-HT1A (h 5-HT1A) receptors by measuring G-protein activation in recombinant C6-glial and HeLa membranes, using agonist-stimulated [35S]GTPgammaS binding. In these two membrane preparations, the density of h 5-HT1A receptors (i.e., 246 to 320 fmol mg(-1) protein) and of their G-proteins, and the receptor: G-protein density ratio (0.08 to 0.18) appeared to be similar. It was found that: (i) the maximal [35S]GTPgammaS binding responses induced by the 5-HT1B/D receptor ligands in the HeLa preparation at 30 microM GDP were comparable to that of the native agonist 5-HT; (ii) as compared to 5-HT (1.00), similar potencies but lower maximal responses were observed in the C6-glial preparation at 0.3 microM GDP for zolmitriptan (0.89), dihydroergotamine (0.81), rizatriptan (0.71), CP122638 (0.69), naratriptan (0.60) and sumatriptan (0.53); and that (iii) maximal [35S]GTPgammaS binding responses induced by 5-HT1B/D ligands in the C6-glial preparation were either unaffected or significantly enhanced by increasing the GDP concentration from 0.3 to 30 microM and higher concentrations. These features differ from those observed with 5-HT1A receptor agonists; the latter display the same rank order of potency and efficacy in both membrane preparations, and increasing the amount of GDP with C6-glial membranes results in an attenuation of both the agonist's maximal effect and the apparent potency of partial agonists. The differential regulation of 5-HT1A and 5-HT1B/D agonist responses by GDP suggests that different G-protein subtypes are involved upon 5-HT1A receptor activation by 5-HT1A and 5-HT1B/D agonists. PMID:9650800

  15. Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5.

    PubMed Central

    Teodoro, J G; Branton, P E

    1997-01-01

    The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E1A gene products to induce cell transformation. E1A proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is believed that the role of the E1B-55kDa protein in transformation is to protect against p53-dependent apoptosis by binding to and inactivating p53. It has been shown previously that the 55-kDa polypeptide abrogates p53-mediated transactivation and that mutants defective in p53 binding are unable to cooperate with E1A in transformation. We have previously mapped phosphorylation sites near the carboxy terminus of the E1B-55kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues greatly reduced transforming activity, and although the mutant 55-kDa protein was found to interact with p53 at normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now report that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to block E1A-induced p53-dependent apoptosis, whereas cells infected by mutant pm490/1/5A, which contains alanine residues at all three phosphorylation sites, exhibited extensive DNA fragmentation and classic apoptotic cell death. The E1B-55kDa product has been shown to exhibit intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absence of p53. Such repression activity was totally absent with mutant pm490/1/5A. These data suggested that inhibition of p53-dependent apoptosis may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carboxy terminus. PMID:9094635

  16. Dictyostelium ribosomal protein genes and the elongation factor 1B gene show coordinate developmental regulation which is under post-transcriptional control.

    PubMed

    Agarwal, A K; Blumberg, D D

    1999-06-01

    Starvation for amino acids initiates the developmental program in the cellular slime mold, Dictyostelium discoideum [19, 20]. One of the earliest developmental events is the decline in ribosomal protein synthesis [2, 17, 29, 30]. The ribosomal protein mRNAs are excluded from polysomes with 20 min to 1 h following the removal of nutrients, and their mRNA levels decline sharply at about 9 h into the 24-h developmental cycle [28, 31, 35, 36]. It has been generally assumed that the decline in r-protein mRNA levels during late development reflected a decline in the transcription rate [12, 32, 43]. Here we demonstrate that this is not the case. The transcription rates of three ribosomal protein genes, rpL11, rpL23 and rpS9 as well as an elongation factor 1B gene have been determined during growth and development in Dictyostelium. Throughout growth and development the transcription rate of the ribosomal protein genes remains relatively constant at 0.2%-0.5% of the rate of rRNA transcription while the elongation factor 1B gene is transcribed at 0.4%-0.6% of the rRNA rate. This low but constant transcription rate is in contrast to a spore coat protein gene Psp D, which is transcribed at 6% of the rRNA rate in late developing cells. The elongation factor 1B gene appears to be co-regulated with the ribosomal protein genes both in terms of its transcription rate and mRNA accumulation. Dictyostelium has been a popular model for understanding signal transduction and the growth to differentiation transition, thus it is of significance that the regulation of ribosome biosynthesis in Dictyostelium resembles that of higher eukaryotes in being regulated largely at the post-transcriptional level in response to starvation as opposed to yeasts where the regulation is largely transcriptional [27]. PMID:10374261

  17. Autism and Intellectual Disability-Associated KIRREL3 Interacts with Neuronal Proteins MAP1B and MYO16 with Potential Roles in Neurodevelopment.

    PubMed

    Liu, Ying F; Sowell, Sarah M; Luo, Yue; Chaubey, Alka; Cameron, Richard S; Kim, Hyung-Goo; Srivastava, Anand K

    2015-01-01

    Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying

  18. Autism and Intellectual Disability-Associated KIRREL3 Interacts with Neuronal Proteins MAP1B and MYO16 with Potential Roles in Neurodevelopment

    PubMed Central

    Liu, Ying F.; Sowell, Sarah M.; Luo, Yue; Chaubey, Alka; Cameron, Richard S.; Kim, Hyung-Goo; Srivastava, Anand K.

    2015-01-01

    Cell-adhesion molecules of the immunoglobulin superfamily play critical roles in brain development, as well as in maintaining synaptic plasticity, the dysfunction of which is known to cause cognitive impairment. Recently dysfunction of KIRREL3, a synaptic molecule of the immunoglobulin superfamily, has been implicated in several neurodevelopmental conditions including intellectual disability, autism spectrum disorder, and in the neurocognitive delay associated with Jacobsen syndrome. However, the molecular mechanisms of its physiological actions remain largely unknown. Using a yeast two-hybrid screen, we found that the KIRREL3 extracellular domain interacts with brain expressed proteins MAP1B and MYO16 and its intracellular domain can potentially interact with ATP1B1, UFC1, and SHMT2. The interactions were confirmed by co-immunoprecipitation and colocalization analyses of proteins expressed in human embryonic kidney cells, mouse neuronal cells, and rat primary neuronal cells. Furthermore, we show KIRREL3 colocalization with the marker for the Golgi apparatus and synaptic vesicles. Previously, we have shown that KIRREL3 interacts with the X-linked intellectual disability associated synaptic scaffolding protein CASK through its cytoplasmic domain. In addition, we found a genomic deletion encompassing MAP1B in one patient with intellectual disability, microcephaly and seizures and deletions encompassing MYO16 in two unrelated patients with intellectual disability, autism and microcephaly. MAP1B has been previously implicated in synaptogenesis and is involved in the development of the actin-based membrane skeleton. MYO16 is expressed in hippocampal neurons and also indirectly affects actin cytoskeleton through its interaction with WAVE1 complex. We speculate KIRREL3 interacting proteins are potential candidates for intellectual disability and autism spectrum disorder. Moreover, our findings provide further insight into understanding the molecular mechanisms underlying

  19. A peptide inhibitor of exportin1 blocks shuttling of the adenoviral E1B 55 kDa protein but not export of viral late mRNAs

    SciTech Connect

    Flint, S.J. . E-mail: sjflint@molbio.princeton.edu; Huang, Wenying; Goodhouse, Joseph; Kyin, Saw

    2005-06-20

    The human subgroup C adenoviral E1B 55 kDa and E4 Orf6 proteins are required for efficient nuclear export of viral late mRNAs, but the cellular pathway that mediates such export has not been identified. As a first step to develop a general approach to address this issue, we have assessed the utility of cell-permeable peptide inhibitors of cellular export receptors. As both E1B and E4 proteins have been reported to contain a leucine-rich nuclear export signal (NES), we synthesized a cell-permeable peptide containing such an NES. This peptide induced substantial inhibition of export of the E1B protein, whereas a control, non-functional peptide did not. However, under the same conditions, the NES peptide had no effect on export of viral late mRNAs. These observations establish that viral late mRNAs are not exported by exportin1, as well as the value of peptide inhibitors in investigation of mRNA export regulation in adenovirus-infected cells.

  20. Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

    PubMed

    Nguyen, Phi-Hung; Ji, Da-Jung; Han, Yu-Ran; Choi, Jae-Sue; Rhyu, Dong-Young; Min, Byung-Sun; Woo, Mi-Hee

    2015-07-01

    As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3-7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5±0.1 to 13.2±0.8μM. Furthermore, the isolates (1-7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia. PMID:25907369

  1. Synthesis, biological evaluation and 3D-QSAR studies of imidazolidine-2,4-dione derivatives as novel protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Wang, Mei-Yan; Jin, Yuan-Yuan; Wei, Hui-Yu; Zhang, Li-Song; Sun, Su-Xia; Chen, Xiu-Bo; Dong, Wei-Li; Xu, Wei-Ren; Cheng, Xian-Chao; Wang, Run-Ling

    2015-10-20

    Protein tyrosine phosphatase 1B (PTP1B) plays a vital role in the regulation of insulin sensitivity and dephosphorylation of the insulin receptor, so PTP1B inhibitors may be potential agents to treat type 2 diabetes. In this work, a series of novel imidazolidine-2,4-dione derivatives were designed, synthesized and assayed for their PTP1B inhibitory activities. These compounds exhibited potent activities with IC50 values at 0.57-172 μM. A 3D-QSAR study using CoMFA and CoMSIA techniques was carried out to explore structure activity relationship of these molecules. The CoMSIA model was more predictive with q(2) = 0.777, r(2) = 0.999, SEE = 0.013 and r(2)pred = 0.836, while the CoMFA model gave q(2) = 0.543, r(2) = 0.998, SEE = 0.029 and r(2)pred = 0.754. The contour maps derived from the best CoMFA and CoMSIA models combined with docking analysis provided good insights into the structural features relevant to the bioactivity, and could be used in the molecular design of novel imidazolidine-2,4-dione derivatives. PMID:26342135

  2. Canine parvovirus NS1 protein exhibits anti-tumor activity in a mouse mammary tumor model.

    PubMed

    Gupta, Shishir Kumar; Yadav, Pavan Kumar; Gandham, Ravi Kumar; Sahoo, A P; Harish, D R; Singh, Arvind Kumar; Tiwari, A K

    2016-02-01

    Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity. PMID:26739427

  3. Molecular Analysis of Aedes aegypti Classical Protein Tyrosine Phosphatases Uncovers an Ortholog of Mammalian PTP-1B Implicated in the Control of Egg Production in Mosquitoes

    PubMed Central

    Moretti, Debora Monteiro; Ahuja, Lalima Gagan; Nunes, Rodrigo Dutra; Cudischevitch, Cecília Oliveira; Daumas-Filho, Carlos Renato Oliveira; Medeiros-Castro, Priscilla; Ventura-Martins, Guilherme; Jablonka, Willy; Gazos-Lopes, Felipe; Senna, Raquel; Sorgine, Marcos Henrique Ferreira; Hartfelder, Klaus; Capurro, Margareth; Atella, Georgia Correa; Mesquita, Rafael Dias; Silva-Neto, Mário Alberto Cardoso

    2014-01-01

    Background Protein Tyrosine Phosphatases (PTPs) are enzymes that catalyze phosphotyrosine dephosphorylation and modulate cell differentiation, growth and metabolism. In mammals, PTPs play a key role in the modulation of canonical pathways involved in metabolism and immunity. PTP1B is the prototype member of classical PTPs and a major target for treating human diseases, such as cancer, obesity and diabetes. These signaling enzymes are, hence, targets of a wide array of inhibitors. Anautogenous mosquitoes rely on blood meals to lay eggs and are vectors of the most prevalent human diseases. Identifying the mosquito ortholog of PTP1B and determining its involvement in egg production is, therefore, important in the search for a novel and crucial target for vector control. Methodology/Principal Findings We conducted an analysis to identify the ortholog of mammalian PTP1B in the Aedes aegypti genome. We identified eight genes coding for classical PTPs. In silico structural and functional analyses of proteins coded by such genes revealed that four of these code for catalytically active enzymes. Among the four genes coding for active PTPs, AAEL001919 exhibits the greatest degree of homology with the mammalian PTP1B. Next, we evaluated the role of this enzyme in egg formation. Blood feeding largely affects AAEL001919 expression, especially in the fat body and ovaries. These tissues are critically involved in the synthesis and storage of vitellogenin, the major yolk protein. Including the classical PTP inhibitor sodium orthovanadate or the PTP substrate DiFMUP in the blood meal decreased vitellogenin synthesis and egg production. Similarly, silencing AAEL001919 using RNA interference (RNAi) assays resulted in 30% suppression of egg production. Conclusions/Significance The data reported herein implicate, for the first time, a gene that codes for a classical PTP in mosquito egg formation. These findings raise the possibility that this class of enzymes may be used as novel

  4. Inhibitory evaluation of oligonol on α-glucosidase, protein tyrosine phosphatase 1B, cholinesterase, and β-secretase 1 related to diabetes and Alzheimer's disease.

    PubMed

    Choi, Jae Sue; Bhakta, Himanshu Kumar; Fujii, Hajime; Min, Byung-Sun; Park, Chan Hum; Yokozawa, Takako; Jung, Hyun Ah

    2016-03-01

    Oligonol is a low-molecular-weight form of polyphenol that is derived from lychee fruit extract and contains catechin-type monomers and oligomers of proanthocyanidins. This study investigates the anti-diabetic activities of oligonol via α-glucosidase and human recombinant protein tyrosine phosphatase 1B (PTP1B) assays, as well as its anti-Alzheimer activities by evaluating the ability of this compound to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein cleaving enzyme 1 (BACE1). Oligonol exhibited potent concentration-dependent anti-diabetic activities by inhibiting α-glucosidase and PTP1B with IC50 values of 23.14 µg/mL and 1.02 µg/mL, respectively. Moreover, a kinetics study revealed that oligonol inhibited α-glucosidase (K i = 22.36) and PTP1B (K i = 8.51) with characteristics typical of a mixed inhibitor. Oligonol also displayed potent concentration-dependent inhibitory activity against AChE and BChE with IC50 values of 4.34 µg/mL and 2.07 µg/mL, respectively. However, oligonol exhibited only marginal concentration-dependent BACE1 inhibitory activity with an IC50 value of 130.45 µg/mL. A kinetics study revealed mixed-type inhibition against AChE (K i = 4.65) and BACE1 (K i = 58.80), and noncompetitive-type inhibition against BChE (K i = 9.80). Furthermore, oligonol exhibited dose-dependent inhibitory activity against peroxynitrite (ONOO(-))-mediated protein tyrosine nitration. These results indicate that oligonol has strong preventative potential in diabetes mellitus and in Alzheimer's disease. PMID:26724817

  5. Protein expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 in young patients with oral squamous cell carcinoma.

    PubMed

    Kaminagakura, E; Caris, A; Coutinho-Camillo, C; Soares, F A; Takahama-Júnior, A; Kowalski, L P

    2016-06-01

    The purpose of this study was to evaluate the expression of the enzymes involved in the biotransformation of tobacco and alcohol. A study group of 41 young patients (≤40 years old) with oral squamous cell carcinoma (OSCC) was compared to 59 control subjects (≥50 years old) with tumours of similar clinical stages and topographies. The immunohistochemical expression of CYP1A1, CYP1B1, ALDH1A1, and ALDH2 was evaluated using the tissue microarray technique. There was a predominance of males, smokers, and alcohol drinkers in both groups. Most tumours were located in the tongue (43.9% vs. 50.8%), were well-differentiated (63.4% vs. 56.6%), and were in clinical stages III or IV (80.5% vs. 78.0%). No difference was observed in the expression of CYP1A1, ALDH1A1, or ALDH2 between the two groups. CYP1A1 and ALDH2 protein expression had no influence on the prognosis. The immunoexpression of CYP1B1 was significantly higher in the control group than in the young group (P<0.001). The 5-year relapse-free survival was better in patients with CYP1B1 overexpression vs. protein underexpression (64% vs. 25%; P<0.05), regardless of age. ALDH1A1 expression improved relapse-free survival in young patients. These results suggest a lower risk of recurrence with increased metabolism of carcinogens by CYP1B1. Further studies involving other genes and proteins are necessary to complement the results of this research. PMID:26944893

  6. The IRE1/bZIP60 pathway are activated by potexvirus and potyvirus small membrane binding proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The endoplasmic reticulum provides an environment for protein synthesis, folding and distribution to all corners of the cell. With respect to protein synthesis and folding, quality production is central to maintaining homeostasis. When conditions occur that disrupt the folding capacity of the ER cau...

  7. Mod(mdg4)-58.8, isoform of mod(mdg4) loci, directly interacts with MTACP1A and MTACP1B proteins of Drosophila melanogaster.

    PubMed

    Golovnin, A K; Kostyuchenko, M V; Georgiev, P G; Melnikova, L S

    2016-01-01

    As a result of experiments in yeast two-hybrid system, coimmunoprecipitation of proteins from D. melanogaster embryo cell lysate, and immunostaining, it was shown for the first time that Mod(mdg4)-58.8 protein (isoform P), a product of mod(mdg4) locus, directly interacts with mtACP1A and mtACP1B proteins. These proteins are involved in de novo biosynthesis of fatty acids in mitochondria and are required for normal gametogenesis in males and females and, possibly, for the trachea development. This result expands the understanding of the role of mod(mdg4) locus products in the regulation of life activity of the eukaryotic cell. PMID:27025476

  8. SARS-CoV ORF1b-encoded nonstructural proteins 12-16: replicative enzymes as antiviral targets.

    PubMed

    Subissi, Lorenzo; Imbert, Isabelle; Ferron, François; Collet, Axelle; Coutard, Bruno; Decroly, Etienne; Canard, Bruno

    2014-01-01

    The SARS (severe acute respiratory syndrome) pandemic caused ten years ago by the SARS-coronavirus (SARS-CoV) has stimulated a number of studies on the molecular biology of coronaviruses. This research has provided significant new insight into many mechanisms used by the coronavirus replication-transcription complex (RTC). The RTC directs and coordinates processes in order to replicate and transcribe the coronavirus genome, a single-stranded, positive-sense RNA of outstanding length (∼27-32kilobases). Here, we review the up-to-date knowledge on SARS-CoV replicative enzymes encoded in the ORF1b, i.e., the main RNA-dependent RNA polymerase (nsp12), the helicase/triphosphatase (nsp13), two unusual ribonucleases (nsp14, nsp15) and RNA-cap methyltransferases (nsp14, nsp16). We also review how these enzymes co-operate with other viral co-factors (nsp7, nsp8, and nsp10) to regulate their activity. These last ten years of research on SARS-CoV have considerably contributed to unravel structural and functional details of one of the most fascinating replication/transcription machineries of the RNA virus world. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". PMID:24269475

  9. Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

    PubMed

    Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

    2016-02-18

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted. PMID:26028028

  10. Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

    PubMed

    Mundell, Stuart J; Pula, Giordano; More, Julia C A; Jane, David E; Roberts, Peter J; Kelly, Eamonn

    2004-06-01

    In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins. PMID:15155843

  11. Dual degradation signals control Gli protein stability and tumor formation

    PubMed Central

    Huntzicker, Erik G.; Estay, Ivette S.; Zhen, Hanson; Lokteva, Ludmila A.; Jackson, Peter K.; Oro, Anthony E.

    2006-01-01

    Regulated protein destruction controls many key cellular processes with aberrant regulation increasingly found during carcinogenesis. Gli proteins mediate the transcriptional effects of the Sonic hedgehog pathway, which is implicated in up to 25% of human tumors. Here we show that Gli is rapidly destroyed by the proteasome and that mouse basal cell carcinoma induction correlates with Gli protein accumulation. We identify two independent destruction signals in Gli1, DN and DC, and show that removal of these signals stabilizes Gli1 protein and rapidly accelerates tumor formation in transgenic animals. These data argue that control of Gli protein accumulation underlies tumorigenesis and suggest a new avenue for antitumor therapy. PMID:16421275

  12. Effect on transformation of mutations in the early region 1b-encoded 21- and 55-kilodalton proteins of adenovirus 5.

    PubMed Central

    Babiss, L E; Fisher, P B; Ginsberg, H S

    1984-01-01

    It is well established that the adenovirus 5 genes responsible for the initiation and maintenance of the transformed cell reside in the early region 1a and 1b genes, but it remains unclear how the polypeptides encoded in these genes mediate their functions. To probe the function of the early region 1b-encoded 55- and 21-kilodalton (kd) polypeptides during this process, a series of viral mutants was engineered so that they contained deletions or insertions at 5.4, 5.7, 7.9, or 9.6 map units. By means of either an overlap recombination procedure involving H5dl314 (delta 3.7 to 4.6 map units) cleaved with ClaI, or a marker rescue procedure involving H5dl312 (delta 1.2 to 3.8 map units), viral mutants were isolated by their ability to produce plaques on KB cell line 18 cells, which constitutively express only viral early region 1b functions. DNA sequence analysis confirmed that the series of mutants generated differed in their abilities to express the 21- or the 55-kd polypeptides, or both. Upon infection of cloned rat embryo fibroblast cells with viruses containing mutations affecting the 55-kd protein, the transformation frequency decreased as the size of the predicted truncated polypeptide decreased. Although all of the foci generated by the 55-kd protein mutants were indistinguishable from the foci induced by wild-type virus, they displayed an inefficient ability to grow in soft agar, again in relation to the size of the truncated polypeptide. In contrast, if cloned rat embryo fibroblast cells were transfected with viral DNA, the defectiveness in transformation observed after infection with virions was not as dramatic. However, all of the viruses containing 21-kd mutations were transformation defective, regardless of the mode by which the viral nucleic acid was introduced into the cell. Images PMID:6333514

  13. The Indeterminate Domain Protein ROC1 Regulates Chilling Tolerance via Activation of DREB1B/CBF1 in Rice

    PubMed Central

    Dou, Mingzhu; Cheng, Shuai; Zhao, Baotian; Xuan, Yuanhu; Shao, Minglong

    2016-01-01

    Abiotic stress, including salinity, drought and cold, severely affect diverse aspects of plant development and production. Rice is an important crop that does not acclimate to cold; therefore, it is relatively sensitive to low temperature stress. Dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat binding factors (CBFs) are well known for their function in cold tolerance, but the transcriptional regulation of CBFs remains elusive, especially in rice. Here, we performed a yeast one-hybrid assay using the promoter of CBF1, a cold-induced gene, to isolate transcriptional regulators of CBF1. Among the seven candidates identified, an indeterminate domain (IDD) protein named ROC1 (a regulator of CBF1) was further analyzed. The ROC1 transcript was induced by exogenously-treated auxin, while it was not altered by cold or ABA stimuli. ROC1-GFP was localized at the nucleus, and ROC1 showed trans-activation activity in yeast. The electrophoretic mobility shift assay (EMSA) and ChIP analyses revealed that ROC1 directly bound to the promoter of CBF1. Furthermore, ROC1 mutants exhibited chilling-sensitive symptoms and inhibited cold-mediated induction of CBF1 and CBF3, indicating that ROC1 is a positive regulator of cold stress responses. Taken together, this study identified the CBF1 regulator, and the results are important for rice plant adaptation to chilling stress. PMID:26927068

  14. The Indeterminate Domain Protein ROC1 Regulates Chilling Tolerance via Activation of DREB1B/CBF1 in Rice.

    PubMed

    Dou, Mingzhu; Cheng, Shuai; Zhao, Baotian; Xuan, Yuanhu; Shao, Minglong

    2016-01-01

    Abiotic stress, including salinity, drought and cold, severely affect diverse aspects of plant development and production. Rice is an important crop that does not acclimate to cold; therefore, it is relatively sensitive to low temperature stress. Dehydration-responsive element-binding protein 1s (DREB1s)/C-repeat binding factors (CBFs) are well known for their function in cold tolerance, but the transcriptional regulation of CBFs remains elusive, especially in rice. Here, we performed a yeast one-hybrid assay using the promoter of CBF1, a cold-induced gene, to isolate transcriptional regulators of CBF1. Among the seven candidates identified, an indeterminate domain (IDD) protein named ROC1 (a regulator of CBF1) was further analyzed. The ROC1 transcript was induced by exogenously-treated auxin, while it was not altered by cold or ABA stimuli. ROC1-GFP was localized at the nucleus, and ROC1 showed trans-activation activity in yeast. The electrophoretic mobility shift assay (EMSA) and ChIP analyses revealed that ROC1 directly bound to the promoter of CBF1. Furthermore, ROC1 mutants exhibited chilling-sensitive symptoms and inhibited cold-mediated induction of CBF1 and CBF3, indicating that ROC1 is a positive regulator of cold stress responses. Taken together, this study identified the CBF1 regulator, and the results are important for rice plant adaptation to chilling stress. PMID:26927068

  15. [Interaction of two tumor suppressors: Phosphatase CTDSPL and Rb protein].

    PubMed

    Beniaminov, A D; Krasnov, G S; Dmitriev, A A; Puzanov, G A; Snopok, B A; Senchenko, V N; Kashuba, V I

    2016-01-01

    Earlier we established that CTDSPL gene encoding small carboxy-terminal domain serine phosphatase can be considered a classical tumor suppressor gene. Besides, transfection of tumor cell line MCF-7 with CTDSPL led to the content decrease of inactive phosphorylated form of another tumor suppressor, retinoblastoma protein (Rb), and subsequently to cell cycle arrest at the G1/S boundary. This result implied that small phosphatase CTDSPL is able to specifically dephosphorylate and activate Rb protein. In order to add some fuel to this hypothesis, in the present work we studied the interaction of two tumor suppressors CTDSPL and Rb in vitro. GST pool-down assay revealed that CTDSPL is able to precipitate Rb protein from MCF-7 cell extracts, while surface plasmon resonance technique showed that interaction of the two proteins is direct. Results of this study reassert that phosphatase CTDSPL and Rb could be involved in the common mechanism of cell cycle regulation. PMID:27414789

  16. Differential regulation of mast cell degranulation versus cytokine secretion by the actin regulatory proteins Coronin1a and Coronin1b

    PubMed Central

    Föger, Niko; Jenckel, André; Orinska, Zane; Lee, Kyeong-Hee; Chan, Andrew C.; Bulfone-Paus, Silvia

    2011-01-01

    Mast cell (MC) activation via aggregation of the high affinity IgE receptor (FcεRI) causes degranulation and release of proinflammatory mediators in a process that involves the reorganization of the actin cytoskeleton. However, the regulatory pathways and the molecular links between cytoskeletal changes and MC function are incompletely understood. In this study, we provide genetic evidence for a critical role of the actin-regulatory proteins Coronin1a (Coro1a) and Coro1b on exocytic pathways in MCs: Coro1a−/− bone marrow–derived MCs exhibit increased FcεRI-mediated degranulation of secretory lysosomes but significantly reduced secretion of cytokines. Hyperdegranulation of Coro1a−/− MCs is further augmented by the additional loss of Coro1b. In vivo, Coro1a−/−Coro1b−/− mice displayed enhanced passive cutaneous anaphylaxis. Functional reconstitution assays revealed that the inhibitory effect of Coro1a on MC degranulation strictly correlates with cortical localization of Coro1a, requires its filamentous actin–binding activity, and is regulated by phosphorylation of Ser2 of Coro1a. Thus, coronin proteins, and in turn the actin cytoskeleton, exhibit a functional dichotomy as differential regulators of degranulation versus cytokine secretion in MC biology. PMID:21844203

  17. The genes encoding for D4Z4 binding proteins HMGB2, YY1, NCL, and MYOD1 are excluded as candidate genes for FSHD1B.

    PubMed

    Bastress, K L; Stajich, J M; Speer, M C; Gilbert, J R

    2005-04-01

    Facioscapulohumeral muscular dystrophy is a disease of skeletal muscle, with symptoms including both facial and shoulder girdle weakness and progression to involve the pelvic girdle and extremities in the majority of cases. For most cases of FSHD, the molecular basis of the disease can be identified as a partial deletion of the D4Z4 repeat array on the end of the long arm of chromosome 4. However, in up to 5% of FSHD families there is no linkage to 4q35. These cases are designated as FSHD1B. Proteins have been identified that bind to the D4Z4 repeats of chromosome 4q35. The genes encoding D4Z4 binding proteins YY1, HMGB2, NCL, and MYOD1 were investigated as candidate genes for FSHD1B. Coding sequences and promoter region were analyzed for HMBG2 and no sequence variations were detected. For YY1, all five exons were analyzed and a polymorphism was detected in both the unaffected and affected populations. In nucleolin (NCL), several SNPs were identified, including a SNP causing the non-synonymous change P515H; however, all polymorphisms either occurred in control samples or were previously reported. A novel polymorphism was also detected in MYOD1, but did not represent a disease-specific variation. These results suggest that HMBG2, YY1, NCL, and MYOD1 are unlikely to represent the genes responsible for FSHD in these families. PMID:15792872

  18. Oncolytic Adenoviral Mutants with E1B19K Gene Deletions Enhance Gemcitabine-induced Apoptosis in Pancreatic Carcinoma Cells and Anti-Tumor Efficacy In vivo

    PubMed Central

    Leitner, Stephan; Sweeney, Katrina; Öberg, Daniel; Davies, Derek; Miranda, Enrique; Lemoine, Nick R.; Halldén, Gunnel

    2010-01-01

    Purpose Pancreatic adenocarcinoma is a rapidly progressive malignancy that is highly resistant to current chemotherapeutic modalities and almost uniformly fatal.We show that a novel targeting strategy combining oncolytic adenoviral mutants with the standard cytotoxic treatment, gemcitabine, can markedly improve the anticancer potency. Experimental Design Adenoviral mutants with the E1B19K gene deleted with and without E3B gene expression (AdΔE1B19K and dl337 mutants, respectively) were assessed for synergistic interactions in combination with gemcitabine. Cell viability, mechanism of cell death, and antitumor efficacy in vivo were determined in the pancreatic carcinoma cells PT45 and Suit2, normal human bronchial epithelial cells, and in PT45 xenografts. Results The ΔE1B19K-deleted mutants synergized with gemcitabine to selectively kill cultured pancreatic cancer cells and xenografts in vivo with no effect in normal cells. The corresponding wild-type virus (Ad5) stimulated drug-induced cell killing to a lesser degree. Gemcitabine blocked replication of all viruses despite the enhanced cell killing activity due to gemcitabine-induced delay in G1/S-cell cycle progression, with repression of cyclin E and cdc25A, which was not abrogated by viral E1A-expression. Synergistic cell death occurred through enhancement of gemcitabine-induced apoptosis in the presence of both AdΔE1B19K and dl337 mutants, shown by increased cell membrane fragmentation, caspase-3 activation, and mitochondrial dysfunction. Conclusions Our data suggest that oncolytic mutants lacking the antiapoptotic E1B19K gene can improve efficacy of DNA-damaging drugs such as gemcitabine through convergence on cellular apoptosis pathways.These findings imply that less toxic doses than currently practicedin the clinic could efficiently target pancreatic adenocarcinomas when combined with adenoviral mutants. PMID:19223497

  19. YKL-40 protein in osteosarcoma tumor tissue.

    PubMed

    Thorn, Andrea Pohly; Daugaard, Søren; Christensen, Lise Hanne; Christensen, Ib Jarle; Petersen, Michael Mørk

    2016-06-01

    YKL-40, a cellular glycoprotein isolated from the human osteosarcoma (OS) cell line MG63, is increased in the blood of patients with various types of cancer, and is found as an independent prognostic variable for survival. YKL-40 is also present with variable intensity in the tumor cells of some cancer types, but survival results have been conflicting. The aim of this study was to investigate the tissue expression of YKL-40 and its possible role as a predictive marker in patients with OS. Forty-eight patients were included in the study. Diagnostic biopsies were analyzed by immunohistochemistry; YKL-staining scores as well as CD14 and CD163 scores were determined, and survival data were determined statistically. A universal intense immunostaining for YKL-40 was found in all tumor cells, but tumor cell/stroma ratio varied, and this ratio (%) served as staining score. Using 24% as mean score to divide the material, patients with tumors of high YKL-40 score had a better survival than patients with low score (p = 0.05). YKL-positive macrophages had no influence on the result. Unexpectedly and contrary to some other findings in cancer tissues, this study has shown a correlation between high YKL-40 tumor cell/matrix ratio and longer overall survival in OS. PMID:26988273

  20. Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes.

    PubMed

    Park, Sun-Ji; Choe, Young-Geun; Kim, Jung-Hak; Chang, Kyu-Tae; Lee, Hyun-Shik; Lee, Dong-Seok

    2016-07-01

    Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells. PMID:27117918

  1. Geranylated 2-arylbenzofurans from Morus alba var. tatarica and their α-glucosidase and protein tyrosine phosphatase 1B inhibitory activities.

    PubMed

    Zhang, Ya-Long; Luo, Jian-Guang; Wan, Chuan-Xing; Zhou, Zhong-Bo; Kong, Ling-Yi

    2014-01-01

    Ten new geranylated 2-arylbenzofuran derivatives, including two monoterpenoid 2-arylbenzofurans (1 and 2), two geranylated 2-arylbenzofuran enantiomers (3a and 3b), and six geranylated 2-arylbenzofurans (4-9), along with four known 2-arylbenzofurans (10-13) were isolated from the root bark of Morus alba var. tatarica. Their structures and relative configurations were established on the basis of spectroscopic data analysis. Compounds 3-7 with one asymmetric carbon at C-7″ were supposed to be enantiomeric mixtures confirmed by chiral HPLC analysis, and the absolute configurations of each enantiomer in 3-7 were determined by Rh2(OCOCF3)4-induced CD and Snatzke's method. The enantiomers with the substituting group at C-2' exhibited better resolutions on a Chiralpak AD-H column than those with the substituting group at C-4'. Compounds 1-7, 10, 11 and 13, showed α-glucosidase inhibitory activities with IC50 values of 11.9-131.9 μM, and compounds 1 and 9-13 inhibited protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 7.9-38.1 μM. PMID:24216050

  2. Chimeric design, synthesis, and biological assays of a new nonpeptide insulin-mimetic vanadium compound to inhibit protein tyrosine phosphatase 1B

    PubMed Central

    Scior, Thomas; Guevara-García, José Antonio; Melendez, FJ; Abdallah, Hassan H; Do, Quoc-Tuan; Bernard, Philippe

    2010-01-01

    Prior to its total synthesis, a new vanadium coordination compound, called TSAG0101, was computationally designed to inhibit the enzyme protein tyrosine phosphatase 1B (PTP1B). The PTP1B acts as a negative regulator of insulin signaling by blocking the active site where phosphate hydrolysis of the insulin receptor takes place. TSAG001, [VVO2(OH)(picolinamide)], was characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy; IR: ν/cm−1 3,570 (NH), 1,627 (C=O, coordinated), 1,417 (C–N), 970/842 (O=V=O), 727 δ̣ (pyridine ring); 13C NMR: 5 bands between 122 and 151 ppm and carbonyl C shifted to 180 ppm; and 1H NMR: 4 broad bands from 7.6 to 8.2 ppm and NH2 shifted to 8.8 ppm. In aqueous solution, in presence or absence of sodium citrate as a biologically relevant and ubiquitous chelator, TSAG0101 undergoes neither ligand exchange nor reduction of its central vanadium atom during 24 hours. TSAG0101 shows blood glucose lowering effects in rats but it produced no alteration of basal- or glucose-induced insulin secretion on β cells during in vitro tests, all of which excludes a direct mechanism evidencing the extrapancreatic nature of its activity. The lethal dose (LD50) of TSAG0101 was determined in Wistar mice yielding a value of 412 mg/kg. This value is one of the highest among vanadium compounds and classifies it as a mild toxicity agent when compared with literature data. Due to its nonsubstituted, small-sized scaffold design, its remarkable complex stability, and low toxicity; TSAG0101 should be considered as an innovative insulin-mimetic principle with promising properties and, therefore, could become a new lead compound for potential nonpeptide PTP1B inhibitors in antidiabetic drug research. In view of the present work, the inhibitory concentration (IC50) and extended solution stability will be tested. PMID:20957214

  3. Matrix metalloproteinase-mediation of tumor targeting human recombinant tumor necrosis factor-α fusion protein.

    PubMed

    Ren, Hui; Shao, Xin; Zeng, Liang; Wang, Fa; Huang, Di-Nan; Hou, Gan

    2015-08-01

    The aim of the present study was to use genetic engineering in order to establish an efficient tumor necrosis factor (TNF)-α fusion protein with low toxicity, which may be used to target tumors. Four types of matrix metalloproteinase (MMP)-mediated tumor targeting human recombinant TNF-α (rhTNF-α) fusion protein vectors were constructed. These were subsequently introduced into Escherichia coli. rhTNF-α fusion protein with a glutathione S-transferase (GST)-tag was purified using GST resin affinity chromatography, and GST-tags were digested using factor Xa. The cytotoxic effects of the fusion protein on L929 cells were determined using MTT assays. At a concentration of 1 pM, the GST-tagged fusion protein exerted no cytotoxic effects on the cells, compared with the negative control cells (P=0.975>0.05). However, at a concentration of 1000 pM, the deblocking fusion protein exerted greater cytotoxic effects on L929 cells, compared with positive control cells (P<0.05). Treatment with the fusion protein also induced cell apoptosis in the nasopharyngeal cancer cell line, CNE-2Z, which secretes high levels of MMP-1. In conclusion, the results of the present study suggested that MMP-mediated rhTNF-α fusion protein induces CNE-2Z cells apoptosis. rhTNF-α exhibits high efficacy and tumor cell targeting capability, with low toxicity effects on healthy cells. PMID:25891416

  4. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption. PMID:26561828

  5. Oncolytic Replication of E1b-Deleted Adenoviruses.

    PubMed

    Cheng, Pei-Hsin; Wechman, Stephen L; McMasters, Kelly M; Zhou, Heshan Sam

    2015-11-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viralmRNAexport, and cell cycle disruption. PMID:26561828

  6. RUNX1 Permits E4orf6-Directed Nuclear Localization of the Adenovirus E1B-55K Protein and Associates with Centers of Viral DNA and RNA Synthesis▿

    PubMed Central

    Marshall, Leslie J.; Moore, Amy C.; Ohki, Misao; Kitabayashi, Issay; Patterson, David; Ornelles, David A.

    2008-01-01

    The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-ΔN variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirus-infected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1. PMID:18417565

  7. Pathway focused protein profiling indicates differential function for IL-1B, -18 and VEGF during initiation and resolution of lung inflammation evoked by carbon nanoparticle exposure in mice

    PubMed Central

    2009-01-01

    Background Carbonaceous nanoparticles possess an emerging source of human exposure due to the massive release of combustion products and the ongoing revolution in nanotechnology. Pulmonary inflammation caused by deposited nanoparticles is central for their adverse health effects. Epidemiological studies suggest that individuals with favourable lung physiology are at lower risk for particulate matter associated respiratory diseases probably due to efficient control of inflammation and repair process. Therefore we selected a mouse strain C3H/HeJ (C3) with robust lung physiology and exposed it to moderately toxic carbon nanoparticles (CNP) to study the elicited pulmonary inflammation and its resolution. Methods 5 μg, 20 μg and 50 μg CNP were intratracheally (i.t.) instilled in C3 mice to identify the optimal dose for subsequent time course studies. Pulmonary inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers. Results 1 day after instillation of CNP, C3 mice exhibited a typical dose response, with the lowest dose (5 μg) representing the 'no effect level' as reflected by polymorphonuclear leucocyte (PMN), and BAL/lung concentrations of pro-inflammatory proteins. Histological analysis and BAL-protein concentration did not reveal any evidence of tissue injury in 20 μg CNP instilled animals. Accordingly time course assessment of the inflammatory response was performed after 3 and 7 days with this dose (20 μg). Compared to day 1, BAL PMN counts were significantly decreased at day 3 and completely returned to normal by day 7. We have identified protein markers related to the acute response and also to the time dependent response in lung and BAL. After complete resolution of PMN influx on day 7, we detected elevated concentrations of 20 markers that included IL1B, IL18, FGF2, EDN1, and VEGF in lung and/or BAL. Biological pathway analysis revealed these factors to be involved in a closely regulated

  8. Proteins and an Inflammatory Network Expressed in Colon Tumors

    PubMed Central

    Zhu, Wenhong; Fang, Changming; Gramatikoff, Kosi; Niemeyer, Christina C.; Smith, Jeffrey W.

    2011-01-01

    The adenomatous polyposis coli (APC) protein is crucial to homeostasis of normal intestinal epithelia because it suppresses the β-catenin/TCF pathway. Consequently, loss or mutation of the APC gene causes colorectal tumors in humans and mice. Here, we describe our use of Multidimensional Protein Identification Technology (MudPIT) to compare protein expression in colon tumors to that of adjacent healthy colon tissue from ApcMin/+ mice. Twenty-seven proteins were found to be up-regulated in colon tumors and twenty-five down-regulated. As an extension of the proteomic analysis, the differentially expressed proteins were used as “seeds” to search for co-expressed genes. This approach revealed a co-expression network of 45 genes that is up-regulated in colon tumors. Members of the network include the antibacterial peptide cathelicidin (CAMP), Toll-like receptors (TLRs), IL-8, and triggering receptor expressed on myeloid cells 1 (TREM1). The co-expression network is associated with innate immunity and inflammation, and there is significant concordance between its connectivity in humans versus mice (Friedman: p value = 0.0056). This study provides new insights into the proteins and networks that are likely to drive the onset and progression of colon cancer. PMID:21366352

  9. High-throughput screen for inhibitors of transglycosylase and/or transpeptidase activities of Escherichia coli penicillin binding protein 1b.

    PubMed

    Chandrakala, B; Shandil, Radha K; Mehra, Upasana; Ravishankar, Sudha; Kaur, Parvinder; Usha, Veeraraghavan; Joe, Bina; deSousa, Sunita M

    2004-01-01

    Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC(50)s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other beta-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC(50)s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors. PMID:14693515

  10. Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

    PubMed

    Lyu, Lei; Whitcomb, Elizabeth A; Jiang, Shuhong; Chang, Min-Lee; Gu, Yumei; Duncan, Melinda K; Cvekl, Ales; Wang, Wei-Lin; Limi, Saima; Reneker, Lixing W; Shang, Fu; Du, Linfang; Taylor, Allen

    2016-03-01

    Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei. PMID:26590164

  11. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    SciTech Connect

    Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  12. The Structural Characterization of Tumor Fusion Genes and Proteins.

    PubMed

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  13. The Structural Characterization of Tumor Fusion Genes and Proteins

    PubMed Central

    Wang, Dandan; Li, Daixi; Qin, Guangrong; Zhang, Wen; Ouyang, Jian; Zhang, Menghuan; Xie, Lu

    2015-01-01

    Chromosomal translocation, which generates fusion proteins in blood tumor or solid tumor, is considered as one of the major causes leading to cancer. Recent studies suggested that the disordered fragments in a fusion protein might contribute to its carcinogenicity. Here, we investigated the sequence feature near the breakpoints in the fusion partner genes, the structure features of breakpoints in fusion proteins, and the posttranslational modification preference in the fusion proteins. Results show that the breakpoints in the fusion partner genes have both sequence preference and structural preference. At the sequence level, nucleotide combination AG is preferred before the breakpoint and GG is preferred at the breakpoint. At the structural level, the breakpoints in the fusion proteins prefer to be located in the disordered regions. Further analysis suggests the phosphorylation sites at serine, threonine, and the methylation sites at arginine are enriched in disordered regions of the fusion proteins. Using EML4-ALK as an example, we further explained how the fusion protein leads to the protein disorder and contributes to its carcinogenicity. The sequence and structural features of the fusion proteins may help the scientific community to predict novel breakpoints in fusion genes and better understand the structure and function of fusion proteins. PMID:26347798

  14. Tumor promotion by caspase-resistant retinoblastoma protein

    PubMed Central

    Borges, Helena L.; Bird, Jeff; Wasson, Katherine; Cardiff, Robert D.; Varki, Nissi; Eckmann, Lars; Wang, Jean Y. J.

    2005-01-01

    The retinoblastoma (RB) protein regulates cell proliferation and cell death. RB is cleaved by caspase during apoptosis. A mutation of the caspase-cleavage site in the RB C terminus has been made in the mouse Rb-1 locus; the resulting Rb-MI mice are resistant to endotoxin-induced apoptosis in the intestine. The Rb-MI mice do not exhibit increased tumor incidence, because the MI mutation does not disrupt the Rb tumor suppressor function. In this study, we show that Rb-MI can promote the formation of colonic adenomas in the p53-null genetic background. Consistent with this tumor phenotype, Rb-MI reduces colorectal epithelial apoptosis and ulceration caused by dextran sulfate sodium. By contrast, Rb-MI does not affect the lymphoma phenotype of p53-null mice, in keeping with its inability to protect thymocytes and splenocytes from apoptosis. The Rb-MI protein is expressed and phosphorylated in the tumors, thereby inactivating its growth suppression function. These results suggest that RB tumor suppressor function, i.e., inhibition of proliferation, is inactivated by phosphorylation, whereas RB tumor promoting function, i.e., inhibition of apoptosis, is inactivated by caspase cleavage. PMID:16227443

  15. Translationally Controlled Tumor Protein in Prostatic Adenocarcinoma: Correlation with Tumor Grading and Treatment-Related Changes

    PubMed Central

    Rocca, Bruno Jim; Ginori, Alessandro; Barone, Aurora; Calandra, Calogera; Crivelli, Filippo; De Falco, Giulia; Gazaneo, Sara; Tripodi, Sergio; Cevenini, Gabriele; del Vecchio, Maria Teresa; Ambrosio, Maria Raffaella; Tosi, Piero

    2015-01-01

    Prostate cancer is the second leading cause of cancer-related death. The androgen deprivation therapy is the standard treatment for advanced stages. Unfortunately, virtually all tumors become resistant to androgen withdrawal. The progression to castration-resistance is not fully understood, although a recent paper has suggested translationally controlled tumor protein to be implicated in the process. The present study was designed to investigate the role of this protein in prostate cancer, focusing on the correlation between its expression level with tumor differentiation and response to treatment. We retrieved 292 prostatic cancer specimens; of these 153 had been treated only by radical prostatectomy and 139 had undergone radical prostatectomy after neoadjuvant treatment with combined androgen blockade therapy. Non-neoplastic controls were represented by 102 prostatic peripheral zone specimens. In untreated patients, the expression of the protein, evaluated by RT-qPCR and immunohistochemistry, was significantly higher in tumor specimens than in non-neoplastic control, increasing as Gleason pattern and score progressed. In treated prostates, the staining was correlated with the response to treatment. An association between protein expression and the main clinicopathological factors involved in prostate cancer aggressiveness was identified. These findings suggest that the protein may be a promising prognostic factor and a target for therapy. PMID:25667934

  16. Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells

    PubMed Central

    Tsioras, Konstantinos; Papastefanaki, Florentia; Politis, Panagiotis K.; Matsas, Rebecca; Gaitanou, Maria

    2013-01-01

    BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2'-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors. PMID:24312406

  17. Neurospecific proteins in the serum of patients with brain tumors.

    PubMed

    Lyubimova, N V; Toms, M G; Popova, E E; Bondarenko, Y V; Krat, V B; Kushlinskii, N E

    2011-04-01

    Neurospecific proteins S-100 and GFAP were measured in the serum of 145 patients with neural tumors and 69 healthy individuals. In patients with glyoblastomas, the concentrations of S-100 and GFAP were significantly higher than in patients with anaplastic astrocytomas, benign meningiomas, and brain metastases and in healthy individuals. Serum S-100 concentrations in patients with anaplastic astrocytomas, benign meningiomas, and brain metastases were similar; significant difference from the control was found only for patients with cerebral metastases. A specific feature of GFAP was high incidence of its detection in patients with glioblastomas (83%) compared to other groups of patients with neural tumors and healthy volunteers who demonstrated practically zero level of this protein. These findings attest to the possibility of using S-100 as an additional biochemical criterion of brain involvement in tumor patients and GFAP as a glioblastoma marker. PMID:22235430

  18. Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Choi, Jeong Uk; Kim, Seong Who; Kim, Sang Yoon; Ahsan, Fakhrul; Kim, In-San

    2016-01-01

    Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis. PMID:26950422

  19. Overexpression of Membrane Proteins in Primary and Metastatic Gastrointestinal Neuroendocrine Tumors

    PubMed Central

    Wang, Donghong; Dahdaleh, Fadi S.; Bellizzi, Andrew M.; O’Dorisio, M. Sue; O’Dorisio, Thomas M.; Howe, James R.

    2014-01-01

    Background Small bowel and pancreatic neuroendocrine tumors (SBNETs and PNETs) are rare tumors whose incidence is increasing. Drugs targeting the somatostatin receptor are beneficial in these tumors. To identify additional cell-surface targets, we recently found receptors and membrane proteins with gene expression significantly different from adjacent normal tissues in a small number of primary SBNETs and PNETs. We set out to validate these expression differences in a large group of primary neuroendocrine tumors and to determine whether they are present in corresponding liver and lymph node metastases. Methods Primary SBNETs and PNETs, normal tissue, nodal, and liver metastases were collected and mRNA expression of six target genes was determined by quantitative PCR. Expression was normalized to GAPDH and POLR2A internal controls, and differences as compared to normal tissue were assessed by Welch’s t test. Results Gene expression was determined in 45 primary PNETs with 20 nodal and 17 liver metastases, and 51 SBNETs with 50 nodal and 29 liver metastases. Compared to normal tissue, the oxytocin receptor (OXTR) showed significant overexpression in both primary and metastatic SBNETs and PNETs. Significant overexpression was observed for MUC13 and MEP1B in PNET primary tumors, and for GPR113 in primary SBNETs and their metastases. SCTR and ADORA1 were significantly underexpressed in PNETs and their metastases. OXTR protein expression was confirmed by immunohistochemistry. Conclusions OXTR is significantly overexpressed relative to normal tissue in primary SBNETs and PNETs, and this overexpression is present in their liver and lymph node metastases, making OXTR a promising target for imaging and therapeutic interventions. PMID:24114056

  20. Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea.

    PubMed

    Liu, Linxia; Zhang, Jun; Chen, Chuan; Teng, Jitao; Wang, Chengshu; Luo, Duqiang

    2015-08-01

    Fumosorinone, isolated from the entomogenous fungus Isaria fumosorosea, is a new 2-pyridone alkaloid which is elucidated by HRESIMS 1D and 2DNMR. Fumosorinone is structurally similar to tenellin and desmethylbassianin but it differs in chain length and degree of methylation. It is characterized by a classic noncompetitive inhibitor of protein tyrosine phosphatase 1B (IC50 14.04μM) which was implicated as a negative regulator of insulin receptor signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. For further study, we identified the biosynthetic gene cluster of fumosorinone from ongoing genome sequencing project, and it was verified by a direct knock-out strategy, reported for the first time in I. fumosorosea, using the Agrobacterium-mediated transformation in conjunction with linear deletion cassettes. The biosynthetic gene cluster includes a hybrid polyketide synthase-nonribosomal peptide synthetase gene, two cytochrome P450 enzyme genes, a trans-enoyl reductase gene, and other two transcription regulatory genes. Comparison of fumosorinone biosynthetic cluster with known gene clusters gives further insight into biosynthesis of pyridone alkaloids and provides the foundation for combinatorial biosynthesis for new fumosorinone derivatives. PMID:25857260

  1. miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs

    PubMed Central

    Xu, Mao-Chun; Gao, Xiu-Fang; Ruan, Changwu; Ge, Zhi-Ru; Lu, Ji-De; Zhang, Jian-Jun; Zhang, Yu; Wang, Lu; Shi, Hai-Ming

    2015-01-01

    Oxidative stress plays a critical role in cardiovascular diseases. Salidroside, a glycoside from Rhodiola rosea, has been used as an antioxidative therapy for oxidative injury in cardiac diseases. However, the mechanism underlying its antioxidant effect needs to be elucidated. Treatment of HUVECs with H2O2 significantly decreased the expression of miR-103 in a dose- and time-dependent manner, whereas pretreatment with salidroside significantly inhibited this decrease. Subsequent analysis showed that overexpression of miR-103 abrogated cell activity and ROS production induced by H2O2. Bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) was determined to be a novel miR-103 target in HUVECs. Interestingly, H2O2 treatment upregulated BNIP3 expression; in turn, this effect was inhibited by pretreatment with salidroside. Further studies confirmed that the knockdown of BNIP3 enhanced cell activity and suppressed the ROS production induced by H2O2. These results demonstrated for the first time that salidroside protects HUVECs in part by upregulating the expression of miR-103, which mediates BNIP3 downregulation and plays an important role in the cytoprotective actions. PMID:26000071

  2. Potent and tumor specific: arming bacteria with therapeutic proteins

    PubMed Central

    Van Dessel, Nele; Swofford, Charles A; Forbes, Neil S

    2015-01-01

    Bacteria are perfect vessels for targeted cancer therapy. Conventional chemotherapy is limited by passive diffusion, and systemic administration causes severe side effects. Bacteria can overcome these obstacles by delivering therapeutic proteins specifically to tumors. Bacteria have been modified to produce proteins that directly kill cells, induce apoptosis via signaling pathways, and stimulate the immune system. These three modes of bacterial treatment have all been shown to reduce tumor growth in animal models. Bacteria have also been designed to convert nontoxic prodrugs to active therapeutic compounds. The ease of genetic manipulation enables creation of arrays of bacteria that release many new protein drugs. This versatility will allow targeting of multiple cancer pathways and will establish a platform for individualized cancer medicine. PMID:25853312

  3. Opposing oncogenic activities of small DNA tumor virus transforming proteins

    PubMed Central

    Chinnadurai, G.

    2011-01-01

    The E1A gene of species C human adenovirus is an intensely investigated model viral oncogene that immortalizes primary cells and mediates oncogenic cell transformation in cooperation with other viral or cellular oncogenes. Investigations using E1A proteins have illuminated important paradigms in cell proliferation and the functions of cellular proteins such as the retinoblastoma protein. Studies with E1A have led to the surprising discovery that E1A also suppresses cell transformation and oncogenesis. Here, I review our current understanding of the transforming and tumor suppressive functions of E1A, and how E1A studies led to the discovery of a related tumor suppressive function in benign human papillomaviruses. The potential role of these opposing functions in viral replication in epithelial cells is also discussed. PMID:21330137

  4. PLU-1/JARID1B overexpression predicts proliferation properties in head and neck squamous cell carcinoma.

    PubMed

    Cui, Zhibin; Song, Liwei; Hou, Zhaoyuan; Han, Yifan; Hu, Yuhua; Wu, Yadi; Chen, Wantao; Mao, Li

    2015-05-01

    PLU-1/JARID1B (jumonji AT rich interactive domain 1B) is one of the testis cancer antigens and functions as a histone demethylase in the regulation of various human types of cancers. However, its functions in head and neck squamous cell carcinoma (HNSCC) are rarely reported. The aim of the present study was to examine PLU-1/JARID1B expression levels in HNSCCs and to investigate its role in cancer cell proliferation. In the present study, we found that PLU-1/JARID1B mRNA was upregulated in all tested HNSCC cell lines. Immunohistochemical staining showed that PLU-1/JARID1B protein expression was detected in 87.8% (87/99) of the HNSCC cases. A positive association was observed between high PLU-1/JARID1B expression and higher Ki-67 labeling in the HNSCC samples (Pearson r=0.6514, P=0.0003). Stable PLU-1/JARID1B knockdown by PLU-1-shRNAs in the HNSCC cell lines suppressed cell growth both in the in vitro and in vivo studies. Moreover, PLU-1/JARID1B knockdown resulted in G1 arrest and early apoptosis by suppressing Bcl-2 family members in the HNSCCs. These data indicate that PLU-1/JARID1B is overexpressed in HNSCCs and is associated with tumor proliferation and apoptosis. Therefore, PLU-1/JARID1B represents a candidate proliferation biomarker for HNSCC diagnosis and treatment. PMID:25777981

  5. The effect of different dosing regimens of motesanib on the gallbladder: a randomized phase 1b study in patients with advanced solid tumors

    PubMed Central

    2013-01-01

    Background Gallbladder toxicity, including cholecystitis, has been reported with motesanib, an orally administered small-molecule antagonist of VEGFRs 1, 2 and 3; PDGFR; and Kit. We assessed effects of motesanib on gallbladder size and function. Methods Patients with advanced metastatic solid tumors ineligible for or progressing on standard-of-care therapies with no history of cholecystitis or biliary disease were randomized 2:1:1 to receive motesanib 125 mg once daily (Arm A); 75 mg twice daily (BID), 14-days-on/7-days-off (Arm B); or 75 mg BID, 5-days-on/2-days-off (Arm C). Primary endpoints were mean change from baseline in gallbladder size (volume by ultrasound; independent review) and function (ejection fraction by CCK-HIDA; investigator assessment). Results Forty-nine patients received ≥1 dose of motesanib (Arms A/B/C, n = 25/12/12). Across all patients, gallbladder volume increased by a mean 22.2 cc (from 38.6 cc at baseline) and ejection fraction decreased by a mean 19.2% (from 61.3% at baseline) during treatment. Changes were similar across arms and appeared reversible after treatment discontinuation. Three patients had cholecystitis (grades 1, 2, 3, n = 1 each) that resolved after treatment discontinuation, one patient developed grade 3 acute cholecystitis requiring cholecystectomy, and two patients had other notable grade 1 gallbladder disorders (gallbladder wall thickening, gallbladder dysfunction) (all in Arm A). Two patients developed de novo gallstones during treatment. Twelve patients had right upper quadrant pain (Arms A/B/C, n = 8/1/3). The incidence of biliary “sludge” in Arms A/B/C was 39%/36%/27%. Conclusions Motesanib treatment was associated with increased gallbladder volume, decreased ejection fraction, biliary sludge, gallstone formation, and infrequent cholecystitis. Trial registration ClinicalTrials.gov NCT00448786 PMID:23679351

  6. Role of ribosomal protein mutations in tumor development (Review).

    PubMed

    Goudarzi, Kaveh M; Lindström, Mikael S

    2016-04-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  7. Role of ribosomal protein mutations in tumor development (Review)

    PubMed Central

    GOUDARZI, KAVEH M.; LINDSTRÖM, MIKAEL S.

    2016-01-01

    Ribosomes are cellular machines essential for protein synthesis. The biogenesis of ribosomes is a highly complex and energy consuming process that initiates in the nucleolus. Recently, a series of studies applying whole-exome or whole-genome sequencing techniques have led to the discovery of ribosomal protein gene mutations in different cancer types. Mutations in ribosomal protein genes have for example been found in endometrial cancer (RPL22), T-cell acute lymphoblastic leukemia (RPL10, RPL5 and RPL11), chronic lymphocytic leukemia (RPS15), colorectal cancer (RPS20), and glioma (RPL5). Moreover, patients suffering from Diamond-Blackfan anemia, a bone marrow failure syndrome caused by mutant ribosomal proteins are also at higher risk for developing leukemia, or solid tumors. Different experimental models indicate potential mechanisms whereby ribosomal proteins may initiate cancer development. In particular, deregulation of the p53 tumor suppressor network and altered mRNA translation are mechanisms likely to be involved. We envisage that changes in expression and the occurrence of ribosomal protein gene mutations play important roles in cancer development. Ribosome biology constitutes a re-emerging vital area of basic and translational cancer research. PMID:26892688

  8. The Bladder Tumor Suppressor Protein TERE1 (UBIAD1)Modulates Cell Cholesterol: Implications for Tumor Progression

    PubMed Central

    McGarvey, Terry; Wang, Huiyi; Lal, Priti; Puthiyaveettil, Raghunath; Tomaszewski, John; Sepulveda, Jorge; Labelle, Ed; Weiss, Jayne S.; Nickerson, Michael L.; Kruth, Howard S.; Brandt, Wolfgang; Wessjohann, Ludger A.; Malkowicz, S. Bruce

    2011-01-01

    Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression. PMID:21740188

  9. Overexpression of BUB1B contributes to progression of prostate cancer and predicts poor outcome in patients with prostate cancer

    PubMed Central

    Fu, Xin; Chen, Guo; Cai, Zhi-duan; Wang, Cong; Liu, Ze-zhen; Lin, Zhuo-yuan; Wu, Yong-ding; Liang, Yu-xiang; Han, Zhao-dong; Liu, Jun-chen; Zhong, Wei-De

    2016-01-01

    BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) is a member of the spindle assembly checkpoint protein family, which has been proven to be associated with many kinds of cancers. The aim of this study was to investigate whether BUB1B was correlated with progression and prognosis in patients with prostate cancer (PCa) and how BUB1B regulated the proliferation, migration, and invasion of PCa cell lines. Compared to benign prostate cells and tissues, both messenger RNA and protein expressions of BUB1B were statistically increased in PCa cell lines and tumor tissues. In vitro studies revealed that BUB1B overexpression enhanced the proliferation, migration, and invasion ability of PCa cell lines, whereas depletion of BUB1B did not affect the cell functions. Microarray analysis showed the positive staining of BUB1B was upregulated in the higher Gleason score group, which also correlated with advanced clinicopathological stage, higher serum prostate-specific antigen, metastasis, overall survival, and prostate-specific antigen failure. Furthermore, the survival analysis indicated that high expression of BUB1B was an independent predictor for shorter biochemical recurrence-free survival, which had no effect on overall survival. BUB1B plays an important role in tumor growth and progression, which can lead to its use as a potential biomarker for the diagnosis and prognosis of PCa. PMID:27143916

  10. Rice phytochrome-interacting factor protein OsPIFff14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a Yeast one Hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bi...

  11. Impact of Protein Stability, Cellular Localization, and Abundance on Proteomic Detection of Tumor-Derived Proteins in Plasma

    PubMed Central

    Faca, Vitor M.; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B.; McIntosh, Martin W.; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  12. Impact of protein stability, cellular localization, and abundance on proteomic detection of tumor-derived proteins in plasma.

    PubMed

    Fang, Qiaojun; Kani, Kian; Faca, Vitor M; Zhang, Wenxuan; Zhang, Qing; Jain, Anjali; Hanash, Sam; Agus, David B; McIntosh, Martin W; Mallick, Parag

    2011-01-01

    Tumor-derived, circulating proteins are potentially useful as biomarkers for detection of cancer, for monitoring of disease progression, regression and recurrence, and for assessment of therapeutic response. Here we interrogated how a protein's stability, cellular localization, and abundance affect its observability in blood by mass-spectrometry-based proteomics techniques. We performed proteomic profiling on tumors and plasma from two different xenograft mouse models. A statistical analysis of this data revealed protein properties indicative of the detection level in plasma. Though 20% of the proteins identified in plasma were tumor-derived, only 5% of the proteins observed in the tumor tissue were found in plasma. Both intracellular and extracellular tumor proteins were observed in plasma; however, after normalizing for tumor abundance, extracellular proteins were seven times more likely to be detected. Although proteins that were more abundant in the tumor were also more likely to be observed in plasma, the relationship was nonlinear: Doubling the spectral count increased detection rate by only 50%. Many secreted proteins, even those with relatively low spectral count, were observed in plasma, but few low abundance intracellular proteins were observed. Proteins predicted to be stable by dipeptide composition were significantly more likely to be identified in plasma than less stable proteins. The number of tryptic peptides in a protein was not significantly related to the chance of a protein being observed in plasma. Quantitative comparison of large versus small tumors revealed that the abundance of proteins in plasma as measured by spectral count was associated with the tumor size, but the relationship was not one-to-one; a 3-fold decrease in tumor size resulted in a 16-fold decrease in protein abundance in plasma. This study provides quantitative support for a tumor-derived marker prioritization strategy that favors secreted and stable proteins over all but the

  13. Endosomal localization of phospholipase D 1a and 1b is defined by the C-termini of the proteins, and is independent of activity.

    PubMed

    Hughes, W E; Parker, P J

    2001-06-15

    The factors regulating the activity of cellular phospholipase D (PLD) have been well characterized; however, the cellular distribution of specific PLD isoforms and the factors defining localization are less clear. Two specific PLD1 isoforms, PLD1a and PLD1b, are shown in the present study to be localized in endosomal compartments with early endosomal autoantigen 1, internalizing epidermal growth factor receptor (ErbB1) and lysobisphosphatidic acid. Novel C-terminal splice variants of PLD1, PLD1a2 and PLD1b2, do not exhibit this endosomal localization. Studies using catalytically inactive and C-terminal deletion mutants of the four PLD1 isoforms led to the conclusion that the C-terminus plays an important part in the catalytic activity of PLD1, but that the endosomal localization of PLD1a and PLD1b is defined by the C-terminus and not catalytic activity. PMID:11389680

  14. Design, synthesis and docking studies on phenoxy-3-piperazin-1-yl-propan-2-ol derivatives as protein tyrosine phosphatase 1B inhibitors.

    PubMed

    Gupta, Swati; Pandey, Gyanendra; Rahuja, Neha; Srivastava, Arvind K; Saxena, Anil K

    2010-10-01

    A series of substituted phenoxy-3-piperazin-1-yl-propan-2-ols has been synthesized and evaluated for PTP1B inhibitory activity in vitro and for antidiabetic activity in vivo. Two molecules viz. 4a and 5b showed PTP1B inhibition of 31.58% and 35.90% at 100 μM concentration. The compound 4a also showed 40.3% normalization of plasma glucose levels at 100mg/kg in Sugar-loaded model (SLM) and 32% activity in Streptozodocin model (STZ). The docking studies of these molecules revealed that hydrogen bond formation with Arg221 is important for activity. PMID:20797859

  15. Triton 2 (1B)

    NASA Technical Reports Server (NTRS)

    Clark, Michelle L.; Meiss, A. G.; Neher, Jason R.; Rudolph, Richard H.

    1994-01-01

    The goal of this project was to perform a detailed design analysis on a conceptually designed preliminary flight trainer. The Triton 2 (1B) must meet the current regulations in FAR Part 23. The detailed design process included the tasks of sizing load carrying members, pulleys, bolts, rivets, and fuselage skin for the safety cage, empennage, and control systems. In addition to the regulations in FAR Part 23, the detail design had to meet established minimums for environmental operating conditions and material corrosion resistance.

  16. Survivin and related proteins in canine mammary tumors: immunohistochemical expression.

    PubMed

    Bongiovanni, L; Romanucci, M; Malatesta, D; D'Andrea, A; Ciccarelli, A; Della Salda, L

    2015-03-01

    Survivin is reexpressed in most human breast cancers, where its expression has been associated with tumor aggressiveness, poor prognosis, and poor response to therapy. Survivin expression was evaluated in 41 malignant canine mammary tumors (CMTs) by immunohistochemistry, in relation to histological grade and stage, and correlated with that of some related molecules (β-catenin, caspase 3, heat shock proteins) to understand their possible role in canine mammary tumorigenesis. An increase in nuclear survivin expression, compared with healthy mammary glands, was observed in CMTs, where nuclear immunolabeling was related to the presence of necrosis. No statistically significant relation was found between the expression of the investigated molecules and the histological grade or stage. The present study may suggest an important involvement of survivin in CMT tumorigenesis. Its overexpression in most of the cases evaluated might suggest that targeting survivin in CMTs may be a valid anticancer therapy. PMID:24686389

  17. RNA binding by the Wilms tumor suppressor zinc finger proteins.

    PubMed Central

    Caricasole, A; Duarte, A; Larsson, S H; Hastie, N D; Little, M; Holmes, G; Todorov, I; Ward, A

    1996-01-01

    The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms. Images Fig. 1 Fig. 2 Fig. 3 PMID:8755514

  18. LincRNA-p21 acts as a mediator of ING1b-induced apoptosis

    PubMed Central

    Tran, U M; Rajarajacholan, U; Soh, J; Kim, T-s; Thalappilly, S; Sensen, C W; Riabowol, K

    2015-01-01

    ING1b is a tumor suppressor that affects transcription, cell cycle control and apoptosis. ING1b is deregulated in disease, and its activity is closely linked to that of p53. In addition to regulating protein-coding genes, we found that ING1b also influences the expression of large intergenic non-coding RNAs (lincRNAs). In particular, lincRNA-p21 was significantly induced after DNA-damage stress or by ING1b overexpression. Furthermore, lincRNA-p21 expression in response to DNA damage was significantly attenuated in cells lacking ING1b. LincRNA-p21 is also a target of p53 and can trigger apoptosis in mouse cell models. We found that this function of lincRNA-p21 is conserved in human cell models. Moreover, ING1b and p53 could function independently to influence lincRNA-p21 expression. However, their effects become more additive under conditions of stress. In particular, ING1b regulates lincRNA-p21 levels by binding to its promoter and is required for induction of lincRNA-p21 by p53. The ability of ING1b to cause apoptosis is also impaired in the absence of lincRNA-p21. Surprisingly, deletion of the ING1b plant homeodomain, which allows it to bind histones and regulate chromatin structure, did not alter regulation of lincRNA-p21. Our findings suggest that ING1b induces lincRNA-p21 expression independently of histone 3 lysine 4 trimethylation mark recognition and that lincRNA-p21 functions downstream of ING1b. Thus, regulation at the level of lincRNA-p21 may represent the point at which ING1b and p53 pathways converge to induce apoptosis under specific stress conditions. PMID:25741593

  19. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of phytochrome B.

    PubMed

    Cordeiro, André M; Figueiredo, Duarte D; Tepperman, James; Borba, Ana Rita; Lourenço, Tiago; Abreu, Isabel A; Ouwerkerk, Pieter B F; Quail, Peter H; Margarida Oliveira, M; Saibo, Nelson J M

    2016-02-01

    DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses. PMID:26732823

  20. Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of Phytochrome B

    PubMed Central

    Cordeiro, André M.; Figueiredo, Duarte D.; Tepperman, James; Borba, Ana Rita; Lourenço, Tiago; Abreu, Isabel A.; Ouwerkerk, Pieter B.F.; Quail, Peter H.; Oliveira, M. Margarida; Saibo, Nelson J. M.

    2016-01-01

    DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a Yeast one Hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a Phytochrome Interacting Factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses. PMID:26732823

  1. IGF-Binding Protein 2 – Oncogene or Tumor Suppressor?

    PubMed Central

    Pickard, Adam; McCance, Dennis J.

    2015-01-01

    The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner. In this mini-review, we discuss the factors influencing the variation in IGFBP2 expression in cancer and our interpretation of these findings. PMID:25774149

  2. High Brain Ammonia Tolerance and Down-Regulation of Na+:K+:2Cl- Cotransporter 1b mRNA and Protein Expression in the Brain of the Swamp Eel, Monopterus albus, Exposed to Environmental Ammonia or Terrestrial Conditions

    PubMed Central

    Ip, Yuen K.; Hou, Zhisheng; Chen, Xiu L.; Ong, Jasmine L. Y.; Chng, You R.; Ching, Biyun; Hiong, Kum C.; Chew, Shit F.

    2013-01-01

    Na+:K+:2Cl- cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g-1 and 2.7 µmol g-1 after 6 days of exposure to 50 mmol l-1 NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance. PMID:24069137

  3. Nucleotide sequences of genes encoding penicillin-binding proteins from Streptococcus pneumoniae and Streptococcus oralis with high homology to Escherichia coli penicillin-binding proteins 1a and 1b.

    PubMed Central

    Martin, C; Briese, T; Hakenbeck, R

    1992-01-01

    The nucleotide sequence of a 3,378-bp DNA fragment of Streptococcus pneumoniae that included the structural gene for penicillin-binding protein (PBP) 1a (ponA), which encodes 719 amino acids, was determined. Homologous DNA fragments from an S. oralis strain were amplified with ponA-specific oligonucleotides. The 2,524-bp S. oralis sequence contained the coding region for the first 636 amino acids of a PBP. The coding sequence differed by 437 nucleotides (27%) and one additional triplet, resulting in 87 amino acid substitutions (14%), from S. pneumoniae PBP 1a. Both PBPs are highly homologous to bifunctional high-M(r) Escherichia coli PBPs 1a and 1b. Images PMID:1624444

  4. Racemosin C, a novel minor bisindole alkaloid with protein tyrosine phosphatase-1B inhibitory activity from the green alga Caulerpa racemosa.

    PubMed

    Yang, Hui; Liu, Ding-Quan; Liang, Tong-Jun; Li, Jia; Liu, Ai-Hong; Yang, Peng; Lin, Kun; Yu, Xiao-Qing; Guo, Yue-Wei; Mao, Shui-Chun; Wang, Bin

    2014-12-01

    A novel minor bisindole alkaloid, racemosin C (1), characterized by a naturally unprecedented 8-hydroxy-2,4,6-cyclooctatrienone ring fused with two indole systems, was isolated from the green alga Caulerpa racemosa, together with one known related metabolite, caulersin (2). The structure of 1 was elucidated on the basis of extensive spectroscopic analysis, and by comparison with the data of related known compounds. A plausible biosynthetic pathway of 1 was proposed. Compounds 1 and 2 exhibited significant PTP1B inhibitory activity with IC50 values of 5.86 ± 0.57 and 7.14 ± 1.00 μM, respectively, compared with the positive control oleanolic acid (IC50 = 3.03 ± 0.20 μM). On the basis of the data obtained, the Caulerpa bisindole alkaloids may be considered as a new class of PTP1B inhibitors. PMID:25296343

  5. Ribosome-inactivating proteins: from plant defense to tumor attack.

    PubMed

    de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

    2010-11-01

    Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

  6. Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

    PubMed Central

    de Virgilio, Maddalena; Lombardi, Alessio; Caliandro, Rocco; Fabbrini, Maria Serena

    2010-01-01

    Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential. PMID:22069572

  7. CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation.

    PubMed

    Kwon, Yeo-Jung; Baek, Hyoung-Seok; Ye, Dong-Jin; Shin, Sangyun; Kim, Donghak; Chun, Young-Jin

    2016-01-01

    Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction. PMID:26981862

  8. CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation

    PubMed Central

    Kwon, Yeo-Jung; Baek, Hyoung-Seok; Ye, Dong-Jin; Shin, Sangyun; Kim, Donghak; Chun, Young-Jin

    2016-01-01

    Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction. PMID:26981862

  9. Expression of extracellular matrix proteins in adenomatoid odontogenic tumor.

    PubMed

    Modolo, Filipe; Biz, Michelle Tillmann; Martins, Marília Trierveiller; Machado de Sousa, Suzana Orsini; de Araújo, Ney Soares

    2010-03-01

    Altered expression of extracellular matrix (ECM) components has been reported in several pathologies; however, few ECM proteins have been evaluated in adenomatoid odontogenic tumor (AOT). The aim of this study was to analyze the expression and distribution of the ECM proteoglycans: biglycan and decorin; and glycoproteins: osteonectin, osteopontin, bone sialoprotein and osteocalcin in the AOT. Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method with the antibodies against the proteins previously cited. Only the osteonectin was expressed in the epithelial cells. The eosinophilic amorphous material and the connective tissue showed expression of all components studied. The calcification foci expressed only osteopontin. In conclusion, the low expression of the components studied in neoplastic epithelial cells suggests that the epithelial cells act probably as stimulators of the expression by the stroma, which in turn can act as agonist or antagonist of the tumor growth. These results suggest that the components studied probably have a key role in the biological behavior of the AOT. PMID:20070486

  10. Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tumor and stromal cells secrete a variety of proteins acting as extracellular signals and creating a supportive microenvironment for tumor development, angiogenesis, and metastasis. We used the Luminex immunoassay platform (including MILLIPLEX® MAP cytokine/chemokine, bone metabolism, adipocyte, M...

  11. Pro-apoptotic function of the retinoblastoma tumor suppressor protein

    PubMed Central

    Ianari, Alessandra; Natale, Tiziana; Calo, Eliezer; Ferretti, Elisabetta; Alesse, Edoardo; Screpanti, Isabella; Haigis, Kevin; Gulino, Alberto; Lees, Jacqueline A.

    2009-01-01

    SUMMARY The retinoblastoma protein (pRB) tumor suppressor blocks cell proliferation by repressing the E2F transcription factors. This inhibition is relieved through mitogen-induced phosphorylation of pRB, triggering E2F release and activation of cell cycle genes. E2F1 can also activate pro-apoptotic genes in response to genotoxic or oncogenic stress. However, pRB’s role in this context has not been established. Here we show that DNA damage and E1A-induced oncogenic stress promotes formation of a pRB-E2F1 complex even in proliferating cells. Moreover, pRB is bound to pro-apoptotic promoters that are transcriptional active and pRB is required for maximal apoptotic response in vitro and in vivo. Together, these data reveal a direct role for pRB in the induction of apoptosis in response to genotoxic or oncogenic stress. SIGNIFICANCE pRB function is disrupted in many human tumors through either inactivation of the Rb gene or alterations in its upstream regulators. pRB’s tumor suppressive activity is at least partially dependent upon its ability to arrest cells through E2F inhibition. Our data now establish a second role for pRB as a stress-induced activator of apoptosis. Notably, pRB’s ability to promote either arrest versus apoptosis seems to be context dependent, with apoptosis being favored in proliferating cells. This finding has the potential to explain why cells are typically more resistant to apoptosis when in the arrested state. Most importantly, our observations suggest that Rb status will influence tumor response to chemotherapy by impairing both the arrest and apoptotic checkpoint responses. PMID:19249677

  12. Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: Requirement for the E1b 58-Kilodalton protein and the products of E4 open reading frames 3 and 6

    SciTech Connect

    Panning, B.; Smiley, J.R. )

    1993-06-01

    Alu elements are the single most abundant class of dispersed repeated sequences in the human genome, comprising 5-10% of the mass of human DNA. This report demonstrates that Ad5 infection strongly stimulates Pol III transcription of human Alu elements in HeLa and 293 cells. In contrast to the cases of Ad5-induced Pol III transcriptional activation, this process requires the E1b 58-kDa protein and the products of E4 open reading frames (ORFs) 3 and 6 in addition to the E1a 289-residue product. These findings suggest novel regulatory properties of the Ad5 E1b and E4 proteins and raise the possibility that analogous cellular trans-acting factors serve to modulate Alu expression in vivo.

  13. An Endocrine-Exocrine Switch in the Activity of the Pancreatic Homeodomain Protein PDX1 through Formation of a Trimeric Complex with PBX1b and MRG1 (MEIS2)

    PubMed Central

    Swift, Galvin H.; Liu, Ying; Rose, Scott D.; Bischof, Larry J.; Steelman, Scott; Buchberg, Arthur M.; Wright, Christopher V. E.; MacDonald, Raymond J.

    1998-01-01

    HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in β-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, β-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the β-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The β-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells. PMID:9710595

  14. Hexabrachion proteins in embryonic chicken tissues and human tumors.

    PubMed

    Erickson, H P; Taylor, H C

    1987-09-01

    Cell cultures of chicken embryo and human fibroblasts produce a large extracellular matrix molecule with a six-armed structure that we called a hexabrachion (Erickson, H. P., and J. L. Iglesias, 1984, Nature (Lond.), 311:267-269. In the present work we have determined that the myotendinous (M1) antigen described by M. Chiquet and D. M. Fambrough in chicken tissues (1984, J. Cell Biol., 98:1926-1936), and the glioma mesenchymal extracellular matrix protein described by Bourdon et al. in human tumors (Bourdon, M. A., C. J. Wikstrand, H. Furthmayr, T. J. Matthews, and D. D. Bigner, 1983, Cancer Res. 43:2796-2805) have the structure of hexabrachions. We also demonstrate that the M1 antigen is present in embryonic brain, where it was previously reported absent, and have purified hexabrachions from brain homogenates. The recently described cytotactin (Grumet, M., S. Hoffman, K. L. Crossin, and G. M. Edelman, 1985, Proc. Natl. Acad. Sci. USA, 82:8075-8079) now appears to be identical to the chicken hexabrachion protein. In a search for functional roles, we looked for a possible cell attachment activity. A strong, fibronectin-like attachment activity was present in (NH4)2SO4 precipitates of cell supernatant and sedimented with hexabrachions in glycerol gradients. Hexabrachions purified by antibody adsorption, however, had lost this activity, suggesting that it was due to a separate factor associated with hexabrachions in the gradient fractions. The combined information in the several, previously unrelated studies suggests that hexabrachions may play a role in organizing localized regions of extracellular matrix. The protein is prominently expressed at specific times and locations during embryonic development, is retained in certain adult tissues, and is reexpressed in a variety of tumors. PMID:3654758

  15. Interferon Gamma-1b Injection

    MedlinePlus

    Interferon gamma-1b injection is used to reduce the frequency and severity of serious infections in people ... with severe, malignant osteopetrosis (an inherited bone disease). Interferon gamma-1b is in a class of medications ...

  16. Aldo-keto Reductase 1B15 (AKR1B15)

    PubMed Central

    Weber, Susanne; Salabei, Joshua K.; Möller, Gabriele; Kremmer, Elisabeth; Bhatnagar, Aruni; Adamski, Jerzy; Barski, Oleg A.

    2015-01-01

    Aldo-keto reductases (AKRs) comprise a superfamily of proteins involved in the reduction and oxidation of biogenic and xenobiotic carbonyls. In humans, at least 15 AKR superfamily members have been identified so far. One of these is a newly identified gene locus, AKR1B15, which clusters on chromosome 7 with the other human AKR1B subfamily members (i.e. AKR1B1 and AKR1B10). We show that alternative splicing of the AKR1B15 gene transcript gives rise to two protein isoforms with different N termini: AKR1B15.1 is a 316-amino acid protein with 91% amino acid identity to AKR1B10; AKR1B15.2 has a prolonged N terminus and consists of 344 amino acid residues. The two gene products differ in their expression level, subcellular localization, and activity. In contrast with other AKR enzymes, which are mostly cytosolic, AKR1B15.1 co-localizes with the mitochondria. Kinetic studies show that AKR1B15.1 is predominantly a reductive enzyme that catalyzes the reduction of androgens and estrogens with high positional selectivity (17β-hydroxysteroid dehydrogenase activity) as well as 3-keto-acyl-CoA conjugates and exhibits strong cofactor selectivity toward NADP(H). In accordance with its substrate spectrum, the enzyme is expressed at the highest levels in steroid-sensitive tissues, namely placenta, testis, and adipose tissue. Placental and adipose expression could be reproduced in the BeWo and SGBS cell lines, respectively. In contrast, AKR1B15.2 localizes to the cytosol and displays no enzymatic activity with the substrates tested. Collectively, these results demonstrate the existence of a novel catalytically active AKR, which is associated with mitochondria and expressed mainly in steroid-sensitive tissues. PMID:25577493

  17. Quantification of Plasmodium falciparum malaria from complex infections in the Peruvian Amazon using quantitative PCR of the merozoite surface protein 1, block 2 (PfMSP1-B2): in vitro dynamics reveal density-dependent interactions.

    PubMed

    Zervos, Thomas M; Hernandez, Jean N; Sutton, Patrick L; Branch, Oralee H

    2012-05-01

    The majority of Plasmodium falciparum field isolates are defined as complex infections because they contain multiple genetically distinct clones. Studying interactions between clones in complex infections in vivo and in vitro could elucidate important phenomena in malaria infection, transmission and treatment. Using quantitative PCR (qPCR) of the P. falciparum merozoite surface protein 1, block 2 (PfMSP1-B2), we provide a sensitive and efficient genotyping method. This is important for epidemiological studies because it makes it possible to study genotype-specific growth dynamics. We compared 3 PfMSP1-B2 genotyping methods by analysing 79 field isolates from the Peruvian Amazon. In vivo observations from other studies using these techniques led to the hypothesis that clones within complex infections interact. By co-culturing clones with different PfMSP1-B2 genotypes, and measuring parasitaemia using qPCR, we found that suppression of clonal expansion was a factor of the collective density of all clones present in a culture. PfMSP1-B2 qPCR enabled us to find in vitro evidence for parasite-parasite interactions and could facilitate future investigations of growth trends in naturally occurring complex infections. PMID:22339946

  18. PML tumor suppressor protein is required for HCV production

    SciTech Connect

    Kuroki, Misao; Ariumi, Yasuo; Hijikata, Makoto; Ikeda, Masanori; Dansako, Hiromichi; Wakita, Takaji; Shimotohno, Kunitada; Kato, Nobuyuki

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer PML tumor suppressor protein is required for HCV production. Black-Right-Pointing-Pointer PML is dispensable for HCV RNA replication. Black-Right-Pointing-Pointer HCV could not alter formation of PML-NBs. Black-Right-Pointing-Pointer INI1 and DDX5, PML-related proteins, are involved in HCV life cycle. -- Abstract: PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.

  19. JARID1B promotes metastasis and epithelial-mesenchymal transition via PTEN/AKT signaling in hepatocellular carcinoma cells.

    PubMed

    Tang, Bo; Qi, Guangying; Tang, Fang; Yuan, Shengguang; Wang, Zhenran; Liang, Xingsi; Li, Bo; Yu, Shuiping; Liu, Jie; Huang, Qi; Wei, Yangchao; Zhai, Run; Lei, Biao; Yu, Hongping; Jiao, Xingyuan; He, Songqing

    2015-05-20

    JARID1B is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). JARID1B has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in hepatocellular carcinoma (HCC) remain unknown. Here we show that JARID1B is elevated in HCC and its expression level is positively correlated with metastasis. In addition Kaplan-Meier survival analysis showed that high expression of JARID1B was associated with decreased overall survival of HCC patients. Overexpression of JARID1B in HCC cells increased proliferation, epithelial-mesenchymal transition, migration and invasion in vitro, and enhanced tumorigenic and metastatic capacities in vivo. In contrast, silencing JARID1B in aggressive and invasive HCC cells inhibited these processes. Mechanistically, we found JARID1B exerts its function through modulation of H3K4me3 at the PTEN gene promoter, which was associated with inactive PTEN transcription. PTEN overexpression blocked JARID1B-driven proliferation, EMT, and metastasis. Our results, for the first time, portray a pivotal role of JARID1B in stimulating metastatic behaviors of HCC cells. Targeting JARID1B may thus be a useful strategy to impede HCC cell invasion and metastasis. PMID:25909289

  20. Wilms Tumor 1 protein is not expressed in oral lymphangiomas.

    PubMed

    Netto, Ana Carolina de Mesquita; de Oliveira, Mariana Batista; Bernardes, Vanessa Fátima; Gomes, Carolina Cavaliéri; Gomez, Ricardo Santiago

    2012-01-01

    Lymphangiomas are benign hamartomatous lesions of lymphatic vessels. Wilms Tumor 1 (WT1) is a transcription factor that is activated in some human neoplasias. WT1 protein expression is observed in endothelial cells during angiogenesis and is a useful marker to distinguish between vascular proliferations and vascular malformations. The purpose of the present study is to report a case series of oral lymphangiomas together with an immunohistochemical investigation of WT1. Seventeen cases of oral lymphangioma were retrieved and reviewed. Immunohistochemical analysis of WT1 protein was performed and pyogenic granuloma samples were used as positive controls. The male/female ratio was 1.125 and most of the lesions occurred in young subjects. While pyogenic granuloma showed positive staining for WT1, the endothelial cells lining the thin-walled dilated lymphatic vessels of lymphangiomas were negative for this protein. The findings strengthen the idea that oral lymphangioma is a vascular malformation characterized by lymphatic dilatation without significant endothelial proliferation. PMID:23338265

  1. Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure.

    PubMed

    Ratovitski, Edward A

    2016-01-01

    Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells. PMID:27537898

  2. Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure

    PubMed Central

    Ratovitski, Edward A.

    2016-01-01

    Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells. PMID:27537898

  3. Targeting mutant p53 protein and the tumor vasculature: an effective combination therapy for advanced breast tumors

    PubMed Central

    Liang, Yayun; Besch-Williford, Cynthia; Benakanakere, Indira; Thorpe, Philip E.

    2010-01-01

    Breast cancer progression depends upon the elaboration of a vasculature sufficient for the nourishment of the developing tumor. Breast tumor cells frequently contain a mutant form of p53 (mtp53), a protein which promotes their survival. The aim of this study was to determine whether combination therapy targeting mtp53 and anionic phospholipids (AP) on tumor blood vessels might be an effective therapeutic strategy for suppressing advanced breast cancer. We examined the therapeutic effects, singly, or in combination, of p53 reactivation and induction of massive apoptosis (PRIMA-1), which reactivates mtp53 and induces tumor cell apoptosis, and 2aG4, a monoclonal antibody that disrupts tumor vasculature by targeting AP on the surface of tumor endothelial cells and causes antibody-dependent destruction of tumor blood vessels, leading to ischemia and tumor cell death. Xenografts from two tumor cell lines containing mtp53, BT-474 and HCC-1428, were grown in nude mice to provide models of advanced breast tumors. After treatment with PRIMA-1 and/or 2aG4, regressing tumors were analyzed for vascular endothelial growth factor (VEGF) expression, blood vessel loss, and apoptotic markers. Individual drug treatment led to partial suppression of breast cancer progression. In contrast, combined treatment with PRIMA-1 and 2aG4 was extremely effective in suppressing tumor growth in both models and completely eradicated approximately 30% of tumors in the BT-474 model. Importantly, no toxic effects were observed in any treatment group. Mechanistic studies determined that PRIMA-1 reactivated mtp53 and also exposed AP on the surface of tumor cells as determined by enhanced 2aG4 binding. Combination treatment led to significant induction of tumor cell apoptosis, loss of VEGF expression, as well as destruction of tumor blood vessels. Furthermore, combination treatment severely disrupted tumor blood vessel perfusion in both tumor models. The observed in vitro PRIMA-1-induced exposure of

  4. Tumor Protein 53-Induced Nuclear Protein 1 Enhances p53 Function and Represses Tumorigenesis.

    PubMed

    Shahbazi, Jeyran; Lock, Richard; Liu, Tao

    2013-01-01

    Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a stress-induced p53-target gene whose expression is modulated by transcription factors such as p53, p73, and E2F1. TP53INP1 gene encodes two isoforms of TP53INP1 proteins, TP53INP1α and TP53INP1β, both of which appear to be key elements in p53 function. In association with homeodomain-interacting protein kinase-2 (HIPK2), TP53INP1 phosphorylates p53 protein at Serine-46. This enhances p53 protein stability and its transcriptional activity, leading to transcriptional activation of p53-target genes such as p21 and PIG3, cell growth arrest and apoptosis upon DNA damage stress. The anti-proliferative and pro-apoptotic activities of TP53INP1 indicate that TP53INP1 has an important role in cellular homeostasis and DNA damage response. Deficiency in TP53INP1 expression results in increased tumorigenesis, whereas TP53INP1 expression is repressed during early stages of cancer by factors such as miR-155. This review aims to summarize the roles of TP53INP1 in blocking tumor progression through p53-dependant and p53-independent pathways, as well as the elements which repress TP53INP1 expression, hence highlighting its potential as a therapeutic target in cancer treatment. PMID:23717325

  5. Impaired Self-Renewal and Increased Colitis and Dysplastic Lesions in Colonic Mucosa of AKR1B8 Deficient Mice

    PubMed Central

    Shen, Yi; Ma, Jun; Yan, Ruilan; Ling, Hongyan; Li, Xiaoning; Yang, Wancai; Gao, John; Huang, Chenfei; Bu, Yiwen; Cao, Yu; He, Yingchun; Wan, Laxiang; Zu, Xuyu; Liu, Jianghua; Huang, Mei Chris; Stenson, William F; Liao, Duan-Fang; Cao, Deliang

    2015-01-01

    Purpose Ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but down-regulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in UC and CAC. Experimental design UC and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8 −/−) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide Exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors. Results AKR1B10 expression was markedly diminished in over 90% of UC and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8 −/− mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8 −/− mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8 −/− mice than in wild type mice. Palpable masses were seen in the AKR1B8 −/− mice only, not in wild type. Conclusion AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of UC and CAC, being a new etiopathogenic factor of these diseases. PMID:25538260

  6. G-protein coupled receptor-associated sorting protein 1 (GASP-1), a ubiquitous tumor marker.

    PubMed

    Zheng, Xiaoyi; Chang, Frank; Zhang, Xinmin; Rothman, Vicki L; Tuszynski, George P

    2012-08-01

    Using an innovative "2-D high performance liquid electrophoresis" (2-D HPLE) technology we identified that a specific fragment of G-protein coupled receptor-associated sorting protein 1 (GASP-1) was present in the sera of breast cancer patients and was over-expressed in early and late stage breast tumors (Tuszynski, G.P. et al., 2011). In this study we further investigated the significance of GASP-1 as a tumor marker by investigating the expression GASP-1 in different kinds of tumors as well as in the sera of patients with various cancers. Over expression of GASP-1 was detected in brain, pancreatic, and breast cancers as compared to their respective normal tissues as assessed by immunohistochemical staining of tissue arrays using a "peptide specific" GASP-1 antibody. We found that across these cancers, GASP-1 was expressed approximately 10 fold more in the cancer as compared to normal tissue. The increase in GASP-1 expression was also seen in hyperplastic and inflammatory lesions of breast and pancreatic cancers as compared to normal tissue. GASP-1 was primarily expressed in the tumor epithelium of the epithelial-derived cancers and in the transformed glial cells of the brain tumors. Using a sensitive "competitive ELISA" for GASP-1, we found that sera from patients with brain, liver, breast and lung cancers expressed 4-7 fold more GASP-1 peptide than sera from normal healthy individuals. These studies identify GASP-1 as a potential new serum and tumor biomarker for several cancers and suggest that GASP-1 may be a novel target for development of cancer therapeutics. PMID:22483848

  7. Altered Splicing of JARID1B in Development of Human Cutaneous Melanoma?

    PubMed

    Kuźbicki, Łukasz; Lange, Dariusz; Strączyńska-Niemiec, Anita; Chwirot, Barbara W

    2016-03-01

    Upregulated expression of histone H3K4 demethylase JARID1B has been found in several types of human cancer, but the expression pattern of this protein in benign naevi and human cutaneous melanomas seems to differ from that described for other tumors. We demonstrate that the apparent contradiction may be because of the fact that the malignant transformation of melanocytes is associated not so much with a general enhancement of a total expression of JARID1B but rather with a change in relative expression levels of individual splicing variants of the protein. Our data indicate that parallel immunohistochemical assays of the expression levels of all the isoforms and of the RBP2-H1 variant of JARID1B may be an efficient technique of differentiating between benign naevi and melanomas. PMID:25789538

  8. A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery.

    PubMed

    Lakra, Nita; Nutan, Kamlesh K; Das, Priyanka; Anwar, Khalid; Singla-Pareek, Sneh L; Pareek, Ashwani

    2015-03-15

    Plants have evolved a number of molecular strategies and regulatory mechanisms to cope with abiotic stresses. Among the various key factors/regulators, transcription factors (TFs) play critical role(s) towards regulating the gene expression patterns in response to stress conditions. Altering the expression of the key TFs can greatly influence plant stress tolerance. OsHBP1b (accession no. KM096571) is one such TF belonging to bZIP family, localized within the Saltol QTL, whose expression is induced upon salinity treatment in the rice seedlings. qRT-PCR based expression studies for OsHBP1b in seedlings of contrasting genotypes of rice showed its differential regulation in response to salinity stress. A GFP based in vivo study showed that the OsHBP1b protein is nuclear localized and possesses the trans-activation activity. As compared to the WT tobacco plants, the transgenic plants ectopically expressing OsHBP1b showed better survival and favourable osmotic parameters (such as germination and survival rate, membrane stability, K(+)/Na(+) ratio, lipid peroxidation, electrolyte leakage and proline contents) under salinity and drought stress. Under salinity conditions, the transgenic plants accumulated lower levels of reactive oxygen species as compared to the WT. It was also accompanied by higher activities of antioxidant enzymes (such as ascorbate peroxidase and superoxide dismutase), thereby demonstrating that transgenic plants are physiologically better adapted towards the oxidative damage. Taken together, our findings suggest that OsHBP1b contributes to abiotic stress tolerance through multiple physiological pathways and thus, may serve as a useful 'candidate gene' for improving multiple stress tolerance in crop plants. PMID:25543954

  9. KIF1B promotes glioma migration and invasion via cell surface localization of MT1-MMP.

    PubMed

    Chen, Songyu; Han, Mingzhi; Chen, Weiliang; He, Ying; Huang, Bin; Zhao, Peng; Huang, Qibing; Gao, Liang; Qu, Xun; Li, Xingang

    2016-02-01

    Malignant glioma is notorious for its aggressiveness and poor prognosis, and the invasiveness of glioma cells is the major obstacle. Accumulating evidence indicates that kinesin superfamily proteins (KIFs) may play key roles in tumor invasiveness, but the mechanisms remained unresolved. Our previous study demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) was involved in Kinesin family member 1B (KIF1B)-modulated invasion of gastric cancer cells. Therefore, the role of KIF1B in glioma cell invasion and its relationship with MT1-MMP were explored in the present study. We found that aberrantly increased expression of KIF1B was associated with worse WHO pathological classification and Karnofsky performance status (KPS), which also showed a trend towards worse prognosis. In the transwell assay, knockdown of KIF1B using siRNA repressed U87MG and A172 glioma cell migration and invasion. Silencing KIF1B inhibited expression of membranal MT1-MMP; however, the amount of MT1-MMP in the whole cell lysate was not affected. In conclusion, targeting KIF1B may be an option for anti-invasive therapies targeting glioma. PMID:26576027

  10. Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection

    PubMed Central

    Manam, Srikanth; Thomas, Joshua D.; Li, Weidang; Maladore, Allison; Schripsema, Justin H.; Ramsey, Kyle H.; Murthy, Ashlesh K.

    2015-01-01

    Background. We demonstrated previously that tumor necrosis factor α (TNF-α)–producing Chlamydia-specific CD8+ T cells cause oviduct pathological sequelae. Methods. In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8+ T cells to study chlamydial pathogenesis. Results. TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8+ T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8+ T cells but not with TNFR2 KO CD8+ T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8+ T cells restored oviduct pathology to WT levels in both KO groups. Conclusions. Collectively, these results demonstrate that TNFR2-bearing CD8+ T cells and TNFR1-bearing non-CD8+ T cells contribute significantly to oviduct pathology following genital chlamydial infection. PMID:25552370

  11. A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV{sub KU-1bMC33}) susceptible to rimantadine

    SciTech Connect

    Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik; Miller, Jean-Marie; Hill, M. Sarah; Stephens, Edward B. . E-mail: estephen@kumc.edu

    2006-05-10

    Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV{sub KU-1bMC33} in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV{sub M2}) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV{sub KU-1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV{sub VpuA19H} replicated with similar kinetics as the parental SHIV{sub KU-1bMC33} and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV{sub KU-1bMC33}. This SHIV{sub VpuA19H} virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV{sub M2}. Electron microscopic examination of SHIV{sub VpuA19H}-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV{sub M2}-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the

  12. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  13. Induction of protein tyrosine phosphorylation in macrophages incubated with tumor cells.

    PubMed

    Sodhi, A; Shrivastava, A; Kumar, R

    1995-03-01

    The cellular and molecular interaction between monocyte/macrophage and tumor cells leading to macrophage activation is not clearly understood. Since protein tyrosine phosphorylation appears to be a major intracellular signalling event, we checked whether the tumor cells alter tyrosine phosphorylation of proteins in macrophages. We found that both L929 and Yac-1 tumor cells induced increased tyrosine phosphorylation of several polypeptides in peritoneal as well as P388D-1 and IC-21 macrophages. Macrophages co-cultured with tumor cells also showed increased fluorescence with anti-phosphotyrosine-FITC antibody. These observations suggest that increased tyrosine phosphorylation plays a role in tumor cell-induced activation of macrophages. PMID:7539664

  14. TNFα increases hypothalamic PTP1B activity via the NFκB pathway in rat hypothalamic organotypic cultures.

    PubMed

    Ito, Yoshihiro; Banno, Ryoichi; Hagimoto, Shigeru; Ozawa, Yoshiharu; Arima, Hiroshi; Oiso, Yutaka

    2012-02-10

    In obesity, levels of tumor necrosis-factor α (TNFα) are well known to be elevated in adipose tissues or serum, and a high-fat diet (HFD) reportedly increases TNFα expression in the hypothalamus. The expression levels of hypothalamic protein tyrosine phosphatase 1B (PTP1B), a negative regulator of leptin and insulin signaling, are also elevated by HFD, and several lines of evidence support a relationship between TNFα and PTP1B. It remains unclear however how TNFα acts locally in the hypothalamus to regulate hypothalamic PTP1B expression and activity. In this study, we examined whether TNFα can regulate PTP1B expression and activity using rat hypothalamic organotypic cultures. Incubation of cultures with TNFα resulted in increases in mRNA expression, protein levels and activity of PTP1B in a dose- and time-dependent manner, respectively compared with controls. TNFα-induced PTP1B protein levels were not influenced by co-incubation with the sodium channel blocker tetrodotoxin, indicating that the action of TNFα is independent of action potentials. TNFα also increased phosphorylation of p65, a subunit of nuclear factor-κB (NFκB), in a dose- and time-dependent manner. While incubation with inhibitors of NFκB did not affect basal levels of either p65 phosphorylation or PTP1B expression, it markedly suppressed both TNFα-induced p65 phosphorylation and PTP1B expression to almost basal levels. These data suggest that TNFα acts on the hypothalamus to increase hypothalamic PTP1B expression and activity via the NFκB pathway, and that TNFα-mediated induction of NFκB in the hypothalamus may cause leptin and insulin resistance in the hypothalamus by increasing hypothalamic PTP1B activity. PMID:22166493

  15. The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase.

    PubMed

    Pi, Hualiang; Patel, Sarju J; Argüello, José M; Helmann, John D

    2016-06-01

    Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection. PMID:26946370

  16. Simulation of the Protein-Shedding Kinetics of a Fully Vascularized Tumor

    PubMed Central

    Frieboes, Hermann B.; Curtis, Louis T.; Wu, Min; Kani, Kian; Mallick, Parag

    2015-01-01

    Circulating biomarkers are of significant interest for cancer detection and treatment personalization. However, the biophysical processes that determine how proteins are shed from cancer cells or their microenvironment, diffuse through tissue, enter blood vasculature, and persist in circulation remain poorly understood. Since approaches primarily focused on experimental evaluation are incapable of measuring the shedding and persistence for every possible marker candidate, we propose an interdisciplinary computational/experimental approach that includes computational modeling of tumor tissue heterogeneity. The model implements protein production, transport, and shedding based on tumor vascularization, cell proliferation, hypoxia, and necrosis, thus quantitatively relating the tumor and circulating proteomes. The results highlight the dynamics of shedding as a function of protein diffusivity and production. Linking the simulated tumor parameters to clinical tumor and vascularization measurements could potentially enable this approach to reveal the tumor-specific conditions based on the protein detected in circulation and thus help to more accurately manage cancer diagnosis and treatment. PMID:26715830

  17. The chimeric genes AML1/DS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties

    SciTech Connect

    Zent, C.S.; Matheiu, C.; Rowley, J.D.

    1996-02-06

    The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the {alpha} subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the {beta} subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AMLI (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression provented tumor growth. These results suggest that expression of AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line. 26 refs., 5 figs.

  18. Induction of monocyte chemoattractant protein-1 and interleukin-10 by TGFbeta1 in melanoma enhances tumor infiltration and immunosuppression.

    PubMed

    Díaz-Valdés, Nancy; Basagoiti, María; Dotor, Javier; Aranda, Fernando; Monreal, Iñaki; Riezu-Boj, José Ignacio; Borrás-Cuesta, Francisco; Sarobe, Pablo; Feijoó, Esperanza

    2011-02-01

    Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFβ1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFβ1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFβ1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFβ1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,β1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFβ1-blocking peptide P144, TGFβ1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFβ1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma. PMID:21159663

  19. Protein tyrosine phosphatase receptor type O expression in the tumor niche correlates with reduced tumor growth, angiogenesis, circulating tumor cells and metastasis of breast cancer.

    PubMed

    Liu, Zhao; Hou, Jiajie; Ren, Lidong; He, Jing; Sun, Beicheng; Sun, Lu-Zhe; Wang, Shui

    2015-04-01

    Protein tyrosine phosphatase receptor type O (PTPRO) has been recognized as a tumor suppressor in various types of cancer cells. However, little attention has been given to the role of PTPRO expression in the tumor microenvironment. We aimed to reveal the role of PTPRO in the breast cancer niche. Py8119 mouse breast cancer cells were implanted orthotopically into female wild-type or ptpro-/- C57Bl/6 mice. We observed that the loss of PTPRO in the tumor niche was correlated with larger tumor volume, more metastases, increased number of circulating tumor cells (CTCs), less apoptosis and reduced necrosis rates in the orthotopic mouse model of breast cancer. The tumor microenvironment in the ptpro-/- mice also showed increased microvessel density. Moreover, an intracardiac injection mouse model was used to determine the role of PTPRO in the pre-metastatic niche. Notably, more metastases were observed in the mice of the ptpro-/- group. Taken together, PTPRO expression in the tumor niche prevents tumor growth and the formation of metastases of breast cancer, in part by attenuating tumor-associated angiogenesis and inducing the apoptosis and necrosis of tumor cells. PMID:25646811

  20. Tumor-specific immunotherapy of murine bladder cancer with butanol-extracted antigens and ethylchlorformate polymerized tumor protein.

    PubMed

    Rochester, M G; Sarosdy, M F; Pickett, S H; Stogdill, B J; Lamm, D L

    1988-09-01

    Successful treatment of superficial bladder cancer using nonspecific immunotherapy with Bacillus Calmette-Guerin (BCG) has been well documented. Investigation of two potential tumor-specific immunotherapeutic agents using a murine transitional-cell carcinoma model (MBT-2) is reported. The survival of mice immunized with tumor proteins obtained by treating tumor cells with either 1-butanol or ethylchlorformate was compared to the survival of animals immunized with BCG. Long-term immunity conferred by each of these agents was also assessed. Significant protection by both agents was noted in all treatment groups compared to controls. Long-term immunity was also found to result from treatment with both investigational agents as well as with BCG. Butanol-extracted antigens and ethylchlorformate polymerized tumor protein may be useful as immunotherapeutic alternatives to BCG. PMID:3411695

  1. Hydroxysteroid Sulfotransferase SULT2B1b Promotes Hepatocellular Carcinoma Cells Proliferation In Vitro and In Vivo

    PubMed Central

    Yang, Xiaoming; Xu, Yali; Guo, Fenghua; Ning, Yanxia; Zhi, Xiuling; Yin, Lianhua; Li, Xiaobo

    2013-01-01

    Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC. PMID:23593328

  2. Inducible 70 kDa Heat Shock Proteins Protect Embryos from Teratogen-Induced Exencephaly: Analysis using Hspa1a/a1b Knockout Mice

    EPA Science Inventory

    Background: It is well known that a variety of teratogens induce neural tube defects in animals; however, less is known about proteins that play a role in protecting embryos from teratogen-induced neural tube defects. Previously, our lab has shown that embryos over-expressing th...

  3. Novel 2,7-Substituted (S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic Acids: Peroxisome Proliferator-Activated Receptor γ Partial Agonists with Protein-Tyrosine Phosphatase 1B Inhibition.

    PubMed

    Otake, Kazuya; Azukizawa, Satoru; Takeda, Shigemitsu; Fukui, Masaki; Kawahara, Arisa; Kitao, Tatsuya; Shirahase, Hiroaki

    2015-01-01

    A novel series of 2,7-substituted 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid derivatives were synthesized and biologically evaluated. (S)-2-(2-Furylacryloyl)-7-[2-(2-methylindane-2-yl)-5-methyloxazol-4-yl]methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid tert-butylamine salt (13jE) was identified as a potent human peroxisome proliferator-activated receptor γ (PPARγ)-selective agonist (EC50=85 nM) and human protein-tyrosine phosphatase 1B (PTP-1B) inhibitor (IC50=1.0 µM). Compound 13jE partially activated PPARγ, but not PPARα or PPARδ, and antagonized farglitazar, a full PPARγ agonist. Cmax after the oral administration of 13jE at 10 mg/kg was 28.6 µg/mL (53 µM) in male Sprague-Dawley (SD) rats. Repeated administration of 13jE and rosiglitazone for 14 d at 10 mg/kg/d decreased plasma glucose and triglyceride levels significantly in male KK-A(y) mice. Rosiglitazone, but not 13jE, significantly increased the plasma volume and liver weight. In conclusion, 13jE showed stronger hypoglycemic and hypolipidemic effects and weaker hemodilution and hepatotoxic effects than rosiglitazone, suggesting that its safer efficacy may be due to its partial PPARγ agonism and PTP-1B inhibition. PMID:26633022

  4. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

    PubMed

    Andruska, Neal D; Zheng, Xiaobin; Yang, Xujuan; Mao, Chengjian; Cherian, Mathew M; Mahapatra, Lily; Helferich, William G; Shapiro, David J

    2015-04-14

    Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration. PMID:25825714

  5. Photodynamic therapy cures green fluorescent protein expressing RIF1 tumors in mice

    NASA Astrophysics Data System (ADS)

    Castano, Ana P.; Liu, Qingde; Hamblin, Michael R.

    2004-07-01

    Cancer is a leading cause of death among modern people, largely due to metastatic disease. The ideal cancer treatment should destroy both the primary tumor and distant metastases with minimal toxicity to normal tissue. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species that eventually cause vascular shutdown and tumor cell apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens are likely to emerge in the future to even further enhance immunity. Green fluorescent protein is used as an optical reporter to non-invasively image the progression of mouse tumors, and in addition, may act as a foreign (jellyfish) antigen. We asked whether the response of tumor bearing mice to PDT differed when a non-immunogenic tumor cell line was transfected with GFP? We injected RIF-1 or RIF1-EGFP cells in the leg of C3H/HeN mice and both the cells and tumors grew equally well. We used two PDT protocols (benzoporphyrin derivative (BPD) with 15-minute interval or Photofrin with 24-hour interval). The results showed significant differences between the responses of RIF1 or RIF1-EGFP tumors after BPD or Photofrin PDT and complete cures and mouse survival when RIF-1 EGFP tumors were treated with BPD. This increased tumor response may be due to antibody-mediated cytotoxicity and the presence of an artificial tumor antigen (GFP) that can produce a CD8 T-cell response against the whole tumor. The presence of antibodies against EGFP in mouse serum correlates with the hypothesis.

  6. Expression of Translationally Controlled Tumor Protein in Human Kidney and in Renal Cell Carcinoma.

    PubMed

    Ambrosio, Maria R; Rocca, Bruno J; Barone, Aurora; Onorati, Monica; Mundo, Lucia; Crivelli, Filippo; Di Nuovo, Franca; De Falco, Giulia; del Vecchio, Maria T; Tripodi, Sergio A; Tosi, Piero

    2015-01-01

    Translationally controlled tumor protein is a multifaceted protein involved in several physiological and biological functions. Its expression in normal kidney and in renal carcinomas, once corroborated by functional data, may add elements to elucidate renal physiology and carcinogenesis. In this study, translationally controlled tumor protein expression was evaluated by quantitative real time polymerase chain reaction and western blotting, and its localization was examined by immunohistochemistry on 84 nephrectomies for cancer. In normal kidney protein expression was found in the cytoplasm of proximal and distal tubular cells, in cells of the thick segment of the loop of Henle, and in urothelial cells of the pelvis. It was also detectable in cells of renal carcinoma with different pattern of localization (membranous and cytoplasmic) depending on tumor histotype. Our data may suggest an involvement of translationally controlled tumor protein in normal physiology and carcinogenesis. However, functional in vitro and in vivo studies are needed to verify this hypothesis. PMID:26425551

  7. Locomotor proteins in tissues of primary tumors and metastases of ovarian and breast cancer

    NASA Astrophysics Data System (ADS)

    Kondakova, I. V.; Yunusova, N. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Kolomiets, L. A.; Slonimskaya, E. M.; Villert, A. B.

    2016-08-01

    The paper discusses the capability for active movement in an extracellular matrix, wherein remodeling of the cytoskeleton by actin binding proteins plays a significant role in metastases formation. We studied the expression of actin binding proteins and β-catenin in tissues of primary tumors and metastases of ovarian and breast cancer. Contents of p45 Ser β-catenin and the actin severing protein gelsolin were decreased in metastases of ovarian cancer relative to primary tumors. The level of the cofilin, functionally similar to gelsolin, was significantly higher in metastases compared to primary ovarian and breast tumor tissue. In breast cancer, significant increase in the number of an actin monomer binder protein thymosin-β4 was observed in metastases as compared to primary tumors. The data obtained suggest the involvement of locomotor proteins in metastases formation in ovarian and breast cancer.

  8. Mass spectrometry based proteomics for absolute quantification of proteins from tumor cells

    PubMed Central

    Wang, Hong; Hanash, Sam

    2015-01-01

    In-depth quantitative profiling of the proteome and sub-proteomes of tumor cells has relevance to tumor classification, the development of novel therapeutics, and of prognostic and predictive markers and to disease monitoring. In particular the tumor cell surface represents a highly relevant compartment for the development of targeted therapeutics and immunotherapy. We have developed a proteomic platform to profile tumor cells that encompasses enrichment of surface membrane proteins, intact protein fractionation and label-free mass spectrometry based absolute quantification. Here we describe the methodology for capture, identification and quantification of cell surface proteins using biotinylation for labeling of the cell surface, avidin for capture of biotinylated proteins and ion mobility mass spectrometry for protein identification and quantification. PMID:25794949

  9. Expression of Translationally Controlled Tumor Protein in Human Kidney and in Renal Cell Carcinoma

    PubMed Central

    Ambrosio, Maria R.; Rocca, Bruno J.; Barone, Aurora; Onorati, Monica; Mundo, Lucia; Crivelli, Filippo; Di Nuovo, Franca; De Falco, Giulia; del Vecchio, Maria T.; Tripodi, Sergio A.; Tosi, Piero

    2015-01-01

    Translationally controlled tumor protein is a multifaceted protein involved in several physiological and biological functions. Its expression in normal kidney and in renal carcinomas, once corroborated by functional data, may add elements to elucidate renal physiology and carcinogenesis. In this study, translationally controlled tumor protein expression was evaluated by quantitative real time polymerase chain reaction and western blotting, and its localization was examined by immunohistochemistry on 84 nephrectomies for cancer. In normal kidney protein expression was found in the cytoplasm of proximal and distal tubular cells, in cells of the thick segment of the loop of Henle, and in urothelial cells of the pelvis. It was also detectable in cells of renal carcinoma with different pattern of localization (membranous and cytoplasmic) depending on tumor histotype. Our data may suggest an involvement of translationally controlled tumor protein in normal physiology and carcinogenesis. However, functional in vitro and in vivo studies are needed to verify this hypothesis. PMID:26425551

  10. Destruction of tumor vasculature and abated tumor growth upon VEGF blockade is driven by proapoptotic protein Bim in endothelial cells.

    PubMed

    Naik, Edwina; O'Reilly, Lorraine A; Asselin-Labat, Marie-Liesse; Merino, Delphine; Lin, Ann; Cook, Michele; Coultas, Leigh; Bouillet, Philippe; Adams, Jerry M; Strasser, Andreas

    2011-07-01

    For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature. PMID:21646395

  11. Multistage skin tumor promotion: involvement of a protein kinase

    SciTech Connect

    Mamrack, M.; Slaga, T. J.

    1980-01-01

    Current information suggests that chemical carcinogenesis is a multistep process with one of the best studied models in this regard being the two-stage carcinogenesis system using mouse skin. The effects of several carcinogens and tumor promoters in various sequences of application were studied to examine the nature of the process. The actions of several tumor inhibitors were compared. (ACR)

  12. Interferon Beta-1b Injection

    MedlinePlus

    ... course of disease where symptoms flare up from time to time) of multiple sclerosis (MS, a disease in which ... interferon beta-1b injection at around the same time of day each time you inject it. Follow ...

  13. Immunohistochemical detection and clinicopathological significance of JARID1B/KDM5B and P16 expression in invasive ductal carcinoma of the breast.

    PubMed

    Zhao, L H; Liu, H G

    2015-01-01

    The aims of this study were to investigate the expression of the H3K4 demethylase, jumonji AT-rich interactive domain 1B (JARID1B/KDM5B) and of p16 (multiple tumor suppressor gene MTS1) in breast cancer tissue and determine its clinicopathological significance. JARID1B/KDM5B and P16 protein expression in 176 resected breast cancer specimens and adjacent normal breast tissue was detected by the streptavidin-peroxidase (S-P) immunohistochemical method. The TNM staging grade was assigned according to the World Health Organization (2012) breast classification system. The positive staining rate of JARID1B/KDM5B and p16 protein in cancer tissue was 74.43 and 35.8%, respectively. JARID1B/KDM5B protein expression was positively associated with T grade, Bloom and Richardson (B&R) score and axillary lymph node metastasis (P < 0.05). p16 protein expression was negatively associated with T grade, B&R score, and axillary lymph node metastasis (P < 0.05). JARID1B/KDM5B and p16 protein expression in breast cancer and adjacent normal breast tissue were negatively correlated (r = -0.303, P < 0.001). The data demonstrated that protein expression of p16 and JARID1B/KDM5B is negatively correlated in invasive ductal carcinoma of the breast. PMID:26125737

  14. Tetramer formation of tumor suppressor protein p53: Structure, function, and applications.

    PubMed

    Kamada, Rui; Toguchi, Yu; Nomura, Takao; Imagawa, Toshiaki; Sakaguchi, Kazuyasu

    2016-11-01

    Tetramer formation of p53 is essential for its tumor suppressor function. p53 not only acts as a tumor suppressor protein by inducing cell cycle arrest and apoptosis in response to genotoxic stress, but it also regulates other cellular processes, including autophagy, stem cell self-renewal, and reprogramming of differentiated cells into stem cells, immune system, and metastasis. More than 50% of human tumors have TP53 gene mutations, and most of them are missense mutations that presumably reduce tumor suppressor activity of p53. This review focuses on the role of the tetramerization (oligomerization), which is modulated by the protein concentration of p53, posttranslational modifications, and/or interactions with its binding proteins, in regulating the tumor suppressor function of p53. Functional control of p53 by stabilizing or inhibiting oligomer formation and its bio-applications are also discussed. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 598-612, 2016. PMID:26572807

  15. Localization of heat shock protein 110 in canine mammary gland tumors.

    PubMed

    Okada, Satoru; Furuya, Masaru; Takenaka, Shigeo; Fukui, Ayano; Matsubayashi, Makoto; Tani, Hiroyuki; Sasai, Kazumi

    2015-10-15

    Heat shock proteins (HSPs) function as molecular chaperones in the regulation of protein folding, conformation, and assembly; in addition, they also protect cells from protein-protein aggregation resulting from cellular stress. Recently, HSPs were shown to be overexpressed in several human cancer cells compared with normal cells. HSPs are considered to be related to apoptosis-associated proteins, and inhibition of apoptosis promotes tumor growth. Canine mammary gland tumors have received a great deal of attention from researchers due to the many common biological and histological characteristics that they share with human tumors. We previously confirmed that HSP110 is a canine mammary gland tumor antigen and reported that HSP110 mRNA expression significantly increased in tumor tissue. We have now created a functional recombinant canine HSP110 protein and a rabbit anti-HSP110 polyclonal antibody. This recombinant protein can refold heat-denatured firefly luciferase at 42°C. Immunohistochemical analysis showed that HSP110 was mainly localized in the cytoplasm of epithelial and interstitial cells in canine mammary gland tumors. Extensive genomic research has revealed genetic similarities between humans and dogs; comparative oncological studies between these species have made remarkable progress. The results reported here contribute valuable oncological knowledge for the development of novel therapeutic methods in both veterinary science and human medicine. PMID:26292766

  16. Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration.

    PubMed

    Ferreira-da-Silva, André; Valacca, Cristina; Rios, Elisabete; Pópulo, Helena; Soares, Paula; Sobrinho-Simões, Manuel; Scorrano, Luca; Máximo, Valdemar; Campello, Silvia

    2015-01-01

    Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1--a papillary thyroid carcinoma derived cell line--and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells' migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype. PMID:25822260

  17. Mitochondrial Dynamics Protein Drp1 Is Overexpressed in Oncocytic Thyroid Tumors and Regulates Cancer Cell Migration

    PubMed Central

    Ferreira-da-Silva, André; Valacca, Cristina; Rios, Elisabete; Pópulo, Helena; Soares, Paula; Sobrinho-Simões, Manuel; Scorrano, Luca; Máximo, Valdemar; Campello, Silvia

    2015-01-01

    Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1 - a papillary thyroid carcinoma derived cell line—and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells’ migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype. PMID:25822260

  18. Structure and Promoter Characterization of Aldo-Keto Reductase Family 1 B10 Gene

    PubMed Central

    Liu, Ziwen; Zhong, Linlin; Krishack, Paulette A; Robbins, Sarah; Cao, Julia X; Zhao, Yupei; Chung, Stephen; Cao, Deliang

    2009-01-01

    Aldo-keto reductase family 1 member B10 (AKR1B10) is overexpressed in human hepatocellular carcinoma, lung squamous carcinoma, and lung adenocarcinoma in smokers. Our recent studies have showed that AKR1B10 plays a critical role in the growth and proliferation of cancer cells by detoxifying reactive carbonyls and regulating fatty acid biosynthesis. However, little is known about the regulatory mechanisms of AKR1B10 expression. In this study, we determined the structure of AKR1B10 gene and characterized its promoter. The results demonstrated that AKR1B10 consists of 10 exons and 9 introns, stretching approximately 13.8 kb. A 5′-RACE study determined the transcriptional start site of AKR1B10 at 320 bp upstream of the ATG translational start codon. A TATA-like (TAATAA) and a CAAT box are present from −145 to −140 bp and −193 to −190 bp upstream of the transcriptional start site, respectively. Motif analysis recognized multiple putative oncogenic and tumor suppressor protein binding sites in the AKR1B10 promoter, including c-Ets-1, C/EBP, AP-1, and p53, but osmolytic response elements were not found. A -4,091 bp of the 5′-flanking fragment of the AKR1B10 gene was capable of driving GFP and luciferase reporter gene expression in HepG2 cells derived from human hepatocellular carcinoma; progressive 5′-deletions revealed that a −255 bp fragment possesses full promoter activity. PMID:19236911

  19. Cyclin K and cyclin D1b are oncogenic in myeloma cells

    PubMed Central

    2010-01-01

    Background Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive. Results To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis. Conclusions Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions. PMID:20459741

  20. Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors

    PubMed Central

    Lechner, Melissa G; Russell, Sarah M; Bass, Rikki S; Epstein, Alan L

    2011-01-01

    In this article, the role of chemokines and costimulatory molecules in the immunotherapy of experimental murine solid tumors and immunotherapy used in ongoing clinical trials are presented. Chemokine networks regulate physiologic cell migration that may be disrupted to inhibit antitumor immune responses or coopted to promote tumor growth and metastasis in cancer. Recent studies highlight the potential use of chemokines in cancer immunotherapy to improve innate and adaptive cell interactions and to recruit immune effector cells into the tumor microenvironment. Another critical component of antitumor immune responses is antigen priming and activation of effector cells. Reciprocal expression and binding of costimulatory molecules and their ligands by antigen-presenting cells and naive lymphocytes ensures robust expansion, activity and survival of tumor-specific effector cells in vivo. Immunotherapy approaches using agonist antibodies or fusion proteins of immunomodulatory molecules significantly inhibit tumor growth and boost cell-mediated immunity. To localize immune stimulation to the tumor site, a series of fusion proteins consisting of a tumor-targeting monoclonal antibody directed against tumor necrosis and chemokines or costimulatory molecules were generated and tested in tumor-bearing mice. While several of these reagents were initially shown to have therapeutic value, combination therapies with methods to delete suppressor cells had the greatest effect on tumor growth. In conclusion, a key conclusion that has emerged from these studies is that successful immunotherapy will require both advanced methods of immunostimulation and the removal of immunosuppression in the host. PMID:22053884

  1. Phylogeography of E1b1b1b-M81 haplogroup and analysis of its subclades in Morocco.

    PubMed

    Reguig, Ahmed; Harich, Nourdin; Barakat, Abdelhamid; Rouba, Hassan

    2014-01-01

    In this study we analyzed 295 unrelated Berber-speaking men from northern, central, and southern Morocco to characterize frequency of the E1b1b1b-M81 haplogroup and to refine the phylogeny of its subclades: E1b1b1b1-M107, E1b1b1b2-M183, and E1b1b1b2a-M165. For this purpose, we typed four biallelic polymorphisms: M81, M107, M183, and M165. A large majority of the Berber-speaking male lineages belonged to the Y-chromosomal E1b1b1b-M81 haplogroup. The frequency ranged from 79.1% to 98.5% in all localities sampled. E1b1b1b2-M183 was the most dominant subclade in our samples, ranging from 65.1% to 83.1%. In contrast, the E1b1b1b1-M107 and E1b1b1b2a-M165 subclades were not found in our samples. Our results suggest a predominance of the E1b1b1b-M81 haplogroup among Moroccan Berber-speaking males with a decreasing gradient from south to north. The most prevalent subclade in this haplogroup was E1b1b1b2-M183, for which diffferences among these three groups were statistically significant between central and southern groups. PMID:25397701

  2. Protein tyrosine phosphatase-1B gene PTPN1: selection of tagging single nucleotide polymorphisms and association with body fat, insulin sensitivity, and the metabolic syndrome in a normal female population.

    PubMed

    Spencer-Jones, Nicola J; Wang, Xiaoling; Snieder, Harold; Spector, Tim D; Carter, Nicholas D; O'Dell, Sandra D

    2005-11-01

    Protein tyrosine phosphatase-1B negatively regulates leptin and insulin signaling, potentially contributing to hormonal resistance. We selected six tagging single nucleotide polymorphisms (SNPs) representing 18 common variants in the protein tyrosine phosphatase-1B gene (PTPN1) and tested their effect on serum leptin, body fat, and measures of insulin sensitivity and the metabolic syndrome in a large sample of normal female Caucasian twins (n = 2,777; mean age, 47.4 +/- 12.5 years) from the St. Thomas' U.K. Adult Twin Registry. SNP rs718049 was significantly associated with waist circumference (P = 0.008) and central fat (P = 0.035) and also with Avignon's insulin sensitivity index (SiM) (P = 0.007), fasting insulin (P = 0.004), fasting glucose (P = 0.022), triglyceride (P = 0.023), and systolic blood pressure (P = 0.046). SNPs rs2282146 and rs1885177 were associated with SiM (P = 0.049 and P = 0.013, respectively), and 1484insG was associated with triglyceride (P = 0.029). A risk haplotype (7.3%) was associated with lower SiM (P = 0.036) and a protective haplotype (5.2%) with higher SiM (P = 0.057), with mean values in homozygotes differing by >1 SD (P = 0.003). The protective haplotype also showed lower triglyceride (P = 0.045) and lower systolic blood pressure (P = 0.006). Fine mapping analyses predicted significant associations with SiM and fasting insulin for several ungenotyped SNPs. PTPN1 variants appear to contribute to central fat and metabolic syndrome traits, secondary to their effect on insulin sensitivity. PMID:16249458

  3. Altered Expression of High Molecular Weight Heat Shock Proteins after OCT4B1 Suppression in Human Tumor Cell Lines

    PubMed Central

    Mirzaei, Mohammad Reza; Kazemi Arababadi, Mohammad; Asadi, Malek Hossein; Mowla, Seyed Javad

    2016-01-01

    Objective OCT4B1, a novel variant of OCT4, is expressed in cancer cell lines and tis- sues. Based on our previous reports, OCT4B1 appears to have a crucial role in regulating apoptosis as well as stress response [heat shock proteins (HSPs)] pathways. The aim of the present study was to determine the effects of OCT4B1 silencing on the expression of high molecular weight HSPs in three different human tumor cell lines. Materials and Methods In this experimental study, OCT4B1 expression was suppressed in AGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cell lines using RNAi strategy. Real-time polymerase chain reaction (PCR) array was em- ployed for expression level analysis and the fold changes were calculated using RT2 Pro- filer PCR array data analysis software version 3.5. Results Our data revealed up-regulation of HSPD1 (from HSP60 family) as well as HSPA14, HSPA1L, HSPA4, HSPA5 and HSPA8 (from HSP70 family) following OCT4B1 knock-down in all three cell lines. In contrast, the expression of HSP90AA1 and HSP90AB1 (from HSP90 family) as well as HSPA1B and HSPA6 (from HSP70 family) was down-regulated under similar conditions. Other stress-related genes showed varying ex- pression pattern in the examined tumor cell lines. Conclusion Our data suggest a direct or indirect correlation between the expression of OCT4B1 and HSP90 gene family. However, OCT4B1 expression was not strongly corre- lated with the expression of HSP70 and HSP60 gene families. PMID:26862520

  4. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins

    PubMed Central

    Zhao, Guoping; Neely, Aaron M.; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G.; Stivers, Nicole S.; Burlison, Joseph A.; White, Carl; Machen, Terry E.; Li, Chi

    2016-01-01

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

  5. N-(3-oxo-acyl) homoserine lactone inhibits tumor growth independent of Bcl-2 proteins.

    PubMed

    Zhao, Guoping; Neely, Aaron M; Schwarzer, Christian; Lu, Huayi; Whitt, Aaron G; Stivers, Nicole S; Burlison, Joseph A; White, Carl; Machen, Terry E; Li, Chi

    2016-02-01

    Pseudomonas aeruginosa produces N-(3-oxododecanoyl)-homoserine lactone (C12) as a quorum-sensing molecule for bacterial communication. C12 has also been reported to induce apoptosis in various types of tumor cells. However, the detailed molecular mechanism of C12-triggerred tumor cell apoptosis is still unclear. In addition, it is completely unknown whether C12 possesses any potential therapeutic effects in vivo. Our data indicate that, unlike most apoptotic inducers, C12 evokes a novel form of apoptosis in tumor cells through inducing mitochondrial membrane permeabilization independent of both pro- and anti-apoptotic Bcl-2 proteins. Importantly, C12 inhibits tumor growth in animals regardless of either pro- or anti-apoptotic Bcl-2 proteins. Furthermore, opposite to conventional chemotherapeutics, C12 requires paraoxonase 2 (PON2) to exert its cytotoxicity on tumor cells in vitro and its inhibitory effects on tumor growth in vivo. Overall, our results demonstrate that C12 inhibits tumor growth independent of both pro- and anti-apoptotic Bcl-2 proteins, and through inducing unique apoptotic signaling mediated by PON2 in tumor cells. PMID:26758417

  6. Selective expression of constitutively active pro-apoptotic protein BikDD gene in primary mammary tumors inhibits tumor growth and reduces tumor initiating cells

    PubMed Central

    Rahal, Omar M; Nie, Lei; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Yi-Hsin; Hsu, Jennifer; Yu, Dihua; Hung, Mien-Chie

    2015-01-01

    Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49fhighCD61high and CD24+Jagged1-. First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in standard

  7. Selective expression of constitutively active pro-apoptotic protein BikDD gene in primary mammary tumors inhibits tumor growth and reduces tumor initiating cells.

    PubMed

    Rahal, Omar M; Nie, Lei; Chan, Li-Chuan; Li, Chia-Wei; Hsu, Yi-Hsin; Hsu, Jennifer; Yu, Dihua; Hung, Mien-Chie

    2015-01-01

    Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49f(high)CD61(high) and CD24(+)Jagged1(-). First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in

  8. The BRCA1-interacting protein, Abraxas, is required for genomic stability and tumor suppression

    PubMed Central

    Castillo, Andy; Paul, Atanu; Sun, Baohua; Huang, Ting Hsiang; Wang, Yucai; Yazinski, Stephanie A.; Tyler, Jessica; Li, Lei; You, M James; Zou, Lee; Yao, Jun; Wang, Bin

    2014-01-01

    Summary Germline mutations of BRCA1 confer hereditary susceptibility to breast and ovarian cancer. However, somatic mutation of BRCA1 is infrequent in sporadic breast cancers. The BRCA1 protein C-terminus BRCT domains interact with multiple proteins and are required for BRCA1's tumor suppressor function. In this study, we demonstrated that Abraxas, a BRCA1 BRCT domain-interacting protein, plays a role in tumor suppression. Abraxas exerts its function through binding to BRCA1 to regulate DNA repair and maintain genome stability. Both homozygous and heterozygous Abraxas knockout mice exhibited decreased survival and increased tumor incidence. The gene encoding Abraxas suffers from gene copy loss and somatic mutations in multiple human cancers including breast, ovarian, and endometrial cancers, suggesting that mutation and loss of function of Abraxas may contribute to tumor development in human patients. PMID:25066119

  9. Fibroblast activation protein α in tumor microenvironment: Recent progression and implications (Review)

    PubMed Central

    ZI, FUMING; HE, JINGSONG; HE, DONGHUA; LI, YI; YANG, LI; CAI, ZHEN

    2015-01-01

    Accumulated evidence has demonstrated that the microenvironment of a given tumor is important in determining its drug resistance, tumorigenesis, progression and metastasis. These microenvironments, like tumor cells, are vital targets for cancer therapy. The cross-talk between tumor cells and cancer-associated fibroblasts (CAFs, alternatively termed activated fibroblasts) is crucial in regulating the drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion and metastasis of a tumor. Fibroblast activation protein α (FAPα) is a transmembrane serine protease and is highly expressed on CAFs present in >90% of human epithelial neoplasms. FAPα activity, alongside that of gelatinase and type I collagenase, has become increasingly important in cancer therapy due to its effectiveness in modulating tumor behavior. In this review, recent progression in the knowledge of the role of FAPα in tumor microenvironments is discussed. PMID:25593080

  10. Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Zazpe, Idoya; Afzal, Mohammad; Sinha, Subrata; Rebhun, Robert B.; Meléndez, Bárbara; Rey, Juan A.

    2016-01-01

    Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease. PMID:25416442

  11. Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein*

    PubMed Central

    Law, Jennifer; Salla, Mohamed; Zare, Alaa; Wong, Yoke; Luong, Le; Volodko, Natalia; Svystun, Orysya; Flood, Kayla; Lim, Jonathan; Sung, Miranda; Dyck, Jason R. B.; Tan, Chong Teik; Su, Yu-Chin; Yu, Victor C.; Mackey, John; Baksh, Shairaz

    2015-01-01

    Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis. PMID:26269600

  12. Chaperone proteins and brain tumors: Potential targets and possible therapeutics1

    PubMed Central

    Graner, Michael W.; Bigner, Darell D.

    2005-01-01

    Chaperone proteins are most notable for the proteo- and cyotoprotective capacities they afford during cellular stress. Under conditions of cellular normalcy, chaperones still play integral roles in the folding of nascent polypeptides into functional entities, in assisting in intracellular/intraorganellar transport, in assembly and maintenance of multi-subunit protein complexes, and in aiding and abetting the degradation of senescent proteins. Tumors frequently have relatively enhanced needs for chaperone number and activity because of the stresses of rapid proliferation, increased metabolism, and overall genetic instability. Thus, it may be possible to take advantage of this reliance that tumor cells have on chaperones by pharmacologic and biologic means. Certain chaperones are abundant in the brain, which implies important roles for them. While it is presumed that the requirements of brain tumors for chaperone proteins are similar to those of any other cell type, tumor or otherwise, very little inquiry has been directed at the possibility of using chaperone proteins as therapeutic targets or even as therapeutic agents against central nervous system malignancies. This review highlights some of the research on the functions of chaperone proteins, on what can be done to modify those functions, and on the physiological responses that tumors and organisms can have to chaperone-targeted or chaperone-based therapies. In particular, this review will also underscore areas of research where brain tumors have been part of the field, although in general those instances are few and far between. This relative dearth of research devoted to chaperone protein targets and therapeutics in brain tumors reveals much untrodden turf to explore for potential treatments of these dreadfully refractive diseases. PMID:16053701

  13. AKR1B10 Induces Cell Resistance to Daunorubicin and Idarubicin by Reducing C13 Ketonic Group

    PubMed Central

    Zhong, Linlin; Shen, Honglin; Huang, Chenfei; Jing, Hongwu; Cao, Deliang

    2011-01-01

    Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at Vmax of 837.42±81.39 nmol/mg/min, Km of 9.317±2.25 mM and kcat/Km of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with Vmax at 460.23±28.12 nmol/mg/min, Km at 0.461±0.09 mM and kcat/Km at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50nM) and idarubicin (30nM) to corresponding alcohols. Within 24 hours, approximately 20±2.7% of daunorubicin (1μM) or 23±2.3% of idarubicin (1μM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7±0.9% and 5±1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggests that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin. PMID:21640744

  14. Increased Reactive Oxygen Species Production and Lower Abundance of Complex I Subunits and Carnitine Palmitoyltransferase 1B Protein Despite Normal Mitochondrial Respiration in Insulin-Resistant Human Skeletal Muscle

    PubMed Central

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T.; Bailowitz, Zachary; De Filippis, Elena A.; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J.

    2010-01-01

    OBJECTIVE The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. RESEARCH DESIGN AND METHODS Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. RESULTS NADH- and FADH2-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-dl-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). CONCLUSIONS We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance. PMID:20682693

  15. Cloning and characterization of human RTVP-1b, a novel splice variant of RTVP-1 in glioma cells

    SciTech Connect

    Xiang Cunli; Sarid, Ronit; Cazacu, Simona; Finniss, Susan; Lee, Hae-Kyung; Ziv-Av, Amotz; Mikkelsen, Tom; Brodie, Chaya

    2007-10-26

    Here, we report the cloning and characterization of RTVP-1b, a novel splice variant of human RTVP-1, which was isolated from the U87 glioma cell line. Sequence analysis revealed that RTVP-1b contains an additional 71 base exon between exons 2 and 3 that is missing in RTVP-1, leading to a frame-shift and a different putative protein. The deduced protein was 237 amino acids in length, sharing the N-terminal 141 amino acids with RTVP-1. RT-PCR analysis demonstrated that RTVP-1b was expressed in a wide range of tissues and that its expression was different from that of RTVP-1. In contrast, RTVP-1 and RTVP-1b showed similar patterns of expression in astrocytic tumors; highly expressed in glioblastomas as compared to normal brains, low-grade astrocytomas and anaplastic oligodendrogliomas. Overexpression of RTVP-1b increased glioma cell proliferation but did not affect cell migration. Our results suggest that RTVP-1b represents a potential prognostic marker and therapeutic target in gliomas.

  16. MAN1B1 Deficiency: An Unexpected CDG-II

    PubMed Central

    Millón, María B.; Race, Valérie; Sturiale, Luisa; Garozzo, Domenico; Mills, Philippa; Clayton, Peter; Asteggiano, Carla G.; Quelhas, Dulce; Cansu, Ali; Martins, Esmeralda; Nassogne, Marie-Cécile; Gonçalves-Rocha, Miguel; Topaloglu, Haluk; Jaeken, Jaak; Foulquier, François; Matthijs, Gert

    2013-01-01

    Congenital disorders of glycosylation (CDG) are a group of rare metabolic diseases, due to impaired protein and lipid glycosylation. In the present study, exome sequencing was used to identify MAN1B1 as the culprit gene in an unsolved CDG-II patient. Subsequently, 6 additional cases with MAN1B1-CDG were found. All individuals presented slight facial dysmorphism, psychomotor retardation and truncal obesity. Generally, MAN1B1 is believed to be an ER resident alpha-1,2-mannosidase acting as a key factor in glycoprotein quality control by targeting misfolded proteins for ER-associated degradation (ERAD). However, recent studies indicated a Golgi localization of the endogenous MAN1B1, suggesting a more complex role for MAN1B1 in quality control. We were able to confirm that MAN1B1 is indeed localized to the Golgi complex instead of the ER. Furthermore, we observed an altered Golgi morphology in all patients' cells, with marked dilatation and fragmentation. We hypothesize that part of the phenotype is associated to this Golgi disruption. In conclusion, we linked mutations in MAN1B1 to a Golgi glycosylation disorder. Additionally, our results support the recent findings on MAN1B1 localization. However, more work is needed to pinpoint the exact function of MAN1B1 in glycoprotein quality control, and to understand the pathophysiology of its deficiency. PMID:24348268

  17. Dendritic-Tumor Fusion Cells Derived Heat Shock Protein70-Peptide Complex Has Enhanced Immunogenicity

    PubMed Central

    Chen, Jun; Liu, Yunyan; Luo, Wen

    2015-01-01

    Tumor-derived heat shock protein70-peptide complexes (HSP70.PC-Tu) have shown great promise in tumor immunotherapy due to numerous advantages. However, large-scale phase III clinical trials showed that the limited immunogenicity remained to be enhanced. In previous research, we demonstrated that heat shock protein 70-peptide complexes (HSP70.PC-Fc) derived from dendritic cell (DC)-tumor fusions exhibit enhanced immunogenicity compared with HSP70.PCs from tumor cells. However, the DCs used in our previous research were obtained from healthy donors and not from the patient population. In order to promote the clinical application of these complexes, HSP70.PC-Fc was prepared from patient-derived DC fused directly with patient-derived tumor cells in the current study. Our results showed that compared with HSP70.PC-Tu, HSP70.PC-Fc elicited much more powerful immune responses against the tumor from which the HSP70 was derived, including enhanced T cell activation, and CTL responses that were shown to be antigen specific and HLA restricted. Our results further indicated that the enhanced immunogenicity is related to the activation of CD4+ T cells and increased association with other heat shock proteins, such as HSP90. Therefore, the current study confirms the enhanced immunogenicity of HSP70.PC derived from DC-tumor fusions and may provide direct evidence promoting their future clinical use. PMID:25961716

  18. Classification of lung cancer tumors based on structural and physicochemical properties of proteins by bioinformatics models.

    PubMed

    Hosseinzadeh, Faezeh; Ebrahimi, Mansour; Goliaei, Bahram; Shamabadi, Narges

    2012-01-01

    Rapid distinction between small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) tumors is very important in diagnosis of this disease. Furthermore sequence-derived structural and physicochemical descriptors are very useful for machine learning prediction of protein structural and functional classes, classifying proteins and the prediction performance. Herein, in this study is the classification of lung tumors based on 1497 attributes derived from structural and physicochemical properties of protein sequences (based on genes defined by microarray analysis) investigated through a combination of attribute weighting, supervised and unsupervised clustering algorithms. Eighty percent of the weighting methods selected features such as autocorrelation, dipeptide composition and distribution of hydrophobicity as the most important protein attributes in classification of SCLC, NSCLC and COMMON classes of lung tumors. The same results were observed by most tree induction algorithms while descriptors of hydrophobicity distribution were high in protein sequences COMMON in both groups and distribution of charge in these proteins was very low; showing COMMON proteins were very hydrophobic. Furthermore, compositions of polar dipeptide in SCLC proteins were higher than NSCLC proteins. Some clustering models (alone or in combination with attribute weighting algorithms) were able to nearly classify SCLC and NSCLC proteins. Random Forest tree induction algorithm, calculated on leaves one-out and 10-fold cross validation) shows more than 86% accuracy in clustering and predicting three different lung cancer tumors. Here for the first time the application of data mining tools to effectively classify three classes of lung cancer tumors regarding the importance of dipeptide composition, autocorrelation and distribution descriptor has been reported. PMID:22829872

  19. The CCN Family Proteins: Modulators of Bone Development and Novel Targets in Bone-Associated Tumors

    PubMed Central

    Chen, Po-Chun; Cheng, Hsu-Chen; Yang, Shun-Fa; Tang, Chih-Hsin

    2014-01-01

    The CCN family of proteins is composed of six extracellular matrix-associated proteins that play crucial roles in skeletal development, wound healing, fibrosis, and cancer. Members of the CCN family share four conserved cysteine-rich modular domains that trigger signal transduction in cell adhesion, migration, proliferation, differentiation, and survival through direct binding to specific integrin receptors and heparan sulfate proteoglycans. In the present review, we discuss the roles of the CCN family proteins in regulating resident cells of the bone microenvironment. In vertebrate development, the CCN family plays a critical role in osteo/chondrogenesis and vasculo/angiogenesis. These effects are regulated through signaling via integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch via direct binding to CCN family proteins. Due to the important roles of CCN family proteins in skeletal development, abnormal expression of CCN proteins is related to the tumorigenesis of primary bone tumors such as osteosarcoma, Ewing sarcoma, and chondrosarcoma. Additionally, emerging studies have suggested that CCN proteins may affect progression of secondary metastatic bone tumors by moderating the bone microenvironment. CCN proteins could therefore serve as potential therapeutic targets for drug development against primary and metastatic bone tumors. PMID:24551846

  20. Screening Bicyclic Peptide Libraries for Protein-Protein Interaction Inhibitors: Discovery of a Tumor Necrosis Factor-alpha Antagonist

    PubMed Central

    Rhodes, Curran A.; Liu, Yusen; Pei, Dehua

    2013-01-01

    Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-alpha (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions. PMID:23865589

  1. Genetically engineered endostatin-lidamycin fusion proteins effectively inhibit tumor growth and metastasis

    PubMed Central

    2013-01-01

    Background Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. Methods In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. Results ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. Conclusions The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor

  2. Characteristic Formation of Hyaluronan-Cartilage Link Protein-Proteoglycan Complex in Salivary Gland Tumors.

    PubMed

    Kuwabara, Hiroko; Nishikado, Akira; Hayasaki, Hana; Isogai, Zenzo; Yoneda, Masahiko; Kawata, Ryo; Hirose, Yoshinobu

    2016-01-01

    Hyaluronan (HA) and its binding molecules, cartilage link protein (LP) and proteoglycan (PG), are structural components of the hydrated extracellular matrix. Because these molecules play important roles in the tumor microenvironment, we examined the distribution of HA, LP, versican, and aggrecan in salivary gland tumors using histochemical and immunohistochemical methods, including double staining. LP was present in pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) tissues, and aggrecan was absent in the malignant tumors that we investigated. LP colocalized with both HA and aggrecan in the chondromyxoid matrix of PA, suggesting the presence of a HA-LP-aggrecan complex. Furthermore, the HA-LP-versican complex could be observed in the pseudocystic space of the cribriform structures in ACC. The characteristic HA-LP-PG complex in PA and ACC might play a role in the behavior of tumors, and immunohistochemical analysis of these molecules could represent a diagnostic adjunct for salivary gland tumors. PMID:26067139

  3. Bifidobacteria Expressing Tumstatin Protein for Antitumor Therapy in Tumor-Bearing Mice.

    PubMed

    Wei, C; Xun, A Y; Wei, X X; Yao, J; Wang, J Y; Shi, R Y; Yang, G H; Li, Y X; Xu, Z L; Lai, M G; Zhang, R; Wang, L-S; Zeng, W S

    2016-06-01

    Tumstatin (Tum) is a powerful angiostatin that inhibits proliferation and induces apoptosis of tumorous vascular endothelial cells. A nonpathogenic and anaerobic bacterium, Bifidobacterium longum (BL), selectively localizes to and proliferates in the hypoxia location within solid tumor. The aims of this study were to develop a novel delivery system for Tum using engineered Bifidobacterium and to investigate the inhibitory effect of Tum on tumor in mice. A vector that enabled the expression of Tum under the control of the pBBADs promoter of BL was constructed and transformed into BL NCC2705 by electroporation. The mouse colon carcinoma cells CT26 (1 × 10(7)/mL) were subcutaneously inserted in the left armpit of BALB/c mice. The tumor-bearing mice were treated with Tum-transformed BL, and green fluorescent protein (GFP)-transformed BL was used as a negative control. The microvessel density (MVD) in the transplanted tumor was determined, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling was used to detect apoptosis of vascular endothelial cells in transplanted tumor. The in vitro expression of Tum was examined in BL after l-arabinose induction. Bifidobacterium longum with pBBAD-Tum (BL-Tum) showed significant antitumor effect in tumor-bearing mice. The weight, volume, growth, and MVD, as well as the percentage of apoptotic vascular endothelial cells of transplanted tumors in the tumor-bearing mice treated with Tum-transformed BL were all significantly lower than those in the GFP negative control group. Intragastric administration, injection in tumor and vena caudalis injection of Tum-transformed BL exerted marked antitumor effects in tumor-bearing mice. This is the first demonstration of the utilization of Tum-transformed BL as a specific gene delivery system for treating tumor. PMID:25969440

  4. Fluorescence imaging of angiogenesis in green fluorescent protein-expressing tumors

    NASA Astrophysics Data System (ADS)

    Yang, Meng; Baranov, Eugene; Jiang, Ping; Li, Xiao-Ming; Wang, Jin W.; Li, Lingna; Yagi, Shigeo; Moossa, A. R.; Hoffman, Robert M.

    2002-05-01

    The development of therapeutics for the control of tumor angiogenesis requires a simple, reliable in vivo assay for tumor-induced vascularization. For this purpose, we have adapted the orthotopic implantation model of angiogenesis by using human and rodent tumors genetically tagged with Aequorea victoria green fluorescent protein (GFP) for grafting into nude mice. Genetically-fluorescent tumors can be readily imaged in vivo. The non-luminous induced capillaries are clearly visible against the bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. Fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. High-level GFP-expressing tumor cell lines made it possible to acquire the high-resolution real-time fluorescent optical images of angiogenesis in both primary tumors and their metastatic lesions in various human and rodent tumor models by means of a light-based imaging system. Intravital images of angiogenesis onset and development were acquired and quantified from a GFP- expressing orthotopically-growing human prostate tumor over a 19-day period. Whole-body optical imaging visualized vessel density increasing linearly over a 20-week period in orthotopically-growing, GFP-expressing human breast tumor MDA-MB-435. Vessels in an orthotopically-growing GFP- expressing Lewis lung carcinoma tumor were visualized through the chest wall via a reversible skin flap. These clinically-relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological micro- environments.

  5. [Role of proteins of the macroglobulin family in regulation of tumor growth].

    PubMed

    Zorin, N A; Zorina, V N; Zorina, R M

    2006-01-01

    Proteins of the macroglobulin family are an ancient and evolutionarily conservative link of the immune system, which is actively involved in both inhibition of tumor growth cells and proliferation of tumor cells. Two basically different binding sites and a great conformational plasticity of all representatives of the macroglobulin family, as well as the presence of two to four representatives of the family in the blood of most species allow them to transport diverse substances and exert various regulatory influences on both the tumor and the entire organism. For example, the capacity of macroglobulins for binding hydrolases makes it possible to inhibit enzyme mediated tumor invasion. At the same time, an excess of macroglobulin/hydrolase complexes can activate apoptosis. The tumor is able of using macroglobulins, especially pregnancy-associated proteins, for its own protection. Specifically, pregnancy-associated alpha2-glycoprotein, which is actively produced by human tumor cells, blocks the histocompatibility complex antigens. On the contrary, the capacity of binding zinc stimulates the thymulin-dependent activation of natural killer cells. Nevertheless, the actively growing tumor expresses many receptors to macroglobulins, which are the main carriers of some cytokines and growth factors essential for proliferation. PMID:16523653

  6. Tumoral Immune Suppression by Macrophages Expressing Fibroblast Activation Protein-Alpha and Heme Oxygenase-1

    PubMed Central

    Arnold, James N.; Magiera, Lukasz; Kraman, Matthew; Fearon, Douglas T.

    2013-01-01

    The depletion of tumor stromal cells that are marked by their expression of the membrane protein fibroblast activation protein-α (FAP) overcomes immune suppression and allows an anti-cancer cell immune response to control tumor growth. In subcutaneous tumors established with immunogenic Lewis lung carcinoma cells expressing ovalbumin (LL2/OVA), the FAP+ population comprises CD45+ and CD45− cells. In the present study, we further characterize the tumoral FAP+/CD45+ population as a minor sub-population of F4/80hi/CCR2+/CD206+ M2 macrophages. Using bone marrow chimeric mice in which the primate diphtheria toxin receptor (DTR) is restricted either to the FAP+/CD45+ or to the FAP+/CD45− subset, we demonstrate by conditionally depleting each subset that both independently contribute to the immune suppressive tumor microenvironment. A basis for the function of the FAP+/CD45+ subset is shown to be the immune inhibitory enzyme, heme oxygenase-1 (HO-1). The FAP+/CD45+ cells are the major tumoral source of HO-1, and an inhibitor of HO-1, Sn mesoporphyrin, causes the same extent of immune-dependent arrest of LL2/OVA tumor growth as does the depletion of these cells. Since this observation of immune suppression by HO-1 expressed by the FAP+/CD45+ stromal cell is replicated in a transplanted model of pancreatic ductal adenocarcinoma, we conclude that pharmacologically targeting this enzyme may improve cancer immunotherapy. PMID:24778275

  7. Peptide aptamer identified by molecular docking targeting translationally controlled tumor protein in leukemia cells.

    PubMed

    Kadioglu, Onat; Efferth, Thomas

    2016-08-01

    Bioinformatics screening and molecular docking analyses were utilized to select high affinity peptides targeting translationally controlled tumor protein (TCTP). Selected peptide aptamers were tested towards cancer cell lines with different levels of TCTP expression. One peptide (WGQWPYHC) revealed specific cytotoxicity according to the TCTP expression in tumor cells without affecting normal cells. Western blot analysis showed peptide-induced down-regulation of TCTP as primary target as well as of cell-cycle related downstream proteins (CDK2, CDK6, Cyclin D3) in MOLT-4 leukemia cells. "WGQWPYHC" deserves further analysis for targeted therapy of TCTP-expressing tumor cells. Graphical abstract Molecular docking on TCTP, cytotoxicity toward MOLT-4 leukemia cell line and downregulation of CDK2, CDK6, CyclinD3 and TCTP proteins. PMID:26972431

  8. AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group

    SciTech Connect

    Zhong Linlin; Shen Honglin; Huang Chenfei; Jing, Hongwu; Cao Deliang

    2011-08-15

    Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V{sub max} of 837.42 {+-} 81.39 nmol/mg/min, K{sub m} of 9.317 {+-} 2.25 mM and k{sub cat}/K{sub m} of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V{sub max} at 460.23 {+-} 28.12 nmol/mg/min, K{sub m} at 0.461 {+-} 0.09 mM and k{sub cat}/K{sub m} at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50 nM) and idarubicin (30 nM) to corresponding alcohols. Within 24 h, approximately 20 {+-} 2.7% of daunorubicin (1 {mu}M) or 23 {+-} 2.3% of idarubicin (1 {mu}M) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7 {+-} 0.9% and 5 {+-} 1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin. - Highlights: > This study defines enzyme activity of AKR1B10 protein towards daunorubicin, idarubicin

  9. Aldo–Keto Reductase 1B10 and Its Role in Proliferation Capacity of Drug-Resistant Cancers

    PubMed Central

    Matsunaga, Toshiyuki; Wada, Yasuhiro; Endo, Satoshi; Soda, Midori; El-Kabbani, Ossama; Hara, Akira

    2011-01-01

    The human aldo–keto reductase AKR1B10, originally identified as an aldose reductase-like protein and human small intestine aldose reductase, is a cytosolic NADPH-dependent reductase that metabolizes a variety of endogenous compounds, such as aromatic and aliphatic aldehydes and dicarbonyl compounds, and some drug ketones. The enzyme is highly expressed in solid tumors of several tissues including lung and liver, and as such has received considerable interest as a relevant biomarker for the development of those tumors. In addition, AKR1B10 has been recently reported to be significantly up-regulated in some cancer cell lines (medulloblastoma D341 and colon cancer HT29) acquiring resistance toward chemotherapeutic agents (cyclophosphamide and mitomycin c), suggesting the validity of the enzyme as a chemoresistance marker. Although the detailed information on the AKR1B10-mediated mechanisms leading to the drug resistance process is not well understood so far, the enzyme has been proposed to be involved in functional regulations of cell proliferation and metabolism of drugs and endogenous lipids during the development of chemoresistance. This article reviews the current literature focusing mainly on expression profile and roles of AKR1B10 in the drug resistance of cancer cells. Recent developments of AKR1B10 inhibitors and their usefulness in restoring sensitivity to anticancer drugs are also reviewed. PMID:22319498

  10. Increased PTP1B expression and phosphatase activity in colorectal cancer results in a more invasive phenotype and worse patient outcome

    PubMed Central

    Hoekstra, Elmer; Das, Asha M.; Swets, Marloes; Cao, Wanlu; van der Woude, C. Janneke; Bruno, Marco J.; Peppelenbosch, Maikel P.; Kuppen, Peter J.K.; ten Hagen, Timo L.M.; Fuhler, Gwenny M.

    2016-01-01

    Cell signaling is dependent on the balance between phosphorylation of proteins by kinases and dephosphorylation by phosphatases. This balance if often disrupted in colorectal cancer (CRC), leading to increased cell proliferation and invasion. For many years research has focused on the role of kinases as potential oncogenes in cancer, while phosphatases were commonly assumed to be tumor suppressive. However, this dogma is currently changing as phosphatases have also been shown to induce cancer growth. One of these phosphatases is protein tyrosine phosphatase 1B (PTP1B). Here we report that the expression of PTP1B is increased in colorectal cancer as compared to normal tissue, and that the intrinsic enzymatic activity of the protein is also enhanced. This suggests a role for PTP1B phosphatase activity in CRC formation and progression. Furthermore, we found that increased PTP1B expression is correlated to a worse patient survival and is an independent prognostic marker for overall survival and disease free survival. Knocking down PTP1B in CRC cell lines results in a less invasive phenotype with lower adhesion, migration and proliferation capabilities. Together, these results suggest that inhibition of PTP1B activity is a promising new target in the treatment of colorectal cancer and the prevention of metastasis. PMID:26942883

  11. Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction

    PubMed Central

    Fonseca, Catia; Soiffer, Robert; Ho, Vincent; Vanneman, Matthew; Jinushi, Masahisa; Ritz, Jerome; Neuberg, Donna; Stone, Richard; DeAngelo, Dan

    2009-01-01

    The identification of cancer antigens that contribute to transformation and are linked with immune-mediated tumor destruction is an important goal for immunotherapy. Toward this end, we screened a murine renal cell carcinoma cDNA expression library with sera from mice vaccinated with irradiated tumor cells engineered to secrete granulocyte macrophage colony-stimulating factor (GM-CSF). Multiple nonmutated, overexpressed proteins that function in tumor cell migration, protein/nucleic acid homeostasis, metabolism, and stress responses were detected. Among these, the most frequently recognized clone was protein disulfide isomerase (PDI). High titer antibodies to human PDI were similarly induced in an acute myeloid leukemia patient who achieved a complete response after vac-cination with irradiated, autologous GM-CSF–secreting tumor cells in the setting of nonmyeloablative allogeneic bone marrow transplantation. Moreover, ERp5, a closely related disulfide isomerase involved in major histocompatibility complex (MHC) class I chain-related protein A (MICA) shedding, also evoked potent humoral reactions in diverse solid and hematologic malignancy patients who responded to GM-CSF–secreting tumor cell vaccines or antibody blockade of cytotoxic T lymphocyte–associated antigen 4 (CTLA-4). Together, these findings reveal the unexpected immunogenicity of PDIs and raise the possibility that these gene products might serve as targets for therapeutic monoclonal antibodies. PMID:19008459

  12. Aldo-Keto Reductases 1B in Adrenal Cortex Physiology

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Martinez, Antoine; Lefrançois-Martinez, A. Marie

    2016-01-01

    Aldose reductase (AKR1B) proteins are monomeric enzymes, belonging to the aldo-keto reductase (AKR) superfamily. They perform oxidoreduction of carbonyl groups from a wide variety of substrates, such as aliphatic and aromatic aldehydes or ketones. Due to the involvement of human aldose reductases in pathologies, such as diabetic complications and cancer, AKR1B subgroup enzymatic properties have been extensively characterized. However, the issue of AKR1B function in non-pathologic conditions remains poorly resolved. Adrenal activities generated large amount of harmful aldehydes from lipid peroxidation and steroidogenesis, including 4-hydroxynonenal (4-HNE) and isocaproaldehyde (4-methylpentanal), which can both be reduced by AKR1B proteins. More recently, some AKR1B isoforms have been shown to be endowed with prostaglandin F synthase (PGFS) activity, suggesting that, in addition to possible scavenger function, they could instigate paracrine signals. Interestingly, the adrenal gland is one of the major sites for human and murine AKR1B expression, suggesting that their detoxifying/signaling activity could be specifically required for the correct handling of adrenal function. Moreover, chronic effects of ACTH result in a coordinated regulation of genes encoding the steroidogenic enzymes and some AKR1B isoforms. This review presents the molecular mechanisms accounting for the adrenal-specific expression of some AKR1B genes. Using data from recent mouse genetic models, we will try to connect their enzymatic properties and regulation with adrenal functions. PMID:27499746

  13. Aldo-Keto Reductases 1B in Endocrinology and Metabolism

    PubMed Central

    Pastel, Emilie; Pointud, Jean-Christophe; Volat, Fanny; Martinez, Antoine; Lefrançois-Martinez, Anne-Marie

    2012-01-01

    The aldose reductase (AR; human AKR1B1/mouse Akr1b3) has been the focus of many research because of its role in diabetic complications. The starting point of these alterations is the massive entry of glucose in polyol pathway where it is converted into sorbitol by this enzyme. However, the issue of AR function in non-diabetic condition remains unresolved. AR-like enzymes (AKR1B10, Akr1b7, and Akr1b8) are highly related isoforms often co-expressed with bona fide AR, making functional analysis of one or the other isoform a challenging task. AKR1B/Akr1b members share at least 65% protein identity and the general ability to reduce many redundant substrates such as aldehydes provided from lipid peroxidation, steroids and their by-products, and xenobiotics in vitro. Based on these properties, AKR1B/Akr1b are generally considered as detoxifying enzymes. Considering that divergences should be more informative than similarities to help understanding their physiological functions, we chose to review specific hallmarks of each human/mouse isoforms by focusing on tissue distribution and specific mechanisms of gene regulation. Indeed, although the AR shows ubiquitous expression, AR-like proteins exhibit tissue-specific patterns of expression. We focused on three organs where certain isoforms are enriched, the adrenal gland, enterohepatic, and adipose tissues and tried to connect recent enzymatic and regulation data with endocrine and metabolic functions of these organs. We presented recent mouse models showing unsuspected physiological functions in the regulation of glucido-lipidic metabolism and adipose tissue homeostasis. Beyond the widely accepted idea that AKR1B/Akr1b are detoxification enzymes, these recent reports provide growing evidences that they are able to modify or generate signal molecules. This conceptually shifts this class of enzymes from unenviable status of scavenger to upper class of messengers. PMID:22876234

  14. Suppression of Lipid Accumulation by Indole-3-Carbinol Is Associated with Increased Expression of the Aryl Hydrocarbon Receptor and CYP1B1 Proteins in Adipocytes and with Decreased Adipocyte-Stimulated Endothelial Tube Formation.

    PubMed

    Wang, Mei-Lin; Lin, Shyh-Hsiang; Hou, Yuan-Yu; Chen, Yue-Hwa

    2016-01-01

    This study investigated the effects of indole-3-carbinol (I3C) on adipogenesis- and angiogenesis-associated factors in mature adipocytes. The cross-talk between mature adipocytes and endothelial cells (ECs) was also explored by cultivating ECs in a conditioned medium (CM) by using I3C-treated adipocytes. The results revealed that I3C significantly inhibited triglyceride accumulation in mature adipocytes in association with significantly increased expression of AhR and CYP1B1 proteins as well as slightly decreased nuclear factor erythroid-derived factor 2-related factor 2, hormone-sensitive lipase, and glycerol-3-phosphate dehydrogenase expression by mature adipocytes. Furthermore, I3C inhibited CM-stimulated endothelial tube formation, which was accompanied by the modulated secretion of angiogenic factors in adipocytes, including vascular endothelial growth factor, interleukin-6, matrix metalloproteinases, and nitric oxide. In conclusion, I3C reduced lipid droplet accumulation in adipocytes and suppressed adipocyte-stimulated angiogenesis in ECs, suggesting that I3C is a potential therapeutic agent for treating obesity and obesity-associated disorders. PMID:27527145

  15. Suppression of Lipid Accumulation by Indole-3-Carbinol Is Associated with Increased Expression of the Aryl Hydrocarbon Receptor and CYP1B1 Proteins in Adipocytes and with Decreased Adipocyte-Stimulated Endothelial Tube Formation

    PubMed Central

    Wang, Mei-Lin; Lin, Shyh-Hsiang; Hou, Yuan-Yu; Chen, Yue-Hwa

    2016-01-01

    This study investigated the effects of indole-3-carbinol (I3C) on adipogenesis- and angiogenesis-associated factors in mature adipocytes. The cross-talk between mature adipocytes and endothelial cells (ECs) was also explored by cultivating ECs in a conditioned medium (CM) by using I3C-treated adipocytes. The results revealed that I3C significantly inhibited triglyceride accumulation in mature adipocytes in association with significantly increased expression of AhR and CYP1B1 proteins as well as slightly decreased nuclear factor erythroid-derived factor 2–related factor 2, hormone-sensitive lipase, and glycerol-3-phosphate dehydrogenase expression by mature adipocytes. Furthermore, I3C inhibited CM-stimulated endothelial tube formation, which was accompanied by the modulated secretion of angiogenic factors in adipocytes, including vascular endothelial growth factor, interleukin-6, matrix metalloproteinases, and nitric oxide. In conclusion, I3C reduced lipid droplet accumulation in adipocytes and suppressed adipocyte-stimulated angiogenesis in ECs, suggesting that I3C is a potential therapeutic agent for treating obesity and obesity-associated disorders. PMID:27527145

  16. Human autoreactive T cells recognize CD1b and phospholipids

    PubMed Central

    Van Rhijn, Ildiko; van Berlo, Twan; Hilmenyuk, Tamara; Cheng, Tan-Yun; Wolf, Benjamin J.; Tatituri, Raju V. V.; Uldrich, Adam P.; Napolitani, Giorgio; Cerundolo, Vincenzo; Altman, John D.; Willemsen, Peter; Huang, Shouxiong; Rossjohn, Jamie; Besra, Gurdyal S.; Brenner, Michael B.; Godfrey, Dale I.; Moody, D. Branch

    2016-01-01

    In contrast with the common detection of T cells that recognize MHC, CD1a, CD1c, or CD1d proteins, CD1b autoreactive T cells have been difficult to isolate in humans. Here we report the development of polyvalent complexes of CD1b proteins and carbohydrate backbones (dextramers) and their use in identifying CD1b autoreactive T cells from human donors. Activation is mediated by αβ T-cell receptors (TCRs) binding to CD1b-phospholipid complexes, which is sufficient to activate autoreactive responses to CD1b-expressing cells. Using mass spectrometry and T-cell responses to scan through the major classes of phospholipids, we identified phosphatidylglycerol (PG) as the immunodominant lipid antigen. T cells did not discriminate the chemical differences that distinguish mammalian PG from bacterial PG. Whereas most models of T-cell recognition emphasize TCR discrimination of differing self and foreign structures, CD1b autoreactive T cells recognize lipids with dual self and foreign origin. PG is rare in the cellular membranes that carry CD1b proteins. However, bacteria and mitochondria are rich in PG, so these data point to a more general mechanism of immune detection of infection- or stress-associated lipids. PMID:26621732

  17. Sequence and structure of the Drosophila melanogaster ovarian tumor gene and generation of an antibody specific for the ovarian tumor protein.

    PubMed Central

    Steinhauer, W R; Walsh, R C; Kalfayan, L J

    1989-01-01

    Sequencing cDNA and genomic DNA from the ovarian tumor gene revealed a gene with seven introns spanning 4.5 kilobases. The proline-rich, hydrophilic otu protein is novel. An antibody prepared to a beta-gal-otu fusion protein recognized a 110-kilodalton ovarian protein which was altered in the ovaries of otu gene mutants. Images PMID:2511440

  18. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  19. Polyetheylenimine-Polyplexes of Spiegelmer NOX-A50 Directed against Intracellular High Mobility Group Protein A1 (HMGA1) Reduce Tumor Growth in Vivo*

    PubMed Central

    Maasch, Christian; Vater, Axel; Buchner, Klaus; Purschke, Werner G.; Eulberg, Dirk; Vonhoff, Stefan; Klussmann, Sven

    2010-01-01

    High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer. PMID:20961861

  20. PRKAR1A gene analysis and protein kinase A activity in endometrial tumors.

    PubMed

    Tsigginou, A; Bimpaki, E; Nesterova, M; Horvath, A; Boikos, S; Lyssikatos, C; Papageorgiou, C; Dimitrakakis, C; Rodolakis, A; Stratakis, C A; Antsaklis, A

    2012-08-01

    PRKAR1A codes for the type 1a regulatory subunit (RIα) of the cAMP-dependent protein kinase A (PKA), an enzyme with an important role in cell cycle regulation and proliferation. PKA dysregulation has been found in various tumors, and PRKAR1A-inactivating mutations have been reported in mostly endocrine neoplasias. In this study, we investigated PKA activity and the PRKAR1A gene in normal and tumor endometrium. Specimens were collected from 31 patients with endometrial cancer. We used as controls 41 samples of endometrium that were collected from surrounding normal tissues or from women undergoing gynecological operations for other reasons. In all samples, we sequenced the PRKAR1A-coding sequence and studied PKA subunit expression; we also determined PKA activity and cAMP binding. PRKAR1A mutations were not found. However, PKA regulatory subunit protein levels, both RIα and those of regulatory subunit type 2b (RIIβ), were lower in tumor samples; cAMP binding was also lower in tumors compared with normal endometrium (P<0.01). Free PKA activity was higher in tumor samples compared with that of control tissue (P<0.01). There are significant PKA enzymatic abnormalities in tumors of the endometrium compared with surrounding normal tissue; as these were not due to PRKAR1A mutations, other mechanisms affecting PKA function ought to be explored. PMID:22461635

  1. The tumor protection effect of high-frequency administration of whole tumor cell vaccine and enhanced efficacy by the protein component from Agrocybe aegerita

    PubMed Central

    Liang, Yi; Sun, Hui

    2015-01-01

    Whole tumor cell vaccines have been widely studied and elicits limited immune responses because of the poor immunogenicity. In the present study, we discovered that high-frequency administration of irradiated whole tumor cell vaccine triggered rejection of tumor cells (90% or 100% of the mice that were vaccinated with irradiated H22 cells or S180 respectively were protected), and provided cross-protection and long-term anti-tumor immunity in BALB/c mouse models. The antitumor activity required CD4+, CD8+ T cells and macrophage that was proved in the nude mice and cell depletion mouse models. The adoptive transfer experiment suggested that repeated whole tumor cell vaccination successfully stimulated the anti-tumor response by activation of the immune cells. A high immunization frequency within a short period of time and the presence of glycosylated molecules and nucleic acids on the surface of intact tumor cells were crucial for the successful prevention of tumor growth by whole tumor cell vaccines. Moreover, Yt, the protein component from fungus Agrocybe aegerita, increased whole tumor cell vaccine-mediated tumor rejection and cross-protection effect. These data indicated that the frequency of administration of whole tumor cell vaccines was of critical importance for the efficacy, which needed to be integrated into vaccine strategies for producing potential vaccines. PMID:26221228

  2. The potential of transferrin-pendant-type polyethyleneglycol liposomes encapsulating decahydrodecaborate-{sup 1}B (GB-10) as {sup 1}B-carriers for boron neutron capture therapy

    SciTech Connect

    Masunaga, Shin-ichiro . E-mail: smasuna@rri.kyoto-u.ac.jp; Kasaoka, Satoshi; Maruyama, Kazuo; Nigg, David; Sakurai, Yoshinori; Nagata, Kenji; Suzuki, Minoru; Kinashi, Yuko; Maruhashi, Akira; Ono, Koji

    2006-12-01

    Purpose: To evaluate GB-10-encapsulating transferrin (TF)-pendant-type polyethyleneglycol (PEG) liposomes as tumor-targeting {sup 1}B-carriers for boron neutron capture therapy. Methods and Materials: A free mercaptoundecahydrododecaborate-{sup 1}B (BSH) or decahydrodecaborate-{sup 1}B (GB-10) solution, bare liposomes, PEG liposomes, or TF-PEG liposomes were injected into SCC VII tumor-bearing mice, and {sup 1}B concentrations in the tumors and normal tissues were measured by {gamma}-ray spectrometry. Meanwhile, tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all intratumor proliferating cells, then injected with these {sup 1}B-carriers containing BSH or GB-10 in the same manner. Right after thermal neutron irradiation, the response of quiescent (Q) cells was assessed in terms of the micronucleus frequency using immunofluorescence staining for BrdU. The frequency in the total tumor cells was determined from the BrdU nontreated tumors. Results: Transferrin-PEG liposomes showed a prolonged retention in blood circulation, low uptake by reticuloendothelial system, and the most enhanced accumulation of {sup 1}B in solid tumors. In general, the enhancing effects were significantly greater in total cells than Q cells. In both cells, the enhancing effects of GB-10-containing {sup 1}B-carriers were significantly greater than BSH-containing {sup 1}B-carriers, whether loaded in free solution or liposomes. In both cells, whether BSH or GB-10 was employed, the greatest enhancing effect was observed with TF-PEG liposomes followed in decreasing order by PEG liposomes, bare liposomes, and free BSH or GB-10 solution. In Q cells, the decrease was remarkable between PEG and bare liposomes. Conclusions: In terms of biodistribution characteristics and tumor cell-killing effect as a whole, including Q cells, GB-10 TF-PEG liposomes were regarded as promising {sup 1}B-carriers.

  3. SIRT3 and SIRT4 are mitochondrial tumor suppressor proteins that connect mitochondrial metabolism and carcinogenesis

    PubMed Central

    2014-01-01

    It is a well-established scientific observation that mammalian cells contain fidelity proteins that appear to protect against and adapt to various forms of endogenous and exogenous cellular conditions. Loss of function or genetic mutation of these fidelity proteins has also been shown to create a cellular environment that is permissive for the development of tumors, suggesting that these proteins also function as tumor suppressors (TSs). While the first identified TSs were confined to either the nucleus and/or the cytoplasm, it seemed logical to hypothesize that the mitochondria may also contain fidelity proteins that serve as TSs. In this regard, it now appears clear that at least two mitochondrial sirtuins function as sensing, watchdog, or TS proteins in vitro, in vivo, and in human tumor samples. In addition, these new results demonstrate that the mitochondrial anti-aging or fidelity/sensing proteins, SIRT3 and SIRT4, respond to changes in cellular nutrient status to alter the enzymatic activity of specific downstream targets to maintain energy production that matches energy availability and ATP consumption. As such, it is proposed that loss of function or genetic deletion of these mitochondrial genes results in a mismatch of mitochondrial energy metabolism, culminating in a cell phenotype permissive for transformation and tumorigenesis. In addition, these findings clearly suggest that loss of proper mitochondrial metabolism, via loss of SIRT3 and SIRT4, is sufficient to promote carcinogenesis. PMID:25332769

  4. SIRT3 and SIRT4 are mitochondrial tumor suppressor proteins that connect mitochondrial metabolism and carcinogenesis.

    PubMed

    Zhu, Yueming; Yan, Yufan; Principe, Daniel R; Zou, Xianghui; Vassilopoulos, Athanassios; Gius, David

    2014-01-01

    It is a well-established scientific observation that mammalian cells contain fidelity proteins that appear to protect against and adapt to various forms of endogenous and exogenous cellular conditions. Loss of function or genetic mutation of these fidelity proteins has also been shown to create a cellular environment that is permissive for the development of tumors, suggesting that these proteins also function as tumor suppressors (TSs). While the first identified TSs were confined to either the nucleus and/or the cytoplasm, it seemed logical to hypothesize that the mitochondria may also contain fidelity proteins that serve as TSs. In this regard, it now appears clear that at least two mitochondrial sirtuins function as sensing, watchdog, or TS proteins in vitro, in vivo, and in human tumor samples. In addition, these new results demonstrate that the mitochondrial anti-aging or fidelity/sensing proteins, SIRT3 and SIRT4, respond to changes in cellular nutrient status to alter the enzymatic activity of specific downstream targets to maintain energy production that matches energy availability and ATP consumption. As such, it is proposed that loss of function or genetic deletion of these mitochondrial genes results in a mismatch of mitochondrial energy metabolism, culminating in a cell phenotype permissive for transformation and tumorigenesis. In addition, these findings clearly suggest that loss of proper mitochondrial metabolism, via loss of SIRT3 and SIRT4, is sufficient to promote carcinogenesis. PMID:25332769

  5. [Suppressive effect of protein kinase C inhibitors on tumor cell function via phosphorylation of p53 protein in mice].

    PubMed

    Nakamura, K; Shinozuka, K; Kunitomo, M

    2000-12-01

    We examined the role of protein kinase C (PKC) in the phosphorylation of a p53 protein. Exposure to a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), increased the phosphorylation of the wild type p53 protein, whereas exposure to a tumor promoter phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), decreased it in vivo after incubation with mouse epidermal JB6 cells for 3 h. Exposure to a cAMP dependent protein kinase (PKA) activator, forskolin, did not decrease the phosphorylation of p53 protein. In the transient transfection/luciferase reporter transactivation assay, H7 slightly increased the mouse double minute (MDM) 2 reporter transactivation activity of the p53 protein after treatment for 24 h, whereas TPA completely blocked it. Exposure to H7 and a specific PKC inhibitor, bisindolylmaleimide (bis), dose-dependently reduced the lung-colonizing potential of highly metastatic B16-F10 mouse melanoma cells in syngeneic mice. These results suggest that the phosphorylation of the wild type p53 protein is inversely related to PKC activation, and also suggest that the phosphorylation of the p53 protein is involved in the function of its transcription factor. The PKC inhibitor may exhibit a potent anti-metastatic effect through the phosphorylation of wild type p53 protein and the activation of its function. PMID:11193387

  6. Establishment of tumor-associated immunity requires interaction of Heat Shock Proteins with CD91

    PubMed Central

    Zhou, Yu Jerry; Messmer, Michelle Nicole; Binder, Robert Julian

    2014-01-01

    Host antitumor adaptive immune responses are generated as a result of the body’s immunosurveillance mechanisms. How the antitumor immune response is initially primed remains unclear, given that soluble tumor antigens generally are quantitatively insufficient for cross-priming and tumors lack the classical pathogen-associated molecular patterns (PAMPs) to activate costimulation and initiate cross-priming. We explored the interaction of the tumor-derived heat-shock proteins (HSP) with their common receptor (CD91) on antigen presenting cells (APCs) as a mechanism for host-priming of T cell-mediated antitumor immunity. Using targeted genetic disruption of the interaction between HSPs and CD19, we demonstrated that specific ablation of CD91 in APCs prevented the establishment of antitumor immunity. The antitumor immunity was also inhibited when the transfer of tumor-derived HSPs to APCs was prevented using an endogenous inhibitor of CD91. Inhibition was manifested in a reduction of cross-presentation of tumor-derived antigenic peptides in the lymph nodes providing a molecular basis for the observed immunity associated with tumor development. Our findings demonstrate that early in tumor development, the HSP-CD91 pathway is critical for the establishment of antitumor immunity. PMID:24778318

  7. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity

    PubMed Central

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP+ microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  8. Establishment of tumor-associated immunity requires interaction of heat shock proteins with CD91.

    PubMed

    Zhou, Yu Jerry; Messmer, Michelle Nicole; Binder, Robert Julian

    2014-03-01

    Host antitumor adaptive immune responses are generated as a result of the body's immunosurveillance mechanisms. How the antitumor immune response is initially primed remains unclear, given that soluble tumor antigens generally are quantitatively insufficient for cross-priming and tumors generally lack the classical pathogen-associated molecular patterns to activate costimulation and initiate cross-priming. We explored the interaction of the tumor-derived heat shock proteins (HSP) with their common receptor (CD91) on antigen-presenting cells (APC) as a mechanism for host-priming of T-cell-mediated antitumor immunity. Using targeted genetic disruption of the interaction between HSPs and CD91, we demonstrated that specific ablation of CD91 in APCs prevented the establishment of antitumor immunity. The antitumor immunity was also inhibited when the transfer of tumor-derived HSPs to APCs was prevented using an endogenous inhibitor of CD91. Inhibition was manifested in a reduction of cross-presentation of tumor-derived antigenic peptides in the lymph nodes, providing a molecular basis for the observed immunity associated with tumor development. Our findings demonstrate that early in tumor development, the HSP-CD91 pathway is critical for the establishment of antitumor immunity. PMID:24778318

  9. In Vivo Visualization of Tumor Antigen-containing Microparticles Generated in Fluorescent-protein-elicited Immunity.

    PubMed

    Yang, Fei; Liu, Shun; Liu, Xiuli; Liu, Lei; Luo, Meijie; Qi, Shuhong; Xu, Guoqiang; Qiao, Sha; Lv, Xiaohua; Li, Xiangning; Fu, Ling; Luo, Qingming; Zhang, Zhihong

    2016-01-01

    In vivo optical spatio-temporal imaging of the tumor microenvironment is useful to explain how tumor immunotherapies work. However, the lack of fluorescent antigens with strong immunogenicity makes it difficult to study the dynamics of how tumors are eliminated by any given immune response. Here, we develop an effective fluorescent model antigen based on the tetrameric far-red fluorescent protein KatushkaS158A (tfRFP), which elicits both humoral and cellular immunity. We use this fluorescent antigen to visualize the dynamic behavior of immunocytes as they attack and selectively eliminate tfRFP-expressing tumors in vivo; swarms of immunocytes rush toward tumors with high motility, clusters of immunocytes form quickly, and numerous antigen-antibody complexes in the form of tfRFP(+) microparticles are generated in the tumor areas and ingested by macrophages in the tumor microenvironment. Therefore, tfRFP, as both a model antigen and fluorescent reporter, is a useful tool to visualize specific immune responses in vivo. PMID:27375792

  10. Modification of an apparatus for tumor-suppressor protein crystal growth in the International Space Station

    NASA Astrophysics Data System (ADS)

    de Morais Mendonca Teles, Antonio

    Some human diseases as tumors are being studied continuously for the development of vaccines against them. And a way of doing that is by means of proteins research. There are some kinds of proteins, like the p53 and p73 proteins, which are tumor suppressors. There are other diseases such as A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases which are protein-related. The determination of how proteins geometrically order themselves, during its biological functions is very necessary to understand how a protein's structure affects its function, to design vaccines that intercede in tumor-protein activities and in other proteins related to those other diseases. The protein crystal growth in microgravity environment produces purer crystallization than on the ground, and it is a powerful tool to produce better vaccines. Several data have already been acquired using ground-based research and in spaceflight experiments aboard the Spacelab and Space Shuttle missions, and in the MIR and in the International Space Station (ISS). Here in this paper, I propose to be performed in the ISS Biological Research Facility (which is being developed), multiple crystal growth of proteins related to cancer (as tumors suppressors and oncoproteins), A.I.D.S., hansenosis, the Parkinson's and Chagas' diseases, for the future obtaining of possible vaccines against them. I also propose a simple and practical equipment, a modification of the crystallization plates (which use a vapor diffusion technique) inside each cylinder of the Protein Crystallization Apparatus in Microgravity (PCAM), with multiple chambers with different sizes. Instead of using some chambers with the same size it is better to use several chambers with different sizes. Why is that? The answer is: the energy associated with the surface tension of the liquid in the chamber is directly related to the circle area of it. So, to minimize the total energy of the surface tension of a proteins liquid -making it more stable

  11. Redistribution of the discs large tumor suppressor protein during mitosis.

    PubMed

    Massimi, Paola; Gardiol, Daniela; Roberts, Sally; Banks, Lawrence

    2003-11-01

    Drosophila discs large (Dlg) has been shown to be an essential regulator of cell polarity and attachment, and is classified as a potential tumour suppressor in higher eukaryotes. Human Dlg is expressed in epithelial cells at sites of cell-cell contact and acts as a negative regulator of cell growth. Although hDlg has been shown to be phosphorylated during mitosis, little is known about its activity during this stage of the cell cycle. To investigate this further we have analysed in detail the pattern of hDlg expression during mitotic cell division. In early mitosis there is a marked increase in membrane-bound hDlg which is then retained throughout mitosis, while during cytokinesis, there is a specific concentration of hDlg at the midbody. Using mutants of Dlg we show that this is mediated by sequences in the carboxy terminal region of Dlg, but it does not require the SH3 or PDZ domains, and is independent of binding to protein 4.1. Finally, using a mutant of Dlg that consists of just this carboxy terminal region of the protein, we show that it can compete with endogenous hDlg for midbody accumulation, and this mutant also gives rise to altered cell growth. We conclude that localisation of Dlg to the midbody indicates a role for Dlg at this critical point in cytokinesis. PMID:14567986

  12. Heat Shock Protein-70 Inhibition by the Small-Molecule 2-phenylethynesulfonamide Impairs Protein Clearance Pathways in Tumor Cells

    PubMed Central

    Leu, J. I-Ju; Pimkina, Julia; Pandey, Pooja; Murphy, Maureen E.; George, Donna L.

    2011-01-01

    The evolutionarily conserved stress-inducible HSP70 molecular chaperone plays a central role in maintaining protein quality control in response to various forms of stress. Constitutively elevated HSP70 expression is a characteristic of many tumor cells and contributes to their survival. We recently identified the small-molecule 2-phenylethyenesulfonamide (PES) as a novel HSP70 inhibitor. Here we present evidence that PES-mediated inhibition of HSP70-family proteins in tumor cells results in an impairment of the two major protein degradation systems, namely the autophagy-lysosome system as well as the proteasome pathway. HSP70-family proteins work closely with the HSP90 molecular chaperone to maintain the stability and activities of their many client proteins and PES causes a disruption in the HSP70/HSP90 chaperone system. As a consequence, many cellular proteins, including known HSP70/HSP90 substrates, accumulate in detergent-insoluble cell fractions, indicative of aggregation and functional inactivation. Overall, PES simultaneously disrupts several cancer-critical survival pathways, supporting the idea of targeting HSP70 as a potential approach for cancer therapeutics. PMID:21636681

  13. ALDH1B1 links alcohol consumption and diabetes.

    PubMed

    Singh, Surendra; Chen, Ying; Matsumoto, Akiko; Orlicky, David J; Dong, Hongbin; Thompson, David C; Vasiliou, Vasilis

    2015-08-01

    Aldehyde dehydrogenase 1B1 (ALDH1B1) is a mitochondrial enzyme sharing 65% and 72% sequence identity with ALDH1A1 and ALDH2 proteins, respectively. Compared to the latter two ALDH isozymes, little is known about the physiological functions of ALDH1B1. Studies in humans indicate that ALDH1B1 may be associated with alcohol sensitivity and stem cells. Our recent in vitro studies using human ALDH1B1 showed that it metabolizes acetaldehyde and retinaldehyde. To investigate the in vivo role of ALDH1B1, we generated and characterized a global Aldh1b1 knockout mouse line. These knockout (KO) mice are fertile and show overtly good health. However, ethanol pharmacokinetic analysis revealed ∼40% increase in blood acetaldehyde levels in KO mice. Interestingly, the KO mice exhibited higher fasting blood glucose levels. Collectively, we show for the first time the functional in vivo role of ALDH1B1 in acetaldehyde metabolism and in maintaining glucose homeostasis. This mouse model is a valuable tool to investigate the mechanism by which alcohol may promote the development of diabetes. PMID:26086111

  14. Cyclin D1b overexpression inhibits cell proliferation and induces cell apoptosis in cervical cancer cells in vitro and in vivo

    PubMed Central

    Wang, Ning; Wei, Heng; Yin, Duo; Lu, Yanming; Zhang, Yao; Jiang, Di; Jiang, Yan; Zhang, Shulan

    2014-01-01

    Cyclin D1b is one of two proteins translated from cyclin D1 transcripts (isoforms a and b) that are generated due to gene polymorphism. Our previous study has reported low cyclin D1b expression in cervical cancer tissue, with an expression level in moderately or poorly differentiated tissues that was significantly lower than that in well-differentiated tissues. However, the functional role of cyclin D1b in cervical cancer remains to be elucidated. In this study, using a cervical cancer cell line with stable expression of cyclin D1b, we found that upregulation of cyclin D1b initiated cell cycle arrest at the G0/G1 phase and induced apoptosis, thereby inhibiting cell proliferation and colony formation. Furthermore, xenograft transplantation experiments in nude mice demonstrated that cyclin D1b upregulation inhibited cancer growth and induce apoptosis in vivo. In conclusion, the present study indicates anti-tumor effects of cyclin D1b in cervical cancer, suggesting that cyclin D1b may represent a potential therapeutic target for cervical cancer. PMID:25120779

  15. Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab

    PubMed Central

    Mukaihara, Kenta; Suehara, Yoshiyuki; Kohsaka, Shinji; Akaike, Keisuke; Tanabe, Yu; Kubota, Daisuke; Ishii, Midori; Fujimura, Tsutomu; Kazuno, Saiko; Okubo, Taketo; Takagi, Tatsuya; Yao, Takashi; Kaneko, Kazuo; Saito, Tsuyoshi

    2016-01-01

    Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression

  16. Tumor

    MedlinePlus

    ... be removed because of their location or harmful effect on the surrounding normal brain tissue. If a tumor is cancer , possible treatments may include: Chemotherapy Radiation Surgery Targeted cancer therapy Biologic therapy Other treatment options

  17. Clinicopathologic significance of immunostaining of α-thalassemia/mental retardation syndrome X-linked protein and death domain-associated protein in neuroendocrine tumors.

    PubMed

    Chen, Shi-Fan; Kasajima, Atsuko; Yazdani, Samaneh; Chan, Monica S M; Wang, Lin; He, Yang-Yang; Gao, Hong-Wen; Sasano, Hironobu

    2013-10-01

    α-Thalassemia/mental retardation syndrome X-linked protein (ATRX) and death domain-associated protein (DAXX) genes are tumor suppressors whose mutations have been identified in sporadic pancreatic neuroendocrine tumors as well as in patients with MEN1. However, it is unknown whether ATRX and DAXX alterations are specific for pancreatic neuroendocrine tumor. In addition, the association of ATRX/DAXX protein loss with tumor cell proliferation has not been examined. We, therefore, immunostained ATRX and DAXX in 10 gastric, 15 duodenal, 20 rectal, 70 pancreatic, and 22 pulmonary neuroendocrine tumors with 15 nonneoplastic pancreases and 27 pancreatic adenocarcinomas to elucidate the site-specific roles of ATRX/DAXX abnormalities. At least 1 loss of ATRX and DAXX immunoreactivity was detected in all neuroendocrine tumor cases but not in any of nonneoplastic pancreatic tissues or pancreatic adenocarcinomas. The loss of DAXX protein was correlated with the Ki-67 index (ATRX, P = .904; DAXX, P = .044). The status of DAXX immunoreactivity correlated positively with World Health Organization histologic grade (P = .026). These results suggest that the status of ATRX or DAXX protein loss in neuroendocrine tumor differed among the organs in which these tumors arose, and these proteins may play site-specific roles in the development of these tumors. PMID:23954140

  18. Immunohistochemical detection of NRAS(Q61R) protein in follicular-patterned thyroid tumors.

    PubMed

    Oishi, Naoki; Kondo, Tetsuo; Vuong, Huy Gia; Nakazawa, Tadao; Mochizuki, Kunio; Kasai, Kazunari; Inoue, Tomohiro; Tahara, Ippei; Hirokawa, Mitsuyoshi; Miyauchi, Akira; Katoh, Ryohei

    2016-07-01

    The NRAS(A182G) mutation, which results in the NRAS(Q61R) protein, is a major driver mutation in follicular-patterned thyroid neoplasms. Although new immunohistochemistry (IHC) for NRAS(Q61R) is now available, its sensitivity, specificity, and diagnostic utility for thyroid tumors are not yet established. We performed IHC for NRAS(Q61R) and direct sequencing for NRAS codon 61 in 4 thyroid cancer-derived cell lines and 98 follicular-patterned thyroid tumors that included 22 follicular thyroid adenomas (FTAs), 35 follicular thyroid carcinomas (FTCs), and 41 cases of nodular hyperplasia (NH). In the tumors with NRAS(Q61R), the expression of BRAF(V600E) was further evaluated immunohistochemically. Two cell lines with NRAS(A182G) showed selective immunoreactivity for NRAS(Q61R). In tumor tissues, NRAS(Q61R) IHC was positive in 18% (4/22), 29% (10/35), and 2% (1/41) of FTAs, FTCs, and NH samples, respectively. The frequencies of the NRAS(Q61R) in FTAs and FTCs were significantly higher than that in NH (P=.046 and P=.001, respectively). All tumors with NRAS(Q61R) expression exhibited uniform cytoplasmic positivity with or without accumulation in their cell membranes. Of the 15 tumors with NRAS(Q61R) expression, 13 cases showed NRAS(A182G) in direct sequencing, whereas all of the tumors without NRAS(Q61R) expression were negative for the mutation. There were no tumors with overlapping expression of NRAS(Q61R) and BRAF(V600E). In reference to the direct sequencing, sensitivity and specificity of the NRAS(Q61R) IHC were 100% and 98%, respectively. In conclusion, NRAS(Q61R) IHC is a highly sensitive and specific tool that is useful for differentiating follicular-patterned thyroid tumors. PMID:26980032

  19. Mass spectrometric imaging of red fluorescent protein in breast tumor xenografts.

    PubMed

    Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T; Greenwood, Tiffany R; Raman, Venu; Bhujwalla, Zaver M; Heeren, Ron M A; Glunde, Kristine

    2013-05-01

    Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters. PMID:23184411

  20. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

    PubMed

    Justus, Calvin R; Dong, Lixue; Yang, Li V

    2013-01-01

    The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention. PMID:24367336

  1. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors

    PubMed Central

    Justus, Calvin R.; Dong, Lixue; Yang, Li V.

    2013-01-01

    The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention. PMID:24367336

  2. Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors

    PubMed Central

    Kang, Xunlei; Kim, Jaehyup; Deng, Mi; John, Samuel; Chen, Heyu; Wu, Guojin; Phan, Hiep; Zhang, Cheng Cheng

    2016-01-01

    ABSTRACT Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology – as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development. PMID:26636629

  3. Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors.

    PubMed

    Kang, Xunlei; Kim, Jaehyup; Deng, Mi; John, Samuel; Chen, Heyu; Wu, Guojin; Phan, Hiep; Zhang, Cheng Cheng

    2016-01-01

    Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1-5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit protein tyrosine phosphatase non-receptor type 6 (PTPN6 or SHP-1), protein tyrosine phosphatase non-receptor type 11 (PTPN11 or SHP-2), or Src homology 2 domain-containing inositol phosphatase (SHIP), leading to negative regulation of immune cell activation. Certain of these receptors also play regulatory roles in neuronal activity and osteoclast development. The activation of LILRBs on immune cells by their ligands may contribute to immune evasion by tumors. Recent studies found that several members of LILRB family are expressed by tumor cells, notably hematopoietic cancer cells, and may directly regulate cancer development and relapse as well as the activity of cancer stem cells. LILRBs thus have dual concordant roles in tumor biology - as immune checkpoint molecules and as tumor-sustaining factors. Importantly, the study of knockout mice indicated that LILRBs do not affect hematopoiesis and normal development. Therefore LILRBs may represent ideal targets for tumor treatment. This review aims to summarize current knowledge on expression patterns, ligands, signaling, and functions of LILRB family members in the context of cancer development. PMID:26636629

  4. Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties*

    PubMed Central

    Lorey, Susan; Fiedler, Erik; Kunert, Anja; Nerkamp, Jörg; Lange, Christian; Fiedler, Markus; Bosse-Doenecke, Eva; Meysing, Maren; Gloser, Manja; Rundfeldt, Chris; Rauchhaus, Una; Hänssgen, Ilka; Göttler, Thomas; Steuernagel, Arnd; Fiedler, Ulrike; Haupts, Ulrich

    2014-01-01

    Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies. PMID:24474690

  5. Tumor suppressor protein SMAR1 modulates the roughness of cell surface: combined AFM and SEM study

    PubMed Central

    2009-01-01

    Background Imaging tools such as scanning electron microscope (SEM) and atomic force microscope (AFM) can be used to produce high-resolution topographic images of biomedical specimens and hence are well suited for imaging alterations in cell morphology. We have studied the correlation of SMAR1 expression with cell surface smoothness in cell lines as well as in different grades of human breast cancer and mouse tumor sections. Methods We validated knockdown and overexpression of SMAR1 using RT-PCR as well as Western blotting in human embryonic kidney (HEK) 293, human breast cancer (MCF-7) and mouse melanoma (B16F1) cell lines. The samples were then processed for cell surface roughness studies using atomic force microscopy (AFM) and scanning electron microscopy (SEM). The same samples were used for microarray analysis as well. Tumors sections from control and SMAR1 treated mice as well as tissues sections from different grades of human breast cancer on poly L-lysine coated slides were used for AFM and SEM studies. Results Tumor sections from mice injected with melanoma cells showed pronounced surface roughness. In contrast, tumor sections obtained from nude mice that were first injected with melanoma cells followed by repeated injections of SMAR1-P44 peptide, exhibited relatively smoother surface profile. Interestingly, human breast cancer tissue sections that showed reduced SMAR1 expression exhibited increased surface roughness compared to the adjacent normal breast tissue. Our AFM data establishes that treatment of cells with SMAR1-P44 results into increase in cytoskeletal volume that is supported by comparative gene expression data showing an increase in the expression of specific cytoskeletal proteins compared to the control cells. Altogether, these findings indicate that tumor suppressor function of SMAR1 might be exhibited through smoothening of cell surface by regulating expression of cell surface proteins. Conclusion Tumor suppressor protein SMAR1 might be

  6. Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide.

    PubMed

    Jeong, Hyeon-Uk; Kwon, Mihwa; Lee, Yongnam; Yoo, Ji Seok; Shin, Dae Hee; Song, Im-Sook; Lee, Hye Suk

    2015-01-01

    We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (K m) =41.5 μM, maximum uptake rate (V max) =46.2 pmol/minute, and intrinsic clearance (CL int) =1.11 μL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CL int values of 0.035 and 0.034 μL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 μM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb-drug interactions of catalposide, although their clinical relevance awaits further evaluation. PMID:25653502

  7. Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide

    PubMed Central

    Jeong, Hyeon-Uk; Kwon, Mihwa; Lee, Yongnam; Yoo, Ji Seok; Shin, Dae Hee; Song, Im-Sook; Lee, Hye Suk

    2015-01-01

    We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (Km) =41.5 μM, maximum uptake rate (Vmax) =46.2 pmol/minute, and intrinsic clearance (CLint) =1.11 μL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CLint values of 0.035 and 0.034 μL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 μM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb–drug interactions of catalposide, although their clinical relevance awaits further evaluation. PMID:25653502

  8. Regulation of protein translation and c-Jun expression by prostate tumor overexpressed 1.

    PubMed

    Marqués, N; Sesé, M; Cánovas, V; Valente, F; Bermudo, R; de Torres, I; Fernández, Y; Abasolo, I; Fernández, P L; Contreras, H; Castellón, E; Celià-Terrassa, T; Méndez, R; Ramón Y Cajal, S; Thomson, T M; Paciucci, R

    2014-02-27

    Prostate tumor overexpressed-1 (PTOV1), a modulator of the Mediator transcriptional regulatory complex, is expressed at high levels in prostate cancer and other neoplasias in association with a more aggressive disease. Here we show that PTOV1 interacts directly with receptor of activated protein C kinase 1 (RACK1), a regulator of protein kinase C and Jun signaling and also a component of the 40S ribosome. Consistent with this interaction, PTOV1 was associated with ribosomes and its overexpression promoted global protein synthesis in prostate cancer cells and COS-7 fibroblasts in a mTORC1-dependent manner. Transfection of ectopic PTOV1 enhanced the expression of c-Jun protein without affecting the levels of c-Jun or RACK1 mRNA. Conversely, knockdown of PTOV1 caused significant declines in global protein synthesis and c-Jun protein levels. High levels of PTOV1 stimulated the motility and invasiveness of prostate cancer cells, which required c-Jun, whereas knockdown of PTOV1 strongly inhibited the tumorigenic and metastatic potentials of PC-3 prostate cancer cells. In human prostate cancer samples, the expression of high levels of PTOV1 in primary and metastatic tumors was significantly associated with increased nuclear localization of active c-Jun. These results unveil new functions of PTOV1 in the regulation of protein translation and in the progression of prostate cancer to an invasive and metastatic disease. PMID:23455324

  9. Gastrointestinal stromal tumors regularly express synaptic vesicle proteins: evidence of a neuroendocrine phenotype.

    PubMed

    Bümming, Per; Nilsson, Ola; Ahlman, Håkan; Welbencer, Anna; Andersson, Mattias K; Sjölund, Katarina; Nilsson, Bengt

    2007-09-01

    Gastrointestinal stromal tumors (GISTs) are thought to originate from the interstitial cells of Cajal, which share many properties with neurons of the gastrointestinal tract. Recently, we demonstrated expression of the hormone ghrelin in GIST. The aim of the present study was therefore to evaluate a possible neuroendocrine phenotype of GIST. Specimens from 41 GISTs were examined for the expression of 12 different synaptic vesicle proteins. Expression of synaptic-like microvesicle proteins, e.g., Synaptic vesicle protein 2 (SV2), synaptobrevin, synapsin 1, and amphiphysin was demonstrated in a majority of GISTs by immunohistochemistry, western blotting, and quantitative reversetranscriptase PCR. One-third of the tumors also expressed the large dense core vesicle protein vesicular monoamine transporter 1. Presence of microvesicles and dense core vesicles in GIST was confirmed by electron microscopy. The expression of synaptic-like microvesicle proteins in GIST was not related to risk profile or to KIT/platelet derived growth factor alpha (PDGFRA) mutational status. Thus, GISTs regularly express a subset of synaptic-like microvesicle proteins necessary for the regulated secretion of neurotransmitters and hormones. Expression of synaptic-like micro-vesicle proteins, ghrelin and peptide hormone receptors in GIST indicate a neuroendocrine phenotype and suggest novel possibilities to treat therapy-resistant GIST. PMID:17914114

  10. The role of endogenous proteins in the protein-free maintenance of 3 distinct tumor-cell lines invitro.

    PubMed

    Watanabe, H; Chigira, M

    1992-09-01

    We established new two protein-free culture subclones from murine well-characterized Ehrlich ascites carcinoma and P815 mastocytoma using intermittent protein-free culture performed previously for a protein-free subclone of fibrosarcoma (Gc-4 PF). The Ehrlich protein-free subclone (Ehrlich PF) grew much more slowly than the original cell line and showed a proliferative response to FCS. On the other hand, like Gc-4 PF, the P815 protein-free subclone (P815 PF) showed a similar growth rate to that of the original counterpart. Interestingly the original P815 mastocytoma cells also grew exponentially in protein-free medium. Although the protein-free culture exhibited cells that were more spheroid and spread less in each of these three cell lines, the major structure protein bands demonstrated on SDS-PAGE were virtually identical between the original and protein-free culture cells. In contrast to the structural peptides, the distribution of the secretory peptide differed among the three protein-free culture cell lines, which may reflect their state of differentiation. Growth-inhibiting activities were detected from the supernatant of all three protein-free culture cells, while no protein-free culture cells secreted predominantly growth-stimulating activity into their cultured media. These results suggest that autonomy in tumor cell proliferation may result from the acquisition of the ability to escape from negative control in multicellular organisms, as shown in monads, rather than an acquisition of further response to growth-stimulating control. PMID:21584570

  11. Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos

    PubMed Central

    Maeda, Ryu; Ishimura, Akihiko; Mood, Kathleen; Park, Eui Kyun; Buchberg, Arthur M.; Daar, Ira O.

    2002-01-01

    Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development. PMID:11960001

  12. Identification of the Homeobox Protein Prx1 (MHox, Prrx-1) as a Regulator of Osterix Expression and Mediator of Tumor Necrosis Factor α Action in Osteoblast Differentiation

    PubMed Central

    Lu, Xianghuai; Beck, George R; Gilbert, Linda C; Camalier, Corinne E; Bateman, Nicholas W; Hood, Brian L; Conrads, Thomas P; Kern, Michael J; You, Shaojin; Chen, Hong; Nanes, Mark S

    2011-01-01

    Tumor necrosis factor α (TNF-α) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging. © 2011 American Society for Bone and Mineral Research. PMID:20683885

  13. A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging.

    PubMed

    Jiang, Han; Moore, Sarah J; Liu, Shuanglong; Liu, Hongguang; Miao, Zheng; Cochran, Frank V; Liu, Yang; Tian, Mei; Cochran, Jennifer R; Zhang, Hong; Cheng, Zhen

    2013-02-01

    A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α(v)β(3) integrin receptor with high affinity and specificity. In the current study, an (18)F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-(18/19)F-fluoropropionate ((18/19)F-NFP) to produce the fluorinated peptide, (18/19)F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and (19)F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α(v)β(3) integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with (18)F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and (19)F-FP-AgRP-7C specifically competed with (125)I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC(50)) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that (18)F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of (18)F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. (18)F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios

  14. Vitamin D Receptor Protein Expression in Tumor Tissue and Prostate Cancer Progression

    PubMed Central

    Hendrickson, Whitney K.; Flavin, Richard; Kasperzyk, Julie L.; Fiorentino, Michelangelo; Fang, Fang; Lis, Rosina; Fiore, Christopher; Penney, Kathryn L.; Ma, Jing; Kantoff, Philip W.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.; Giovannucci, Edward

    2011-01-01

    Purpose Data suggest that circulating 25-hydroxyvitamin D [25(OH)D] interacts with the vitamin D receptor (VDR) to decrease proliferation and increase apoptosis for some malignancies, although evidence for prostate cancer is less clear. How VDR expression in tumor tissue may influence prostate cancer progression has not been evaluated in large studies. Patients and Methods We examined protein expression of VDR in tumor tissue among 841 patients with prostate cancer in relation to risk of lethal prostate cancer within two prospective cohorts, the Physicians' Health Study and Health Professionals Follow-Up Study. We also examined the association of VDR expression with prediagnostic circulating 25(OH)D and 1,25-dihydroxyvitamin D levels and with two VDR single nucleotide polymorphisms, FokI and BsmI. Results Men whose tumors had high VDR expression had significantly lower prostate-specific antigen (PSA) at diagnosis (P for trend < .001), lower Gleason score (P for trend < .001), and less advanced tumor stage (P for trend < .001) and were more likely to have tumors harboring the TMPRSS2:ERG fusion (P for trend = .009). Compared with the lowest quartile, men whose tumors had the highest VDR expression had significantly reduced risk of lethal prostate cancer (hazard ratio [HR], 0.17; 95% CI, 0.07 to 0.41). This association was only slightly attenuated after adjustment for Gleason score and PSA at diagnosis (HR, 0.33; 95% CI, 0.13 to 0.83) or, additionally, for tumor stage (HR, 0.37; 95% CI, 0.14 to 0.94). Neither prediagnostic plasma vitamin D levels nor VDR polymorphisms were associated with VDR expression. Conclusion High VDR expression in prostate tumors is associated with a reduced risk of lethal cancer, suggesting a role of the vitamin D pathway in prostate cancer progression. PMID:21537045

  15. Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.

    PubMed

    Benseñor, Lorena B; Barlan, Kari; Rice, Sarah E; Fehon, Richard G; Gelfand, Vladimir I

    2010-04-20

    The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function. PMID:20368450

  16. Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants

    PubMed Central

    Benseñor, Lorena B.; Barlan, Kari; Rice, Sarah E.; Fehon, Richard G.; Gelfand, Vladimir I.

    2010-01-01

    The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function. PMID:20368450

  17. Cell Cycle Restriction Is More Important Than Apoptosis Induction for RASSF1A Protein Tumor Suppression*

    PubMed Central

    Donninger, Howard; Clark, Jennifer A.; Monaghan, Megan K.; Schmidt, M. Lee; Vos, Michele; Clark, Geoffrey J.

    2014-01-01

    The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle. In part, cell cycle regulation appears to be dependent upon the ability of RASSF1A to complex with microtubules and regulate their dynamics. Which property of RASSF1A, apoptosis induction or microtubule regulation, is responsible for its tumor suppressor function is not known. We have identified a short conserved motif that is essential for the binding of RASSF family proteins with microtubule-associated proteins. By making a single point mutation in the motif, we were able to generate a RASSF1A variant that retains wild-type apoptotic properties but completely loses the ability to bind microtubule-associated proteins and complex with microtubules. Comparison of this mutant to wild-type RASSF1A showed that, despite retaining its proapoptotic properties, the mutant was completely unable to induce cell cycle arrest or suppress the tumorigenic phenotype. Therefore, it appears that the cell cycle/microtubule effects of RASSF1A are key to its tumor suppressor function rather than its apoptotic effects. PMID:25225292

  18. Transient exposure to proteins SOX2, Oct-4, and NANOG immortalizes exhausted tumor-infiltrating CTLs.

    PubMed

    Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian; Wang, Jianjun; Xiao, Zhengguo

    2016-05-13

    Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokine and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. PMID:27084449

  19. Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation

    SciTech Connect

    Mantovani, G.; Lania, A.G.; Bondioni, S.; Peverelli, E.; Pedroni, C.; Ferrero, S.; Pellegrini, C.; Vicentini, L.; Arnaldi, G.; Bosari, S.; Beck-Peccoz, P.; Spada, A.

    2008-01-01

    The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing's syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n = 16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n = 5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-cAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.

  20. Application of Protein Microarrays for Multiplexed Detection of Antibodies to Tumor Antigens in Breast Cancer

    PubMed Central

    Anderson, Karen S.; Ramachandran, Niroshan; Wong, Jessica; Raphael, Jacob V.; Hainsworth, Eugenie; Demirkan, Gokhan; Cramer, Daniel; Aronzon, Diana; Hodi, F. Stephen; Harris, Lyndsay; Logvinenko, Tanya; LaBaer, Joshua

    2012-01-01

    There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum, may exist in greater concentrations than their cognate antigens, and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies. PMID:18311903

  1. The role of stat1b in zebrafish hematopoiesis

    PubMed Central

    Song, Hao; Yan, Yi-lin; Titus, Tom; He, Xinjun; Postlethwait, John H.

    2011-01-01

    STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by subfunctionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development. PMID:21914475

  2. Identification of Bidentate Salicylic Acid Inhibitors of PTP1B.

    PubMed

    Haftchenary, Sina; Jouk, Andriana O; Aubry, Isabelle; Lewis, Andrew M; Landry, Melissa; Ball, Daniel P; Shouksmith, Andrew E; Collins, Catherine V; Tremblay, Michel L; Gunning, Patrick T

    2015-09-10

    PTP1B is a master regulator in the insulin and leptin metabolic pathways. Hyper-activated PTP1B results in insulin resistance and is viewed as a key factor in the onset of type II diabetes and obesity. Moreover, inhibition of PTP1B expression in cancer cells dramatically inhibits cell growth in vitro and in vivo. Herein, we report the computationally guided optimization of a salicylic acid-based PTP1B inhibitor 6, identifying new and more potent bidentate PTP1B inhibitors, such as 20h, which exhibited a > 4-fold improvement in activity. In CHO-IR cells, 20f, 20h, and 20j suppressed PTP1B activity and restored insulin receptor phosphorylation levels. Notably, 20f, which displayed a 5-fold selectivity for PTP1B over the closely related PTPσ protein, showed no inhibition of PTP-LAR, PRL2 A/S, MKPX, or papain. Finally, 20i and 20j displayed nanomolar inhibition of PTPσ, representing interesting lead compounds for further investigation. PMID:26396684

  3. New protein kinase from plasma membrane of Ehrlich ascites tumor cells activated by natural polypeptides.

    PubMed Central

    Racker, E; Abdel-Ghany, M; Sherrill, K; Riegler, C; Blair, E A

    1984-01-01

    A polypeptide-dependent protein kinase was purified about 80-fold from an extract of plasma membranes of Ehrlich ascites tumor cells. The membranes were extracted with Nonidet P-40, and the extract was purified by ammonium sulfate fractionation and hydroxylapatite and affinity chromatography. The activity was stimulated 10-fold or more by polypeptide preparations from a variety of tissues, including placenta and hypothalamus. Polypeptide-dependent protein kinase had a pH optimum of about 7.5 and required Mg2+ for activity. Mn2+ at low concentrations (200 microM) stimulated enzyme activity somewhat but inhibited activity strongly at higher concentrations. The best available substrate for polypeptide-dependent protein kinase was beta-casein, and little or no phosphorylation was observed with alpha-casein, kappa-casein, phosvitin, alpha-lactalbumin, alpha-lactoglobulin, and histone. However, several endogenous substrates from plasma membranes of Ehrlich ascites tumor cells were phosphorylated. Polypeptide-dependent protein kinase activity was not inhibited by 10 mM N-ethylmaleimide, and this resistance was useful in differentiating this protein kinase from other protein kinases that were present in crude fractions and sensitive to the inhibitor. Images PMID:6589591

  4. The expression of retinoblastoma tumor suppressor protein in oral cancers and precancers: A clinicopathological study

    PubMed Central

    Thomas, Sunila; Balan, Anita; Balaram, Prabha

    2015-01-01

    Background: The role of retinoblastoma (Rb) protein in cell cycle regulation prompted us to take up this study with the aim of assessing its role in the progression of oral cancer and to correlate with various clinicopathological parameters, including habits such as smoking, Paan chewing, and alcoholism. Materials and Methods: This observational study included surgical specimens from 10 apparently normal oral mucosa, 14 oral reactive lesions (ORL), 29 precancerous lesions and 43 oral cancers. The expression of Rb protein in tissue samples were evaluated by immunohistochemistry and correlated with clinicopathological data. The percentage and mean expression of Rb protein were statistically analyzed using Student's t-test and P < 0.05 was considered as statistically significant difference. Results: The expression of Rb protein was found to increase from normal, ORL, precancerous lesions to cancers. A consistently high expression of Rb protein was seen in oral cancers, with an increase in well-differentiated and moderately differentiated tumors. Patients with combined habits of Paan chewing, smoking, and alcohol consumption had a higher expression compared with those without habits. Conclusion: Within the limitations of this study, it seems that overexpression of Rb protein noted in oral cancer, with an increase in well and moderately differentiated tumors suggest a possible role of Rb in differentiation. The high expression of Rb in patients with combined habits of Paan chewing, smoking and alcohol consumption indicates that Rb pathway may be altered in habit-related oral malignancies. PMID:26288619

  5. Role of G protein-coupled receptor kinase 2 in tumoral angiogenesis

    PubMed Central

    Rivas, Verónica; Nogués, Laura; Reglero, Clara; Mayor, Federico; Penela, Petronila

    2014-01-01

    Downregulation of G protein-coupled receptor kinase 2 (GRK2) in endothelial cells has recently been identified as a relevant event in the tumoral angiogenic switch. Based on the effects of altering GRK2 dosage in cell and animal models, this kinase appears to act as a hub in key signaling pathways involved in vascular stabilization and remodeling. Accordingly, decreased GRK2 expression in endothelial cells accelerates tumor growth in mice by impairing the pericytes ensheathing the vessels, thereby promoting hypoxia and macrophage infiltration. These results raise new questions regarding the mechanisms by which transformed cells trigger the decrease in GRK2 observed in human breast cancer vessels and how GRK2 modulates the interactions between different cell types that occur in the tumor microenvironment. PMID:27308373

  6. Mediastinal Yolk Sac Tumor Producing Protein Induced by Vitamin K Absence or Antagonist-II.

    PubMed

    Akutsu, Noriyuki; Adachi, Yasushi; Isosaka, Mai; Mita, Hiroaki; Takagi, Hideyasu; Sasaki, Shigeru; Yamamoto, Hiroyuki; Arimura, Yoshiaki; Ishii, Yoshifumi; Masumori, Naoya; Endo, Takao; Shinomura, Yasuhisa

    2015-01-01

    Extragonadal yolk sac tumors (YSTs) are rare. We herein report the case of a 66-year-old man with mediastinal, lung and liver tumors. The largest mass was located in the liver and contained a high concentration of protein induced by vitamin K absence or antagonist-II (PIVKA-II) and alpha-fetoprotein. Therefore, the lesion was difficult to distinguish from hepatocellular carcinoma. Finally, YST was diagnosed based on the results of a liver biopsy. Although chemotherapy was effective, the patient died of respiratory failure. The autopsy revealed primary mediastinal YST. In the current report, we describe this case of PIVKA-II-producing YST and review previous cases of PIVKA-II-producing tumors other than hepatoma. PMID:26073245

  7. 18 CFR 1b.5 - Formal investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Formal investigations. 1b.5 Section 1b.5 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.5 Formal investigations....

  8. 18 CFR 1b.12 - Transcripts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Transcripts. 1b.12 Section 1b.12 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.12 Transcripts. Transcripts, if any,...

  9. 18 CFR 1b.2 - Scope.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Scope. 1b.2 Section 1b.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.2 Scope. This part applies to...

  10. 18 CFR 1b.1 - Definitions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Definitions. 1b.1 Section 1b.1 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.1 Definitions. For purposes of this part—...

  11. 18 CFR 1b.6 - Preliminary investigations.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Preliminary investigations. 1b.6 Section 1b.6 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.6 Preliminary investigations....

  12. 18 CFR 1b.21 - Enforcement hotline.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Enforcement hotline. 1b.21 Section 1b.21 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.21 Enforcement hotline. (a)...

  13. 18 CFR 1b.14 - Subpoenas.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Subpoenas. 1b.14 Section 1b.14 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.14 Subpoenas. (a) Service of a...

  14. 18 CFR 1b.19 - Submissions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Submissions. 1b.19 Section 1b.19 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES RULES RELATING TO INVESTIGATIONS § 1b.19 Submissions. In the event...

  15. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 1 2012-01-01 2012-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of the various USDA agencies shall be planned, developed, and implemented so as to achieve the goals...

  16. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of the various USDA agencies shall be planned, developed, and implemented so as to achieve the goals...

  17. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of the various USDA agencies shall be planned, developed, and implemented so as to achieve the goals...

  18. 7 CFR 1b.2 - Policy.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 1 2014-01-01 2014-01-01 false Policy. 1b.2 Section 1b.2 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.2 Policy. (a) All policies and programs of the various USDA agencies shall be planned, developed, and implemented so as to achieve the goals...

  19. 7 CFR 1b.3 - Categorical exclusions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Categorical exclusions. 1b.3 Section 1b.3 Agriculture Office of the Secretary of Agriculture NATIONAL ENVIRONMENTAL POLICY ACT § 1b.3 Categorical exclusions. (a) The following are categories of activities which have been determined not to have a significant individual or cumulative effect on the...

  20. Serial Analysis of 38 Proteins during the Progression of Human Breast Tumor in Mice Using an Antibody Colocalization Microarray*

    PubMed Central

    Li, Huiyan; Bergeron, Sébastien; Annis, Matthew G.; Siegel, Peter M.; Juncker, David

    2015-01-01

    Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models. PMID:25680959

  1. Serial analysis of 38 proteins during the progression of human breast tumor in mice using an antibody colocalization microarray.

    PubMed

    Li, Huiyan; Bergeron, Sébastien; Annis, Matthew G; Siegel, Peter M; Juncker, David

    2015-04-01

    Proteins in serum or plasma hold great potential for use in disease diagnosis and monitoring. However, the correlation between tumor burden and protein biomarker concentration has not been established. Here, using an antibody colocalization microarray, the protein concentration in serum was measured and compared with the size of mammary xenograft tumors in 11 individual mice from the time of injection; seven blood samples were collected from each tumor-bearing mouse as well as control mice on a weekly basis. The profiles of 38 proteins detected in sera from these animals were analyzed by clustering, and we identified 10 proteins with the greatest relative increase in serum concentration that correlated with growth of the primary mammary tumor. To evaluate the diagnosis of cancer based on these proteins using either an absolute threshold (i.e. a concentration cutoff) or self-referenced differential threshold based on the increase in concentration before cell injection, receiver operating characteristic curves were produced for 10 proteins with increased concentration, and the area under curve was calculated for each time point based on a single protein or on a panel of proteins, in each case showing a rapid increase of the area under curve. Next, the sensitivity and specificity of individual and optimal protein panels were calculated, showing high accuracy as early as week 2. These results provide a foundation for studies of tumor growth through measuring serial changes of protein concentration in animal models. PMID:25680959

  2. CYP1B1 expression, a potential risk factor for breast cancer

    SciTech Connect

    Goth-Goldstein, Regine; Erdmann, Christine A.; Russell, Marion

    2001-05-31

    CYP1B1 expression in non-tumor breast tissue from breast cancer patients and cancer-free individuals was determined to test the hypothesis that high CYP1B1 expression is a risk factor for breast cancer. Large interindividual variations in CYP1B1 expression were found with CYP1B1 levels notably higher in breast cancer patients than cancer-free individuals. The results indicate that CYP1B1 might play a role in breast cancer either through increased PAH activation or through metabolism of endogenous estrogen to a carcinogenic derivative.

  3. Bromodomain-containing protein 7 (BRD7) as a potential tumor suppressor in hepatocellular carcinoma

    PubMed Central

    Pan, Qiu-Zhong; Tang, Yan; Wang, Qi-Jing; Pan, Ke; Huang, Li-Xi; He, Jia; Zhao, Jing-Jing; Jiang, Shan-Shan; Zhang, Xiao-Fei; Zhang, Hong-Xia; Zhou, Zi-Qi; Weng, De-Sheng; Xia, Jian-Chuan

    2016-01-01

    Bromodomain-containing protein 7 (BRD7) is a subunit of the PBAF complex, which functions as a transcriptional cofactor for the tumor suppressor protein p53. Down-regulation of BRD7 has been demonstrated in multiple types of cancer. This study aimed to investigate BRD7 expression and its tumor suppressive effect in hepatocellular carcinoma (HCC). The expression of BRD7 was examined in clinical specimens of primary HCC and in HCC cell lines through real-time quantitative PCR, western blot and immunohistochemistry. The prognostic value of BRD7 expression and its correlation with the clinicopathological features of HCC patients were statistically analyzed. The effect of BRD7 on the tumorigenicity of HCC was also examined using proliferation and colony-formation assays, cell-cycle assays, migration and cell-invasion assays, and xenograft nude mouse models. BRD7 was down-regulated in tumor tissues and HCC cell lines. BRD7 protein expression was strongly associated with clinical stage and tumor size. Kaplan-Meier survival curves revealed higher survival rates in patients with higher BRD7 expression levels compared to those with lower BRD7 levels. A multivariate analysis indicated that BRD7 expression was an independent prognostic marker. The re-introduction of BRD7 expression significantly inhibited proliferation, colony formation, migration and invasion and led to cell cycle arrest in HCC cells in vitro. Furthermore, experiments in mice suggested that BRD7 overexpression suppresses HCC tumorigenicity in vivo. In conclusions, our data indicated that BRD7 may serve as a tumor suppressor in HCC and may be a novel molecular target for the treatment of HCC. PMID:26919247

  4. Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29.

    PubMed

    Ebert, Bettina; Kisiela, Michael; Wsól, Vladimir; Maser, Edmund

    2011-05-30

    Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the

  5. Downregulation of key regulatory proteins in androgen dependent prostate tumor cells by oncolytic reovirus.

    PubMed

    Gupta-Saraf, Pooja; Meseke, Tyler; Miller, Cathy L

    2015-11-01

    As prostate tumor cell growth depends on hormones, androgen ablation is an effective therapy for prostate cancer (PCa). However, progression of PCa cells to androgen independent growth (castrate resistant prostate cancer, CRPC) results in relapse and mortality. Hypoxia, a microenvironment of low oxygen that modifies the activity of PCa regulatory proteins including the androgen receptor (AR), plays a critical role in progression to CRPC. Therapies targeting hypoxia and the AR may lengthen the time to CRPC progression thereby increasing survival time of PCa patients. Mammalian Orthoreovirus (MRV) has shown promise for the treatment of prostate tumors in vitro and in vivo. In this study, we found that MRV infection induces downregulation of proteins implicated in CRPC progression, interferes with hypoxia-induced AR activity, and induces apoptosis in androgen dependent cells. This suggests MRV possesses traits that could be exploited to create novel therapies for the inhibition of progression to CRPC. PMID:26264969

  6. Activation of the Proapoptotic Bcl-2 Protein Bax by a Small Molecule Induces Tumor Cell Apoptosis

    PubMed Central

    Zhao, Guoping; Zhu, Yanglong; Eno, Colins O.; Liu, Yanlong; DeLeeuw, Lynn; Burlison, Joseph A.; Chaires, Jonathan B.; Trent, John O.

    2014-01-01

    The proapoptotic Bcl-2 protein Bax by itself is sufficient to initiate apoptosis in almost all apoptotic paradigms. Thus, compounds that can facilitate disruptive Bax insertion into mitochondrial membranes have potential as cancer therapeutics. In our study, we have identified small-molecule compounds predicted to associate with the Bax hydrophobic groove by a virtual-screen approach. Among these, one lead compound (compound 106) promotes Bax-dependent but not Bak-dependent apoptosis. Importantly, this compound alters Bax protein stability in vitro and promotes the insertion of Bax into mitochondria, leading to Bax-dependent permeabilization of the mitochondrial outer membrane. Furthermore, as a single agent, compound 106 inhibits the growth of transplanted tumors, probably by inducing apoptosis in tumors. Our study has revealed a compound that activates Bax and induces Bax-dependent apoptosis, which may lead to the development of new therapeutic agents for cancer. PMID:24421393

  7. The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

    PubMed Central

    Hung, Pei-Fang; Hong, Tse-Ming; Hsu, Yi-Chiung; Chen, Hsuan-Yu; Chang, Yih-Leong; Wu, Chen-Tu; Chang, Gee-Chen; Jou, Yuh-Shan

    2013-01-01

    The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas. PMID:23626713

  8. The motor protein KIF14 inhibits tumor growth and cancer metastasis in lung adenocarcinoma.

    PubMed

    Hung, Pei-Fang; Hong, Tse-Ming; Hsu, Yi-Chiung; Chen, Hsuan-Yu; Chang, Yih-Leong; Wu, Chen-Tu; Chang, Gee-Chen; Jou, Yuh-Shan; Pan, Szu-Hua; Yang, Pan-Chyr

    2013-01-01

    The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas. PMID:23626713

  9. The p53 tumor suppressor protein protects against chemotherapeutic stress and apoptosis in human medulloblastoma cells

    PubMed Central

    Parasido, Erika; Tricoli, Lucas; Sivakumar, Angiela; Mikhaiel, John P.; Yenugonda, Venkata; Rodriguez, Olga C.; Karam, Sana D.; Rood, Brian R.; Avantaggiati, Maria Laura; Albanese, Chris

    2015-01-01

    Medulloblastoma (MB), a primitive neuroectodermal tumor, is the most common malignant childhood brain tumor and remains incurable in about a third of patients. Currently, survivors carry a significant burden of late treatment effects. The p53 tumor suppressor protein plays a crucial role in influencing cell survival in response to cellular stress and while the p53 pathway is considered a key determinant of anti-tumor responses in many tumors, its role in cell survival in MB is much less well defined. Herein, we report that the experimental drug VMY-1-103 acts through induction of a partial DNA damage-like response as well induction of non-survival autophagy. Surprisingly, the genetic or chemical silencing of p53 significantly enhanced the cytotoxic effects of both VMY and the DNA damaging drug, doxorubicin. The inhibition of p53 in the presence of VMY revealed increased late stage apoptosis, increased DNA fragmentation and increased expression of genes involved in apoptosis, including CAPN12 and TRPM8, p63, p73, BIK, EndoG, CIDEB, P27Kip1 and P21cip1. These data provide the groundwork for additional studies on VMY as a therapeutic drug and support further investigations into the intriguing possibility that targeting p53 function may be an effective means of enhancing clinical outcomes in MB. PMID:26540407

  10. A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

    PubMed Central

    Redkar, Amey; Hoser, Rafal; Schilling, Lena; Zechmann, Bernd; Krzymowska, Magdalena; Walbot, Virginia; Doehlemann, Gunther

    2015-01-01

    The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize. PMID:25888589

  11. Human Hemochromatosis Protein (HFE) Immunoperoxidase Stain Highlights Choriocarcinoma within Mixed Germ Cell Tumors.

    PubMed

    Cox, Jesse L; Talmon, Geoffrey A; Koepsell, Scott A

    2016-01-01

    Identification of choriocarcinoma within a germ cell tumor can have major implications for the subsequent staging and treatment of testicular neoplasms. Immunoperoxidase staining greatly enhances the speed and sensitivity of identifying occult, though clinically significant, tumor components. In mixed germ cell tumors, staining for beta-human chorionic gonadotropin (β-hCG) has been historically used to assess for the presence and burden of choriocarcinoma. However, current β-hCG stains produce variable, intense staining of trophoblastic elements and surrounding tissues, clouding the assessment of true-positive staining. Human hemochromatosis protein (HFE) is a membrane bound mediator of iron transport expressed at high levels within placenta. Additionally, previous reports have demonstrated that choriocarcinoma cell lines express HFE, although in vivo expression had not been examined. To address whether HFE can stain trophoblastic elements, HFE immunohistochemistry was conducted in choriocarcinoma (n = 4), mixed germ cell tumors (n = 11), seminoma (n = 4), and placenta (n = 11). HFE consistently demonstrated cytoplasmic and membranous staining, highlighting both syncytiotrophoblasts and cytotrophoblasts within choriocarcinoma and placenta. Staining of intratumoral white blood cells was observed within seminomas and mixed germ cell tumors, corroborating prior reports stating that HFE highlights monocytes and macrophages. Taken together, HFE may serve as an alternative target from β-hCG for immunoperoxidase studies when highlighting choriocarcinoma. PMID:27034532

  12. Human Hemochromatosis Protein (HFE) Immunoperoxidase Stain Highlights Choriocarcinoma within Mixed Germ Cell Tumors

    PubMed Central

    Talmon, Geoffrey A.; Koepsell, Scott A.

    2016-01-01

    Identification of choriocarcinoma within a germ cell tumor can have major implications for the subsequent staging and treatment of testicular neoplasms. Immunoperoxidase staining greatly enhances the speed and sensitivity of identifying occult, though clinically significant, tumor components. In mixed germ cell tumors, staining for beta-human chorionic gonadotropin (β-hCG) has been historically used to assess for the presence and burden of choriocarcinoma. However, current β-hCG stains produce variable, intense staining of trophoblastic elements and surrounding tissues, clouding the assessment of true-positive staining. Human hemochromatosis protein (HFE) is a membrane bound mediator of iron transport expressed at high levels within placenta. Additionally, previous reports have demonstrated that choriocarcinoma cell lines express HFE, although in vivo expression had not been examined. To address whether HFE can stain trophoblastic elements, HFE immunohistochemistry was conducted in choriocarcinoma (n = 4), mixed germ cell tumors (n = 11), seminoma (n = 4), and placenta (n = 11). HFE consistently demonstrated cytoplasmic and membranous staining, highlighting both syncytiotrophoblasts and cytotrophoblasts within choriocarcinoma and placenta. Staining of intratumoral white blood cells was observed within seminomas and mixed germ cell tumors, corroborating prior reports stating that HFE highlights monocytes and macrophages. Taken together, HFE may serve as an alternative target from β-hCG for immunoperoxidase studies when highlighting choriocarcinoma. PMID:27034532

  13. Prostate-specific membrane antigen protein expression in tumor tissue and risk of lethal prostate cancer

    PubMed Central

    Kasperzyk, Julie L.; Finn, Stephen P.; Flavin, Richard; Fiorentino, Michelangelo; Lis, Rosina; Hendrickson, Whitney K.; Clinton, Steven K.; Sesso, Howard D.; Giovannucci, Edward L.; Stampfer, Meir J.; Loda, Massimo; Mucci, Lorelei A.

    2013-01-01

    Background Over-expression of prostate-specific membrane antigen (PSMA) in tumor tissue and serum has been linked to increased risk of biochemical recurrence in surgically treated prostate cancer patients, but no studies have assessed its association with disease-specific mortality. Methods We examined whether high PSMA protein expression in prostate tumor tissue was associated with lethal disease, and with tumor biomarkers of progression, among participants of two US-based cohorts (n=902, diagnosed 1983–2004). We used Cox proportional hazards regression to calculate multivariable hazard ratios (HR) and 95% confidence intervals (CI) of lethal prostate cancer, defined as disease-specific death or development of distant metastases (n=95). Partial Spearman rank correlation coefficients were used to correlate PSMA with tumor biomarkers. Results During an average 13 years of follow-up, higher PSMA expression at prostatectomy was significantly associated with lethal prostate cancer (age-adjusted HRQuartile(Q)4vs.Q1=2.42; p-trend<0.01). This association was attenuated and non-significant (multivariable-adjusted HRQ4vs.Q1=1.01; p-trend=0.52) after further adjusting for Gleason score and PSA at diagnosis. High PSMA expression was significantly (p<0.05) correlated with higher Gleason score and PSA at diagnosis, increased tumor angiogenesis, lower vitamin D receptor and androgen receptor expression, and absence of ERG expression. Conclusions High tumor PSMA expression was not an independent predictor of lethal prostate cancer in the current study. PSMA expression likely captures, in part, malignant features of Gleason grade and tumor angiogenesis. Impact PSMA is not a strong candidate biomarker for predicting prostate cancer-specific mortality in surgically treated patients. PMID:24130224

  14. Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth.

    PubMed

    Ucar, Deniz A; Kurenova, Elena; Garrett, Timothy J; Cance, William G; Nyberg, Carl; Cox, Audrey; Massoll, Nicole; Ostrov, David A; Lawrence, Nicholas; Sebti, Said M; Zajac-Kaye, Maria; Hochwald, Steven N

    2012-09-01

    FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2-31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2-31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2-31 significantly inhibited cell proliferation and viability (range 0.05-10 μM). More importantly, 15 mg/kg of INT2-31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2-31) has potential anti-neoplastic therapeutic effects in human melanoma. PMID:22894899

  15. Is Melanoma a stem cell tumor? Identification of neurogenic proteins in trans-differentiated cells

    PubMed Central

    Rasheed, Suraiya; Mao, Zisu; Chan, Jane MC; Chan, Linda S

    2005-01-01

    Background Although several genes and proteins have been implicated in the development of melanomas, the molecular mechanisms involved in the development of these tumors are not well understood. To gain a better understanding of the relationship between the cell growth, tumorigenesis and differentiation, we have studied a highly malignant cat melanoma cell line that trans-differentiates into neuronal cells after exposure to a feline endogenous retrovirus RD114. Methods To define the repertoire of proteins responsible for the phenotypic differences between melanoma and its counterpart trans-differentiated neuronal cells we have applied proteomics technology and compared protein profiles of the two cell types and identified differentially expressed proteins by 2D-gel electrophoresis, image analyses and mass spectrometry. Results The melanoma and trans-differentiated neuronal cells could be distinguished by the presence of distinct sets of proteins in each. Although approximately 60–70% of the expressed proteins were shared between the two cell types, twelve proteins were induced de novo after infection of melanoma cells with RD114 virus in vitro. Expression of these proteins in trans-differentiated cells was significantly associated with concomitant down regulation of growth promoting proteins and up-regulation of neurogenic proteins (p = < 0.001). Based on their physiologic properties, >95% proteins expressed in trans-differentiated cells could be associated with the development, differentiation and regulation of nervous system cells. Conclusion Our results indicate that the cat melanoma cells have the ability to differentiate into distinct neuronal cell types and they express proteins that are essential for self-renewal. Since melanocytes arise from the neural crest of the embryo, we conclude that this melanoma arose from embryonic precursor stem cells. This model system provides a unique opportunity to identify domains of interactions between the expressed

  16. Tumor-promoting function and prognostic significance of the RNA-binding protein T-cell intracellular antigen-1 in esophageal squamous cell carcinoma

    PubMed Central

    Fujita, Yuji; Naruto, Takuya; Kohmoto, Tomohiro; Miyakami, Yuko; Watanabe, Miki; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Otsuji, Eigo; Imoto, Issei

    2016-01-01

    T-cell intracellular antigen-1 (TIA1) is an RNA-binding protein involved in many regulatory aspects of mRNA metabolism. Here, we report previously unknown tumor-promoting activity of TIA1, which seems to be associated with its isoform-specific molecular distribution and regulation of a set of cancer-related transcripts, in esophageal squamous cell carcinoma (ESCC). Immunohistochemical overexpression of TIA1 ectopically localized in the cytoplasm of tumor cells was an independent prognosticator for worse overall survival in a cohort of 143 ESCC patients. Knockdown of TIA1 inhibited proliferation of ESCC cells. By exogenously introducing each of two major isoforms, TIA1a and TIA1b, only TIA1a, which was localized to both the nucleus and cytoplasm, promoted anchorage-dependent and anchorage-independent ESCC cell proliferation. Ribonucleoprotein immunoprecipitation, followed by microarray analysis or massive-parallel sequencing, identified a set of TIA1-binding mRNAs, including SKP2 and CCNA2. TIA1 increased SKP2 and CCNA2 protein levels through the suppression of mRNA decay and translational induction, respectively. Our findings uncover a novel oncogenic function of TIA1 in esophageal tumorigenesis, and implicate its use as a marker for prognostic evaluation and as a therapeutic target in ESCC. PMID:26958940

  17. Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity.

    PubMed

    Lv, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lu, Yan-Hua; Zhao, Jian; Wang, Fu-Jun

    2015-07-01

    MAP30 (Momordica Antiviral Protein 30 Kd), a single-stranded type-I ribosome inactivating protein, possesses versatile biological activities including anti-tumor abilities. However, the low efficiency penetrating into tumor cells hampers the tumoricidal effect of MAP30. This paper describes MAP30 fused with a human-derived cell penetrating peptide HBD which overcome the low uptake efficiency by tumor cells and exhibits higher anti-tumor bioactivity. MAP30 gene was cloned from the genomic DNA of Momordica charantia and the recombinant plasmid pET28b-MAP30-HBD was established and transferred into Escherichia coli BL21 (DE3). The recombinant MAP30-HBD protein (rMAP30-HBD) was expressed in a soluble form after being induced by 0.5mM IPTG for 14h at 15°C. The recombinant protein was purified to greater than 95% purity with Ni-NTA affinity chromatography. The rMAP30-HBD protein not only has topological inactivation and protein translation inhibition activity but also showed significant improvements in cytotoxic activity compared to that of the rMAP30 protein without HBD in the tested tumor cell lines, and induced higher apoptosis rates in HeLa cells analyzed by Annexin V-FITC with FACS. This paper demonstrated a new method for improving MAP30 protein anti-tumor activity and might have potential applications in cancer therapy area. PMID:25797209

  18. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

    PubMed

    Wu, Yi-Ju; Jan, Yee-Jee; Ko, Bor-Sheng; Liang, Shu-Man; Liou, Jun-Yang

    2015-01-01

    There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3's regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation. PMID:26083935

  19. Modulation of primary antibody response by protein A in tumor bearing mice.

    PubMed

    Zaidi, S I; Singh, K P; Raisuddin, S; Jafri, A; Saxena, A K; Choudhary, S; Ray, P K

    1995-11-01

    Protein A (PA) is a cell wall glycoprotein of Staphylococcus aureus Cowan I, which possess a number of immunomodulatory and antitumor properties. We have previously shown that PA suppresses the anti-sheep erythrocyte primary antibody response in normal mice. The present investigation evaluates the effect of protein A on the anti-sheep erythrocyte primary antibody response in tumor-bearing mice. The primary antibody response in tumor-bearing mice immunized with sheep red blood cells (SRBC) was suppressed by the intraperitoneal administration of PA in a dose-dependent fashion. The plaque forming cell (PFC) assay was used to assess this response. Maximum suppression of the PFC response was observed at 12 micrograms PA/animal (p < 0.001) and could be observed at doses as low as 1 microgram PA/animal (p < 0.01). The amount of suppression was proportional to the number of PA doses administered. In addition this effect was critically dependent on the timing of PA administration. PA showed no significant effect on PFC when injected after immunization, but it produced pronounced suppression when injected prior to the immunization with SRBC. Maximum suppression of the PFC response was observed when PA was administered one day before the antigen challenge. PA also reduced splenic localization of 51Cr labeled SRBC to 42% (p < 0.01). The altered localization of antigen in spleen may be responsible for reduced PFC response in tumor-bearing mice. Depletion of B-lymphocyte is reported to exhibit tumor inhibition. Therefore, we propose that the suppression of the primary antibody response by PA helps in tumor regression by reducing the soluble immunosuppressive immune complexes. PMID:8537611

  20. Inhibition of tumor angiogenesis by angiostatin: from recombinant protein to gene therapy.

    PubMed

    Dell'Eva, Raffaella; Pfeffer, Ulrich; Indraccolo, S; Albini, Adriana; Noonan, Douglas

    2002-01-01

    Tumor growth, local invasion, and metastatic dissemination are dependent on the formation of new microvessels. The process of angiogenesis is regulated by a balance between pro-angiogenic and anti-angiogenic factors, and the shift to an angiogenic phenotype (the "angiogenic switch") is a key event in tumor progression. The use of anti-angiogenic agents to restore this balance represents a promising approach to cancer treatment. Known physiological inhibitors include trombospondin, several interleukins, and the proteolytic break-down products of several proteins. Angiostatin, an internal fragment of plasminogen, is one of the more potent of this latter class of angiogenesis inhibitors. Like endostatin, another anti-angiogenic peptide derived from collagen XVIII, angiostatin can induce tumor vasculature regression, leading to a complete cessation of tumor growth. Inhibitors of angiogenesis target normal endothelial cells, therefore the development of resistance to these drugs is unlikely. The efficacy of angiostatin has been demonstrated in animal models for many different types of solid tumors. Anti-angiogenic cancer therapy with angiostatin requires prolonged administration of the peptide. The production of the functional polypeptides is expensive and technical problems related to physical properties and purity are frequently encountered. Gene transfer represents an alternative method to deliver angiostatin. Gene therapy has the potential to produce the therapeutic agent in high concentrations in a local area for a sustained period, thereby avoiding the problems encountered with long-term administration of recombinant proteins, monoclonal antibodies, or anti-angiogenic drugs. In this review we compare the different gene therapy strategies that have been applied to angiostatin, with special regard to their ability to provide sufficient angiostatin at the target site. PMID:12901356

  1. Surface-enhanced Raman spectroscopy of saliva proteins for the noninvasive differentiation of benign and malignant breast tumors

    PubMed Central

    Feng, Shangyuan; Huang, Shaohua; Lin, Duo; Chen, Guannan; Xu, Yuanji; Li, Yongzeng; Huang, Zufang; Pan, Jianji; Chen, Rong; Zeng, Haishan

    2015-01-01

    The capability of saliva protein analysis, based on membrane protein purification and surface-enhanced Raman spectroscopy (SERS), for detecting benign and malignant breast tumors is presented in this paper. A total of 97 SERS spectra from purified saliva proteins were acquired from samples obtained from three groups: 33 healthy subjects; 33 patients with benign breast tumors; and 31 patients with malignant breast tumors