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Sample records for alk protein expression

  1. ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

    PubMed

    Muñoz-Félix, José M; Perretta-Tejedor, Nuria; Eleno, Nélida; López-Novoa, José M; Martínez-Salgado, Carlos

    2014-06-01

    Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

  2. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1)

    PubMed Central

    Marzec, Michal; Zhang, Qian; Goradia, Ami; Raghunath, Puthiyaveettil N.; Liu, Xiaobin; Paessler, Michele; Wang, Hong Yi; Wysocka, Maria; Cheng, Mangeng; Ruggeri, Bruce A.; Wasik, Mariusz A.

    2008-01-01

    The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells strongly express the immunosuppressive cell-surface protein CD274 (PD-L1, B7-H1), as determined on the mRNA and protein level. The CD274 expression is strictly dependent on the expression and enzymatic activity of NPM/ALK, as demonstrated by inhibition of the NPM/ALK function in ALK+TCL cells by the small molecule ALK inhibitor CEP-14083 and by documenting CD274 expression in IL-3-depleted BaF3 cells transfected with the wild-type NPM/ALK, but not the kinase-inactive NPM/ALK K210R mutant or empty vector alone. NPM/ALK induces CD274 expression by activating its key signal transmitter, transcription factor STAT3. STAT3 binds to the CD274 gene promoter in vitro and in vivo, as shown in the gel electromobility shift and chromatin immunoprecipitation assays, and is required for the PD-L1 gene expression, as demonstrated by siRNA-mediated STAT3 depletion. These findings identify an additional cell-transforming property of NPM/ALK and describe a direct link between an oncoprotein and an immunosuppressive cell-surface protein. These results also provide an additional rationale to therapeutically target NPM/ALK and STAT3 in ALK+TCL. Finally, they suggest that future immunotherapeutic protocols for this type of lymphoma may need to include the inhibition of NPM/ALK and STAT3 to achieve optimal clinical efficacy. PMID:19088198

  3. Consequences of heat shock protein 72 (Hsp72) expression and activity on stress-induced apoptosis in CD30+ NPM-ALK+ anaplastic large-cell lymphomas.

    PubMed

    Bonvini, P; Zorzi, E; Mussolin, L; Pillon, M; Romualdi, C; Peron, M; d'Amore, E S G; Lamant, L; Rosolen, A

    2012-06-01

    Understanding the mechanisms that control stress-induced apoptosis is critical to explain how tumours respond to treatment, as cancer cells frequently escape drug toxicity by regulating stress response through heat shock protein (HSP) expression. The overexpression of Hsp72, in particular, results in increased incidence of cell transformation, and correlates with poor prognosis in a wide range of cancers. We have shown that Hsp72 assists folding of oncogenic NPM-ALK kinase in anaplastic large-cell lymphomas (ALCLs), but its role in the maintenance of the malignant phenotype remains uncertain. Therefore, we assessed Hsp72 expression in ALCLs, investigating more in detail the mechanisms that regulate its status and activity. We found that Hsp72 is unique among the HSPs involved in tumourigenesis to be overexpressed in ALK(+) tumours and cell lines and to be induced by stress. Different from other HSPs, Hsp72 prevents cell injury, Bax activation and death by apoptosis in ALK(+) cells, acting both upstream and downstream of mitochondria. Conversely, Hsp72 is underexpressed in ALK(-) ALCL cells, and it is unable to protect cells from apoptosis under stress. Moreover, when Hsp72 expression is reduced following NPM-ALK inhibition, lymphoma cells undergo apoptosis, demonstrating the importance of Hsp72 in regulating ALCL stress response and drug sensitivity.

  4. ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

    PubMed

    Kim, Eun Kyung; Kim, Sewha

    2017-01-01

    Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

  5. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers

    PubMed Central

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  6. The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity.

    PubMed Central

    Chen, B J; Carroll, P; Samson, L

    1994-01-01

    Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage. The alkylation-sensitive phenotype of E. coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents. Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance. However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se. We expressed the E. coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E. coli. AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome. We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell. Images PMID:7928996

  7. Alk1 and Alk2 are two new cell cycle-regulated haspin-like proteins in budding yeast.

    PubMed

    Nespoli, Alessandro; Vercillo, Raffaella; di Nola, Lisa; Diani, Laura; Giannattasio, Michele; Plevani, Paolo; Muzi-Falconi, Marco

    2006-07-01

    Haspin is a protein kinase identified in mouse and human cells, and genes coding for haspin-like proteins are present in virtually all eukaryotic genomes sequenced so far. Two haspin homologues, called Alk1 and Alk2, are present in the yeast Saccharomyces cerevisiae. Both Alk1 and Alk2 exhibit a weak auto-kinase activity in vitro, are phosphoproteins in vivo and are hyperphosphorylated in response to DNA damage. The amount and modification of the two proteins is greatly regulated during the cell cycle. In fact, Alk1 and Alk2 levels peak in mitosis and late-S/G2, respectively, and phosphorylation of both proteins is maximal in mitosis. Control of protein stability plays a major role in Alk2 regulation. The half-life of Alk2 is particularly short in G1; mutagenesis and genetic analysis indicate that its degradation is controlled by the APC pathway. Overexpression of ALK2, but not of ALK1, causes a mitotic arrest, which is correlated to the kinase activity of the protein. This finding, together with its cell cycle regulation, suggests a role for Alk2 in the control of mitosis.

  8. The type I BMP receptor Alk3 is required for the induction of hepatic hepcidin gene expression by interleukin-6.

    PubMed

    Mayeur, Claire; Lohmeyer, Lisa K; Leyton, Patricio; Kao, Sonya M; Pappas, Alexandra E; Kolodziej, Starsha A; Spagnolli, Ester; Yu, Binglan; Galdos, Rita L; Yu, Paul B; Peterson, Randall T; Bloch, Donald B; Bloch, Kenneth D; Steinbicker, Andrea U

    2014-04-03

    Increased IL-6 production induces, via STAT3 phosphorylation, hepatic transcription of the gene encoding the iron-regulatory hormone, hepcidin, leading to development of anemia of chronic disease (ACD). Inhibition of bone morphogenetic protein (BMP) signaling prevents the induction of hepcidin gene expression by IL-6 and ameliorates ACD. Using mice with hepatocyte-specific deficiency of Alk2 or Alk3, we sought to identify the BMP type I receptor that participates in IL-6-mediated induction of hepcidin gene expression. Mice were injected with adenovirus specifying IL-6 (Ad.IL-6) or control adenovirus. Seventy-two hours later, serum iron concentrations and hepatic levels of STAT3 phosphorylation and hepcidin messenger RNA were measured. Additional mice were injected with recombinant murine IL-6 (mIL-6) or vehicle, and hepatic hepcidin gene expression was measured 4 hours later. Deficiency of Alk2 or Alk3 did not alter the ability of Ad.IL-6 injection to induce hepatic STAT3 phosphorylation. Ad.IL-6 increased hepatic hepcidin messenger RNA levels and decreased serum iron concentrations in Alk2- but not Alk3-deficient mice. Similarly, administration of mIL-6 induced hepatic hepcidin gene expression in Alk2- but not Alk3-deficient mice. These results demonstrate that the ability of IL-6 to induce hepatic hepcidin gene expression and reduce serum iron concentrations is dependent on the BMP type I receptor Alk3.

  9. Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1

    PubMed Central

    Menotti, Matteo; Poggio, Teresa; Panizza, Elena; Wang, Qi; Minero, Valerio G.; Fagoonee, Sharmila; Compagno, Mara; Altruda, Fiorella; Monti, Stefano; Chiarle, Roberto

    2016-01-01

    A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy. PMID:27119231

  10. Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins

    SciTech Connect

    Kang, Chung Hyo; Yun, Jeong In; Lee, Kwangho; Lee, Chong Ock; Lee, Heung Kyoung; Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho; Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un; Jeong, Hye Gwang; Kim, Hyoung Rae; Park, Chi Hoon

    2015-08-28

    Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151-L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15–20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. - Highlights: • We synthesized KRCA-0008 derivatives having trifluoromethyl instead of chloride. • KRCA-0080 shows superior activity against several ALK mutants to KRCA-0008. • Cellular assays show our ALK inhibitors suppress only EML4-ALK positive cells. • Our ALK inhibitors induce G1/S arrest to lead apoptosis in H3122 cells. • KRCA-0080 has superior in vivo efficacy to crizotinib and KRCA-0008 by 15–20%.

  11. For staining of ALK protein, the novel D5F3 antibody demonstrates superior overall performance in terms of intensity and extent of staining in comparison to the currently used ALK1 antibody.

    PubMed

    Taheri, Diana; Zahavi, David J; Del Carmen Rodriguez, Maria; Meliti, Abdelrazak; Rezaee, Neda; Yonescu, Raluca; Ricardo, Bernardo F P; Dolatkhah, Shahaboddin; Ning, Yi; Bishop, Justin A; Netto, George J; Sharma, Rajni

    2016-09-01

    Inflammatory myofibroblastic tumor (IMT) is a rare neoplasm. Approximately 50 % of IMTs show an anaplastic lymphoma kinase (ALK) gene fusion resulting in ALK overexpression on immunohistochemistry (IHC). A novel anti-ALK monoclonal antibody (D5F3) has been suggested to be of superior sensitivity to the ALK1 antibody which is currently used. We compared the performance of D5F3 in detecting ALK protein expression in IMTs from various anatomic sites compared to the currently utilized ALK1. We selected 25 IMTs from our surgical pathology files (2005-2015). The novel rabbit monoclonal anti-human CD246 (clone D5F3) and the currently used mouse monoclonal anti-human CD246 (clone ALK1) were used for immunohistochemical staining (IHC) in an automated slide stainer. The percentage of immunoreactive tumor cells (0, <5 %, 5-50 %, >50 %) and cytoplasmic staining intensity (graded 0-3) were assessed and compared between the two antibodies. Fluorescence in situ hybridization (FISH) studies for ALK gene rearrangement were performed on 11 tumors. D5F3 antibody stained 76 % and ALK1 antibody stained 72 % of IMTs (p = 0.747). Compared to staining with ALK1, D5F3 stained a higher proportion of cases extensively (>50 % cells) (76 vs. 28 %, p < 0.001) and with high intensity (grade 3 76 % vs 0; p < 0.001). FISH and IHC findings (for both antibodies) were concordant in 9/10 (90 %) IMTs, in which results were informative. The novel anti-ALK rabbit monoclonal antibody (D5F3 clone) demonstrates superior overall performance in term of intensity and extent of staining of ALK protein in IMT. We found IHC staining with both antibody clones to correlate equally well with FISH results for detection of ALK rearrangement.

  12. Evaluation of EML4-ALK Fusion Proteins in Non-Small Cell Lung Cancer Using Small Molecule Inhibitors12

    PubMed Central

    Li, Yongjun; Ye, Xiaofen; Liu, Jinfeng; Zha, Jiping; Pei, Lin

    2011-01-01

    The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non-small cell lung cancer and is mutually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non-small cell lung cancer (NSCLC). We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mechanism of EML4-ALK inhibition by a small molecule inhibitor. PMID:21245935

  13. Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients.

    PubMed

    Hong, Shaodong; Chen, Nan; Fang, Wenfeng; Zhan, Jianhua; Liu, Qing; Kang, Shiyang; He, Xiaobo; Liu, Lin; Zhou, Ting; Huang, Jiaxing; Chen, Ying; Qin, Tao; Zhang, Yaxiong; Ma, Yuxiang; Yang, Yunpeng; Zhao, Yuanyuan; Huang, Yan; Zhang, Li

    2016-03-01

    Driver mutations were reported to upregulate programmed death-ligand 1 (PD-L1) expression. However, how PD-L1 expression and immune function was affected by ALK-TKIs and anti-PD-1/PD-L1 treatment in ALK positive non-small-cell lung cancer (NSCLC) remains poorly understood. In the present study, western-blot, real-time PCR, flow cytometry and immunofluorescence were employed to explore how PD-L1 was regulated by ALK fusion protein. ALK-TKIs and relevant inhibitors were used to identify the downstream signaling pathways involved in PD-L1 regulation. Cell apoptosis, viability and Elisa test were used to study the immune suppression by ALK activation and immune reactivation by ALK-TKIs and/or PD-1 blocking in tumor cells and DC-CIK cells co-culture system. We found that PD-L1 expression was associated with EGFR mutations and ALK fusion genes in NSCLC cell lines. Over-expression of ALK fusion protein increased PD-L1 expression. PD-L1 mediated by ALK fusion protein increased the apoptosis of T cells in tumor cells and DC-CIK cells co-culture system. Inhibiting ALK by sensitive TKIs could enhance the production of IFNγ. Anti-PD-1 antibody was effective in both crizotinib sensitive and resistant NSCLC cells. Synergistic tumor killing effects were not observed with ALK-TKIs and anti-PD-1 antibody combination in co-culture system. ALK-TKIs not only directly inhibited tumor viability but also indirectly enhanced the antitumor immunity via the downregulation of PD-L1. Anti-PD-1/PD-L1 antibodies could be an optional therapy for crizotinib sensitive, especially crizotinib resistant NSCLC patients with ALK fusion gene. Combination of ALK-TKIs and anti-PD-1/PD-L1 antibodies treatment for ALK positive NSCLC warrants more data before moving into clinical practice.

  14. AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

    PubMed Central

    Ueda, Yuko; Ooshio, Ikumi; Fusamae, Yasuyuki; Kitae, Kaori; Kawaguchi, Megumi; Jingushi, Kentaro; Hase, Hiroaki; Harada, Kazuo; Hirata, Kazumasa; Tsujikawa, Kazutake

    2017-01-01

    The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion. PMID:28205560

  15. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

    PubMed Central

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

  16. NeuroD1 promotes neuroblastoma cell growth by inducing the expression of ALK.

    PubMed

    Lu, Fangjin; Kishida, Satoshi; Mu, Ping; Huang, Peng; Cao, Dongliang; Tsubota, Shoma; Kadomatsu, Kenji

    2015-04-01

    Neuroblastoma is derived from the sympathetic neuronal lineage of neural crest cells, and is the most frequently observed of the extracranial pediatric solid tumors. The neuronal differentiation factor, NeuroD1, has previously been shown to promote cell motility in neuroblastoma by suppressing the expression of Slit2. Here we report that NeuroD1 is also involved in the proliferation of neuroblastoma cells, including human cell lines and primary tumorspheres cultured from the tumor tissues of model mice. Interestingly, the growth inhibition of neuroblastoma cells induced by knockdown of NeuroD1 was accompanied by a reduction of ALK expression. ALK is known to be one of the important predisposition genes for neuroblastoma. The phenotype resulting from knockdown of NeuroD1 was suppressed by forced expression of ALK and, therefore, NeuroD1 appears to act mainly through ALK to promote the proliferation of neuroblastoma cells. Furthermore, we showed that NeuroD1 directly bound to the promoter region of ALK gene. In addition, the particular E-box in the promoter was responsible for NeuroD1-mediated ALK expression. These results indicate that ALK should be a direct target gene of NeuroD1. Finally, the expressions of NeuroD1 and ALK in the early tumor lesions of neuroblastoma model mice coincided in vivo. We conclude that the novel mechanism would regulate the expression of ALK in neuroblastoma and that NeuroD1 should be significantly involved in neuroblastoma tumorigenesis.

  17. ALK gene expression status in pleural effusion predicts tumor responsiveness to crizotinib in Chinese patients with lung adenocarcinoma

    PubMed Central

    Wang, Zheng; Wu, Xiaonan; Han, Xiaohong; Cheng, Gang; Mu, Xinlin; Zhang, Yuhui; Cui, Di; Liu, Chang; Liu, Dongge; Shi, Yuankai

    2016-01-01

    Objective The relationship between anaplastic lymphoma kinase (ALK) expression in malignant pleural effusion (MPE) samples detected only by Ventana immunohistochemistry (IHC) ALK (D5F3) and the efficacy of ALK-tyrosine kinase inhibitor therapy is uncertain. Methods Ventana anti-ALK (D5F3) rabbit monoclonal primary antibody testing was performed on 313 cell blocks of MPE samples from Chinese patients with advanced lung adenocarcinoma, and fluorescence in situ hybridization (FISH) was used to verify the ALK gene status in Ventana IHC ALK (D5F3)-positive samples. The follow-up clinical data on patients who received crizotinib treatment were recorded. Results Of the 313 MPE samples, 27 (8.6%) were confirmed as ALK expression-positive, and the Ventana IHC ALK (D5F3)-positive rate was 17.3% (27/156) in wild-type epidermal growth factor receptor (EGFR) MPE samples. Twenty-three of the 27 IHC ALK (D5F3)-positive samples were positive by FISH. Of the 11 Ventana IHC ALK (D5F3)-positive patients who received crizotinib therapy, 2 patients had complete response (CR), 5 had partial response (PR) and 3 had stable disease (SD). Conclusions The ALK gene expression status detected by the Ventana IHC ALK (D5F3) platform in MPE samples may predict tumor responsiveness to crizotinib in Chinese patients with advanced lung adenocarcinoma. PMID:28174489

  18. Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells.

    PubMed

    Wilson, T; Wu, X Y; Juengel, J L; Ross, I K; Lumsden, J M; Lord, E A; Dodds, K G; Walling, G A; McEwan, J C; O'Connell, A R; McNatty, K P; Montgomery, G W

    2001-04-01

    The Booroola fecundity gene (FecB) increases ovulation rate and litter size in sheep and is inherited as a single autosomal locus. The effect of FecB is additive for ovulation rate (increasing by about 1.6 corpora lutea per cycle for each copy) and has been mapped to sheep chromosome 6q23-31, which is syntenic to human chromosome 4q21-25. Bone morphogenetic protein IB (BMP-IB) receptor (also known as ALK-6), which binds members of the transforming growth factor-beta (TGF-beta) superfamily, is located in the region containing the FecB locus. Booroola sheep have a mutation (Q249R) in the highly conserved intracellular kinase signaling domain of the BMP-IB receptor. The mutation segregated with the FecB phenotype in the Booroola backcross and half-sib flocks of sheep with no recombinants. The mutation was not found in individuals from a number of sheep breeds not derived from the Booroola strain. BMPR-IB was expressed in the ovary and in situ hybridization revealed its specific location to the oocyte and the granulosa cell. Expression of mRNA encoding the BMP type II receptor was widespread throughout the ovary. The mutation in BMPR-IB found in Booroola sheep is the second reported defect in a gene from the TGF-beta pathway affecting fertility in sheep following the recent discovery of mutations in the growth factor, GDF9b/BMP15.

  19. Therapeutic strategies and mechanisms of drug resistance in Anaplastic Lymphoma Kinase (ALK)-rearranged lung cancer.

    PubMed

    Katayama, Ryohei

    2017-02-06

    Anaplastic lymphoma kinase (ALK) gene encoding the receptor tyrosine kinase ALK is expressed as a fusion gene in a variety of carcinomas. The expression of ALK is nearly undetectable in adults, and its activation is normally regulated by its ligands, FAM150A/B. However, ALK gene rearrangements result in different ALK fusion proteins that are constitutively expressed via the active promoter of fusion partner genes. ALK fusion proteins dimerize in a ligand-independent manner and lead to the dysregulation of cell proliferation via abnormal constitutive activation of ALK tyrosine kinase. Many ALK tyrosine kinase inhibitors (TKIs) have been developed to date, are three of which are currently in clinical use for the treatment of ALK-rearranged non-small cell lung cancer (NSCLC). ALK TKIs often achieve marked tumor regression in NSCLC patients with ALK rearrangements; however, ALK TKI-resistant tumors inevitably emerge within a few years in most cases. In this review, we summarize diverse ALK TKI resistance mechanisms identified in NSCLC with ALK rearrangements, and review potential therapeutic strategies to overcome ALK TKI resistance in these patients.

  20. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

    PubMed Central

    Guan, J.; Tucker, E. R.; Wan, H.; Chand, D.; Danielson, L. S.; Ruuth, K.; El Wakil, A.; Witek, B.; Jamin, Y.; Umapathy, G.; Robinson, S. P.; Johnson, T. W.; Smeal, T.; Martinsson, T.; Chesler, L.; Palmer, R. H.

    2016-01-01

    ABSTRACT The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients. PMID:27483357

  1. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins

    PubMed Central

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø.; Rizzo, Carmelo J.; Guengerich, F. Peter; Tudek, Barbara

    2015-01-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno (ε)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ε-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ε-adducts, 1,N6-ethenoadenine (εA), 3,N4-ethenocytosine (εC) and 1,N2-ethenoguanine (1,N2-εG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ε-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed εA and εC from ds and ssDNA but were inactive toward 1,N2-εG. SC-1A repaired only εA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ε-adducts in dsDNA, while only εA and εC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only εC in ssDNA Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N2-εG and that ALKBH3 removes only εC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  2. A causal link from ALK to hexokinase II overexpression and hyperactive glycolysis in EML4-ALK-positive lung cancer

    PubMed Central

    Ma, Yibao; Yu, Chunrong; Mohamed, Esraa M.; Shao, Huanjie; Wang, Li; Sundaresan, Gobalakrishnan; Zweit, Jamal; Idowu, Michael; Fang, Xianjun

    2016-01-01

    A high rate of aerobic glycolysis is a hallmark of malignant transformation. Accumulating evidence suggests that diverse regulatory mechanisms mediate this cancer-associated metabolic change seen in a wide spectrum of cancer. The echinoderm microtubule associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion protein is found in approximately 3-7% of non-small cell lung carcinomas (NSCLC). Molecular evidence and therapeutic effectiveness of FDA-approved ALK inhibitors indicated that EML4-ALK is a driving factor of lung tumorigenesis. A recent clinical study showed that NSCLC harboring EML4-ALK rearrangements displayed higher glucose metabolism compared to EML4-ALK-negative NSCLC. In the current work, we presented evidence that EML4-ALK is coupled to overexpression of hexokinase II (HK2), one of the rate-limiting enzymes of the glycolytic pathway. The link from EML4-ALK to HK2 upregulation is essential for a high rate of glycolysis and proliferation of EML4-ALK-rearranged NSCLC cells. We identified hypoxia-inducible factor 1α (HIF1α) as a key transcription factor to drive HK2 gene expression in normoxia in these cells. EML4-ALK induced hypoxia-independent but glucose-dependent accumulation of HIF1α protein via both transcriptional activation of HIF1α mRNA and the PI3K-AKT pathway to enhance HIF1α protein synthesis. The EML4-ALK-mediated upregulation of HIF1α, HK2 and glycolytic metabolism was also highly active in vivo as demonstrated by FDG-PET imaging of xenografts grown from EML4-ALK-positive NSCLC cells. Our data reveal a novel EML4-ALK-HIF1α-HK2 cascade to enhance glucose metabolism in EML4-ALK-positive NSCLC. PMID:27132509

  3. SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer.

    PubMed

    Papadakis, Andreas I; Sun, Chong; Knijnenburg, Theo A; Xue, Yibo; Grernrum, Wipawadee; Hölzel, Michael; Nijkamp, Wouter; Wessels, Lodewyk F A; Beijersbergen, Roderick L; Bernards, Rene; Huang, Sidong

    2015-04-01

    Recurrent inactivating mutations in components of SWI/SNF chromatin-remodeling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signaling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance to MET and ALK inhibitors in non-small cell lung cancers (NSCLCs). We found that SMARCE1 binds to regulatory regions of the EGFR locus and suppresses EGFR transcription in part through regulating expression of Polycomb Repressive Complex component CBX2. Addition of the EGFR inhibitor gefitinib restores the sensitivity of SMARCE1-knockdown cells to MET and ALK inhibitors in NSCLCs. Our findings link SMARCE1 to EGFR oncogenic signaling and suggest targeted treatment options for SMARCE1-deficient tumors.

  4. Anaplastic lymphoma kinase (ALK) inhibitors in the treatment of ALK-driven lung cancers.

    PubMed

    Roskoski, Robert

    2017-03-01

    Anaplastic lymphoma kinase is expressed in two-thirds of the anaplastic large-cell lymphomas as an NPM-ALK fusion protein. Physiological ALK is a receptor protein-tyrosine kinase within the insulin receptor superfamily of proteins that participates in nervous system development. The EML4-ALK fusion protein and four other ALK-fusion proteins play a fundamental role in the development in about 5% of non-small cell lung cancers. The amino-terminal portions of the ALK fusion proteins result in dimerization and subsequent activation of the ALK protein kinase domain that plays a key role in the pathogenesis of various tumors. Downstream signaling from the ALK fusion protein leads to the activation of the Ras/Raf/MEK/ERK1/2 cell proliferation module and the JAK/STAT cell survival pathways. Moreover, nearly two dozen ALK activating mutations are involved in the pathogenesis of childhood neuroblastomas. The occurrence of oncogenic ALK-fusion proteins, particularly in non-small cell lung cancer, has fostered considerable interest in the development of ALK inhibitors. Crizotinib was the first such inhibitor approved by the US Food and Drug Administration for the treatment of ALK-positive non-small cell lung cancer in 2011. The median time for the emergence of crizotinib drug resistance is 10.5 months after the initiation of therapy. Such resistance prompted the development of second-generation drugs including ceritinib and alectinib, which are approved for the treatment of non-small cell lung cancer. Unlike the single gatekeeper mutation that occurs in drug-resistant epidermal growth factor receptor in lung cancer, nearly a dozen different mutations in the catalytic domain of ALK fusion proteins have been discovered that result in crizotinib resistance. Crizotinib, ceritinib, and alectinib form a complex within the front cleft between the small and large lobes of an inactive ALK protein-kinase domain with a compact activation segment. These drugs are classified as type I½ B

  5. ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts.

    PubMed

    Thompson, Katherine; Murphy-Marshman, Hannah; Leask, Andrew

    2014-03-01

    The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.

  6. ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

    PubMed Central

    Lux, Andreas; Salway, Fiona; Dressman, Holly K; Kröner-Lux, Gabriele; Hafner, Mathias; Day, Philip JR; Marchuk, Douglas A; Garland, John

    2006-01-01

    Background TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). Methods The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. Results After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. Conclusion Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling. PMID:16594992

  7. The expression of SALL4 is significantly associated with EGFR, but not KRAS or EML4-ALK mutations in lung cancer

    PubMed Central

    Jia, Xiangbo; Qian, Rulin; Zhang, Binbin

    2016-01-01

    Background Lung cancer is the leading cause of cancer-related deaths worldwide; unfortunately, its prognosis is still very poor. Therefore, developing the target molecular is very important for lung cancer diagnosis and treatment, especially in the early stage. With this in view, spalt-like transcription factor 4 (SALL4) is considered a potential biomarker for diagnosis and prognosis in cancers, including lung cancer. Methods In order to better investigate the association between the expression of SALL4 and driver genes mutation, 450 histopathologically diagnosed patients with lung cancer and 11 non-cancer patients were enrolled to test the expression of SALL4 and the status of driver genes mutation. This investigation included epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene homolog (KRAS), and a fusion gene of the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK). Results The results of the study showed that females harbored more EGFR mutation in adenocarcinoma (ADC). The mutation rate of KRAS and EML4-ALK was about 5%, and the double mutations of EGFR/EML4-ALK were higher than EGFR/KRAS. In the expression analysis, the expression of SALL4 was much higher in cancer tissues than normally expected, especially in tissues that carried EGFR mutation (P<0.05), however, there were no significant differences between different mutation types. Likewise, there were no significant differences between expression of SALL4 and KRAS and EML4-ALK mutations. Conclusions SALL4 is up regulated in lung cancer specimens and harbors EGFR mutation; this finding indicates that SALL4 expression may be relevant with EGFR, which could provide a new insight to lung cancer therapy. The mechanism needs further investigation and analysis. PMID:27867542

  8. A New Protein Architecture for Processing Alkylation Damaged DNA: The Crystal Structure of DNA Glycosylase AlkD

    SciTech Connect

    Rubinson, Emily H.; Metz, Audrey H.; O'Quin, Jami; Eichman, Brandt F.

    2008-12-15

    DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA. AlkD is a recently discovered bacterial DNA glycosylase that removes positively charged methylpurines from DNA, and was predicted to adopt a protein fold distinct from from those of other DNA repair proteins. The crystal structure of Bacillus cereus AlkD presented here shows that the protein is composed exclusively of helical HEAT-like repeats, which form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface. Structural analysis of the variant HEAT repeats in AlkD provides a rationale for how this protein scaffolding motif has been modified to bind DNA. We report 7mG excision and DNA binding activities of AlkD mutants, along with a comparison of alkylpurine DNA glycosylase structures. Together, these data provide important insight into the requirements for alkylation repair within DNA and suggest that AlkD utilizes a novel strategy to manipulate DNA in its search for alkylpurine bases.

  9. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE.

  10. Expression of the BMP Receptor Alk3 in the Second Heart Field is Essential for Development of the Dorsal Mesenchymal Protrusion and Atrioventricular Septation

    PubMed Central

    Briggs, Laura E.; Phelps, Aimee L.; Brown, Elizabeth; Kakarla, Jayant; Anderson, Robert H.; van den Hoff, Maurice J. B.; Wessels, Andy

    2013-01-01

    Rationale The Dorsal Mesenchymal Protrusion (DMP) is a prong of mesenchyme derived from the Second Heart Field (SHF) located at the venous pole of the developing heart. Recent studies have shown that perturbation of its development is associated with the pathogenesis of atrioventricular septal defect (AVSD). Although the importance of the DMP to AV septation is now established, the molecular and cellular mechanisms underlying its development are far from fully understood. Prior studies have demonstrated that bone morphogenetic protein (BMP) signaling is essential for proper formation of the AV endocardial cushions and the cardiac outflow tract. A role for BMP signaling in regulation of DMP development remained to be elucidated. Objective To determine the role of BMP signaling in DMP development. Methods and Results Conditional deletion of the BMP receptor Alk3 from venous pole SHF cells leads to impaired formation of the DMP and a completely penetrant phenotype of ostium primum defect, a hallmark feature of AVSDs. Analysis of mutants revealed decreased proliferative index of SHF cells and, consequently, reduced number of SHF cells at the cardiac venous pole. In contrast, volume and expression of markers associated with proliferation and active BMP/TGFβ signaling was not significantly altered in the AV cushions of SHF-Alk3 mutants. Conclusions BMP signaling is required for expansion of the SHF-derived DMP progenitor population at the cardiac venous pole. Perturbation of Alk3-mediated BMP signaling from the SHF results in impaired development of the DMP and ostium primum defects. PMID:23584254

  11. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    SciTech Connect

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  12. Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5.

    PubMed

    Zeeh, Franziska; Witte, David; Gädeken, Thomas; Rauch, Bernhard H; Grage-Griebenow, Evelin; Leinung, Nadja; Fromm, Sofie Joline; Stölting, Stephanie; Mihara, Koichiro; Kaufmann, Roland; Settmacher, Utz; Lehnert, Hendrik; Hollenberg, Morley D; Ungefroren, Hendrik

    2016-07-05

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2-/- mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types.

  13. Proteinase-activated receptor 2 promotes TGF-β-dependent cell motility in pancreatic cancer cells by sustaining expression of the TGF-β type I receptor ALK5

    PubMed Central

    Gädeken, Thomas; Rauch, Bernhard H.; Grage-Griebenow, Evelin; Leinung, Nadja; Fromm, Sofie Joline; Stölting, Stephanie; Mihara, Koichiro; Kaufmann, Roland; Settmacher, Utz; Lehnert, Hendrik; Hollenberg, Morley D.; Ungefroren, Hendrik

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is characterized by high expression of transforming growth factor (TGF)-β and the G protein-coupled receptor proteinase-activated receptor 2 (PAR2), the latter of which functions as a cell-surface sensor for serine proteinases asscociated with the tumour microenvironment. Since TGF-β and PAR2 affect tumourigenesis by regulating migration, invasion and metastasis, we hypothesized that there is signalling crosstalk between them. Depleting PDAC and non-PDAC cells of PAR2 by RNA interference strongly decreased TGF-β1-induced activation of Smad2/3 and p38 mitogen-activated protein kinase, Smad dependent transcriptional activity, expression of invasion associated genes, and cell migration/invasion in vitro. Likewise, the plasminogen activator-inhibitor 1 gene in primary cultures of aortic smooth muscle cells from PAR2−/− mice displayed a greatly attenuated sensitivity to TGF-β1 stimulation. PAR2 depletion in PDAC cells resulted in reduced protein and mRNA levels of the TGF-β type I receptor activin receptor-like kinase 5 (ALK5). Forced expression of wild-type ALK5 or a kinase-active ALK5 mutant, but not a kinase-active but Smad-binding defective ALK5 mutant, was able to rescue TGF-β1-induced Smad3 activation, Smad dependent transcription, and cell migration in PAR2-depleted cells. Together, our data show that PAR2 is crucial for TGF-β1-induced cell motility by its ability to sustain expression of ALK5. Therapeutically targeting PAR2 may thus be a promising approach in preventing TGF-β-dependent driven metastatic dissemination in PDAC and possibly other stroma-rich tumour types. PMID:27248167

  14. Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand–receptor interactions

    PubMed Central

    Reshetnyak, Andrey V.; Murray, Phillip B.; Shi, Xiarong; Mo, Elizabeth S.; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

    2015-01-01

    Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states. PMID:26630010

  15. ALK oncoproteins in atypical inflammatory myofibroblastic tumours: novel RRBP1-ALK fusions in epithelioid inflammatory myofibroblastic sarcoma.

    PubMed

    Lee, Jen-Chieh; Li, Chien-Feng; Huang, Hsuan-Ying; Zhu, Mei-Jun; Mariño-Enríquez, Adrián; Lee, Chung-Ta; Ou, Wen-Bin; Hornick, Jason L; Fletcher, Jonathan A

    2017-02-01

    ALK oncogenic activation mechanisms were characterized in four conventional spindle-cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which had ALK genomic perturbations. Constitutively activated ALK oncoproteins were purified by ALK immunoprecipitation and electrophoresis, and were characterized by mass spectrometry. The four conventional IMT had TPM3/4-ALK fusions (two cases) or DCTN1-ALK fusions (two cases), whereas two atypical spindle-cell IMT had TFG-ALK and TPM3-ALK fusion in one case each, and three epithelioid inflammatory myofibroblastic sarcomas had RANBP2-ALK fusions in two cases, and a novel RRBP1-ALK fusion in one case. The epithelioid inflammatory myofibroblastic sarcoma with RRBP1-ALK fusion had cytoplasmic ALK expression with perinuclear accentuation, different from the nuclear membranous ALK localization in epithelioid inflammatory myofibroblastic sarcomas with RANBP2-ALK fusions. Evaluation of three additional uncharacterized epithelioid inflammatory myofibroblastic sarcomas with ALK cytoplasmic/perinuclear- accentuation expression demonstrated RRBP1-ALK fusion in two cases. These studies show that atypical spindle-cell IMT can utilize the same ALK fusion mechanisms described previously in conventional IMT, whereas in clinically aggressive epithelioid inflammatory myofibroblastic sarcoma we identify a novel recurrent ALK oncogenic mechanism, resulting from fusion with the RRBP1 gene. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  16. Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells.

    PubMed

    Oh, Se Jin; Noh, Kyung Hee; Lee, Young-Ho; Hong, Soon-Oh; Song, Kwon-Ho; Lee, Hyo-Jung; Kim, Soyeon; Kim, Tae Min; Jeon, Ju-Hong; Seo, Jae Hong; Kim, Dong-Wan; Kim, Tae Woo

    2015-11-24

    The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.

  17. EML4-ALK induces epithelial–mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells

    SciTech Connect

    Guo, Fuchun; Liu, Xiaoke Qing, Qin Sang, Yaxiong Feng, Chengjun Li, Xiaoyu Jiang, Li Su, Pei Wang, Yongsheng

    2015-04-10

    The echinoderm microtubule-associated protein-like 4(EML4) – anaplastic lymphoma kinase (ALK) fusion gene has been identified as a driver mutation in non-small-cell lung cancer (NSCLC). However, the role of EML4-ALK in malignant transformation is not entirely clear. Here, for the first time, we showed that H1299 NSCLC cells stably expressing EML4-ALK acquire EMT phenotype, associated with enhanced invasive migration and increased expression of EMT-inducing transcription factors. H1299-EML4-ALK cells also displayed cancer stem cell-like properties with a concomitant up-regulation of CD133 and enhanced ability of mammospheres formation. Moreover, we found that inhibition of ERK1/2 reversed EMT induced by EML4-ALK in H1299 cells. Taken together, these results suggested that EML4-ALK induced ERK activation is mechanistically associated with EMT phenotype. Thus, inhibition of ERK signaling pathway could be a potential strategy in treatment of NSCLC patients with EML4-ALK translocation. - Highlights: • EML4-ALK induced epithelial–mesenchymal transition in H1299 cells. • Expression of EML4-ALK promotes invasion and migration in vitro. • EML4-ALK enhanced sphere formation and stem cell-like properties in H1299 cells. • Blockage of ERK1/2 reverse Epithelial–Mesenchymal transition induced by EML4-ALK.

  18. Novel CAD-ALK gene rearrangement is drugable by entrectinib in colorectal cancer

    PubMed Central

    Amatu, Alessio; Somaschini, Alessio; Cerea, Giulio; Bosotti, Roberta; Valtorta, Emanuele; Buonandi, Pasquale; Marrapese, Giovanna; Veronese, Silvio; Luo, David; Hornby, Zachary; Multani, Pratik; Murphy, Danielle; Shoemaker, Robert; Lauricella, Calogero; Giannetta, Laura; Maiolani, Martina; Vanzulli, Angelo; Ardini, Elena; Galvani, Arturo; Isacchi, Antonella; Sartore-Bianchi, Andrea; Siena, Salvatore

    2015-01-01

    Background: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. Methods: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. Results: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1–35 of CAD with exons 20–29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. Conclusions: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC. PMID:26633560

  19. Novel ALK fusion partners in lung cancer.

    PubMed

    Iyevleva, Aglaya G; Raskin, Grigory A; Tiurin, Vladislav I; Sokolenko, Anna P; Mitiushkina, Natalia V; Aleksakhina, Svetlana N; Garifullina, Aigul R; Strelkova, Tatiana N; Merkulov, Valery O; Ivantsov, Alexandr O; Kuligina, Ekatherina Sh; Pozharisski, Kazimir M; Togo, Alexandr V; Imyanitov, Evgeny N

    2015-06-28

    Detection of ALK rearrangements in patients with non-small cell lung cancer (NSCLC) presents a significant technical challenge due to the existence of multiple translocation partners and break-points. To improve the performance of PCR-based tests, we utilized the combination of 2 assays, i.e. the variant-specific PCR for the 5 most common ALK rearrangements and the test for unbalanced 5'/3'-end ALK expression. Overall, convincing evidence for the presence of ALK translocation was obtained for 34/400 (8.5%) cases, including 14 EML4ex13/ALKex20, 12 EML4ex6/ALKex20, 3 EML4ex18/ALKex20, 2 EML4ex20/ALKex20 variants and 3 tumors with novel translocation partners. 386 (96.5%) out of 400 EGFR mutation-negative NSCLCs were concordant for both tests, being either positive (n = 26) or negative (n = 360) for ALK translocation; 49 of these samples (6 ALK+, 43 ALK-) were further evaluated by FISH, and there were no instances of disagreement. Among the 14 (3.5%) "discordant" tumors, 5 demonstrated ALK translocation by the first but not by the second PCR assay, and 9 had unbalanced ALK expression in the absence of known ALK fusion variants. 5 samples from the latter group were subjected to FISH, and the presence of translocation was confirmed in 2 cases. Next generation sequencing analysis of these 2 samples identified novel translocation partners, DCTN1 and SQSTM1; furthermore, the DCTN1/ALK fusion was also found in another NSCLC sample with unbalanced 5'/3'-end ALK expression, indicating a recurrent nature of this translocation. We conclude that the combination of 2 different PCR tests is a viable approach for the diagnostics of ALK rearrangements. Systematic typing of ALK fusions is likely to reveal new NSCLC-specific ALK partners.

  20. Alk5 inhibition increases delivery of macromolecular and protein-bound contrast agents to tumors

    PubMed Central

    Daldrup-Link, Heike E.; Mohanty, Suchismita; Ansari, Celina; Ito, Ken; Hong, Su Hyun; Hoffmann, Matthias; Pisani, Laura; Boudreau, Nancy; Gambhir, Sanjiv Sam; Coussens, Lisa M.

    2016-01-01

    Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies. PMID:27182558

  1. Diversity of alkane degrading bacteria associated with plants in a petroleum oil-contaminated environment and expression of alkane monooxygenase (alkB) genes

    NASA Astrophysics Data System (ADS)

    Andria, V.; Yousaf, S.; Reichenauer, T. G.; Smalla, K.; Sessitsch, A.

    2009-04-01

    Among twenty-six different plant species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo), and the combination of both plants performed well in a petroleum oil contaminated soil. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Higher numbers of culturable, degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Our results indicated that alkB genes have spread through horizontal gene transfer, particularly in the Italian ryegrass rhizosphere, and suggested mobility of catabolic genes between Gram-negative and Gram-positive bacteria. We furthermore studied the colonization behaviour of selected hydrocarbon-degrading strains (comprising an endopyhte and a rhizosphere strain) as well as the expression of their alkane monooxygenase genes in association with Italian ryegrass. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.

  2. CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

    PubMed Central

    Voeller, Donna; Gower, Arjan; Kim, In-Kyu; Zhang, Yu-Wen; Giaccone, Giuseppe

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene rearrangements are oncogenic drivers in a small subset of patients with non-small-cell lung cancer (NSCLC). The ALK inhibitors are highly effective in NSCLC patients harboring ALK rearrangements; however, most patients acquire resistance to the therapy following an initial response. Mechanisms of acquired resistance are complex. We used LC-MS/MS-based phosphotyrosine-peptide profiling in the EML4-ALK rearranged H3122 and H2228 cells treated with ALK inhibitors, to identify downstream effectors of ALK. We then used Western blot, siRNA experiments, cell proliferation, viability and migration assays to validate our findings. We identified CRKL as a novel downstream effector of ALK signaling. We demonstrated that CRKL tyrosine phosphorylation was repressed by pharmacological inhibition or small interfering RNA (siRNA) knockdown of ALK in the ALK-rearranged cells. More importantly, CRKL knockdown attenuated their cell proliferation, viability, and migration, but it had no effect on ALK phosphorylation and expression in these cells. Furthermore, CRKL tyrosine phosphorylation was inhibited by dasatinib (an inhibitor of ABL and SRC kinases), which in combination with the ALK inhibitor crizotinib displayed a synergistic inhibitory effect in vitro. In conclusion, our study suggests that CRKL is a key downstream effector of ALK, and combined inhibition of ALK and CRKL may represent an effective strategy for treating ALK-rearranged NSCLC patients. PMID:27078848

  3. Developmental expression of BMP4/ALK3/SMAD5 signaling pathway in the mouse testis: a potential role of BMP4 in spermatogonia differentiation.

    PubMed

    Pellegrini, Manuela; Grimaldi, Paola; Rossi, Pellegrino; Geremia, Raffaele; Dolci, Susanna

    2003-08-15

    It is well established that the c-kit gene plays an essential role in the proliferation of differentiating spermatogonia in prepuberal mice. However, the mechanisms that regulate the onset of spermatogenesis, i.e. differentiation of spermatogonial stem cells and c-kit expression, are poorly understood. Here we identify a novel signal transduction system in mouse prepuberal testis regulating this developmental event, involving bone morphogenetic protein 4 (BMP4) and its transduction machinery. BMP4 is produced by Sertoli cells very early in the postnatal life and is successively down regulated in peri-puberal Sertoli cells. Its receptor Alk3 and the R-Smad Smad5 are specifically expressed both in proliferating primordial germ cells and in postnatal spermatogonia. BMP4 stimulation of cultured spermatogonia induces Smad4/5 nuclear translocation and the formation of a DNA-binding complex with the transcriptional coactivator p300/CBP. In vitro exposure of undifferentiated spermatogonia to BMP4 exerts both mitogenic and differentiative effects, inducing [3H]thymidine incorporation and Kit expression. As a result of the latter event, Kit-negative spermatogonia acquire sensitivity to Stem Cell Factor.

  4. Targeting Neuroblastoma Cell Surface Proteins: Recommendations for Homology Modeling of hNET, ALK, and TrkB

    PubMed Central

    Haddad, Yazan; Heger, Zbyněk; Adam, Vojtech

    2017-01-01

    Targeted therapy is a promising approach for treatment of neuroblastoma as evident from the large number of targeting agents employed in clinical practice today. In the absence of known crystal structures, researchers rely on homology modeling to construct template-based theoretical structures for drug design and testing. Here, we discuss three candidate cell surface proteins that are suitable for homology modeling: human norepinephrine transporter (hNET), anaplastic lymphoma kinase (ALK), and neurotrophic tyrosine kinase receptor 2 (NTRK2 or TrkB). When choosing templates, both sequence identity and structure quality are important for homology modeling and pose the first of many challenges in the modeling process. Homology modeling of hNET can be improved using template models of dopamine and serotonin transporters instead of the leucine transporter (LeuT). The extracellular domains of ALK and TrkB are yet to be exploited by homology modeling. There are several idiosyncrasies that require direct attention throughout the process of model construction, evaluation and refinement. Shifts/gaps in the alignment between the template and target, backbone outliers and side-chain rotamer outliers are among the main sources of physical errors in the structures. Low-conserved regions can be refined with loop modeling method. Residue hydrophobicity, accessibility to bound metals or glycosylation can aid in model refinement. We recommend resolving these idiosyncrasies as part of “good modeling practice” to obtain highest quality model. Decreasing physical errors in protein structures plays major role in the development of targeting agents and understanding of chemical interactions at the molecular level. PMID:28163672

  5. ALK-positive anaplastic large cell lymphoma limited to the skin: clinical, histopathological and molecular analysis of 6 pediatric cases. A report from the ALCL99 study.

    PubMed

    Oschlies, Ilske; Lisfeld, Jasmin; Lamant, Laurence; Nakazawa, Atsuko; d'Amore, Emanuele S G; Hansson, Ulrika; Hebeda, Konnie; Simonitsch-Klupp, Ingrid; Maldyk, Jadwiga; Müllauer, Leonhard; Tinguely, Marianne; Stücker, Markus; Ledeley, Marie-Cecile; Siebert, Reiner; Reiter, Alfred; Brugières, Laurence; Klapper, Wolfram; Woessmann, Wilhelm

    2013-01-01

    Anaplastic large cell lymphomas are peripheral T-cell lymphomas that are characterized by a proliferation of large anaplastic blasts expressing CD30. In children, systemic anaplastic large cell lymphomas often present at advanced clinical stage and harbor translocations involving the anaplastic lymphoma kinase (ALK) gene leading to the expression of chimeric anaplastic lymphoma kinase (ALK)-fusion proteins. Primary cutaneous anaplastic large cell lymphoma is regarded as an ALK-negative variant confined to the skin and is part of the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders. Thirty-three of 487 pediatric patients registered within the Anaplastic Large Cell Lymphoma-99 trial (1999 to 2006) presented with a skin limited CD30-positive lympho-proliferative disorder. In 23 of the 33 patients, material for international histopathological review was available, and the cases were studied for histopathological, immunophenotypical and clinical features as well as for breaks within the ALK gene. Five of 23 cases and one additional case (identified after closure of the trial) expressed ALK-protein. Complete staging excluded any other organ involvement in all children. Expression of ALK proteins was demonstrated by immunohistochemistry in all cases and the presence of breaks of the ALK gene was genetically confirmed in 5 evaluable cases. The histopathological and clinical picture of these skin-restricted ALK-positive lymphomas was indistinguishable from that of cutaneous anaplastic large cell lymphoma. Five children presented with a single skin lesion that was completely resected in 4 and incompletely resected in one. Three of these patients received no further therapy, 2 additional local radiotherapy, and one chemotherapy. All children remain in complete remission with a median follow up of seven years (range 1-8 years). We present 6 pediatric cases of ALK-positive primary cutaneous anaplastic large cell lymphomas. After thorough

  6. Application of AlkBGT and AlkL from Pseudomonas putida GPo1 for Selective Alkyl Ester ω-Oxyfunctionalization in Escherichia coli

    PubMed Central

    Eggink, Gerrit; Weusthuis, Ruud A.

    2016-01-01

    ABSTRACT The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (gcdw), 67% of the initial activity on methyl nonanoate. In time-lapse conversions with 5 mM NAEE the main product was 9-hydroxy NAEE (3.6 mM), but also 9-oxo NAEE (0.1 mM) and 9-carboxy NAEE (0.6 mM) were formed. AlkBGT also ω-oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C6 to C10. Increasing the length of the alkyl chain improved the ω-oxidation activity of AlkBGT on esters of C6 and C7 fatty acids. From these esters, application of butyl hexanoate resulted in the highest ω-oxidation activity, 82 U/gcdw. Coexpression of AlkL only had a positive effect on ω-functionalization of substrates with a total length of C11 or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for ω-functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids. IMPORTANCE Fatty acid esters are promising renewable starting materials for the production of ω-hydroxy fatty acid esters (ω-HFAEs). ω-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to ω-HFAEs is challenging, as it generates by-products and needs harsh reaction conditions. Biocatalytic production is a promising alternative. In this study, biocatalytic conversion of fatty acid esters toward ω-HFAEs was investigated using whole cells. This was achieved with recombinant Escherichia coli cells that produce the AlkBGT enzymes. These enzymes can produce ω-HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C

  7. Expression level of CRKL and AXL combined with exon 19 deletion in EGFR and ALK status confer differential prognosis of lung adenocarcinoma subtypes

    PubMed Central

    Cai, Yi-Ran; Dong, Yu-Jie; Wu, Hong-Bo; Yu, Da-Ping; Zhou, Li-Juan; Su, Dan; Zhang, Li; Chen, Xue-Jing

    2016-01-01

    Non-small cell lung cancer (NSCLC) is a lethal cancer-related disease in population. Adenocarcinoma (AC) is subclassified into several subtypes based on the new classification by the International Association for the Study of Lung Cancer, American Thoracic Society and European Respiratory Society in 2011. Correlation between original expression of Crk-like (CRKL) and anaplastic lymphoma receptor tyrosine kinase in diverse histological components of AC and epidermal growth factor receptor (EGFR) or ALK status was evaluated by immunohistochemistry and sequencing in present study. A total of 106 cases, including 83 patients (78.3%) with mixed-type ACs, were assessed in the present study using eligible follow-up data. The ACs consisted of 32 acinar, 12 papillary, 5 mucinous, 11 micropapillary and 46 solid-predominant ACs. In total, 69.8% samples were composed of 2 or 3 histological components, with different expression levels of CRKL and AXL. ACs with EGFR mutation had a higher level of AXL expression compared with ACs without mutation (P=0.019). Multivariate survival analysis showed that AC subtypes and EGFR mutation subtypes were significantly associated with the progression-free survival (PFS) time. Acinar AC was the subtype with the most notable PFS time (30.6 months), which was significantly different from the PFS time of papillary, mucinous, micropapillary and solid-predominant ACs (hazard ratio, 0.4; 95% CI, 0.21–0.75; P=0.005). Among the ACs with exon 19 mutation, the median PFS time (28.8 months) of patients with a lower level of AXL protein expression was increased compared with the PFS time of patients with the L858R mutation and wild-type EGFR (9.1 months and 11 months, respectively; P=0.03), whereas no significant difference in ACs with an increased level of AXL expression. However, AC patients with higher level of CRKL expression had better PFS (28.8 months) than patients with the L858R mutation and wild-type EGFR (9.1 months and 11.3 months

  8. Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK

    PubMed Central

    Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases. PMID:25727400

  9. The Drosophila midkine/pleiotrophin homologues Miple1 and Miple2 affect adult lifespan but are dispensable for alk signaling during embryonic gut formation.

    PubMed

    Hugosson, Fredrik; Sjögren, Camilla; Birve, Anna; Hedlund, Ludmilla; Eriksson, Therese; Palmer, Ruth H

    2014-01-01

    Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

  10. EML4-ALK induces epithelial-mesenchymal transition consistent with cancer stem cell properties in H1299 non-small cell lung cancer cells.

    PubMed

    Guo, Fuchun; Liu, Xiaoke; Qing, Qin; Sang, Yaxiong; Feng, Chengjun; Li, Xiaoyu; Jiang, Li; Su, Pei; Wang, Yongsheng

    2015-04-10

    The echinoderm microtubule-associated protein-like 4(EML4)--anaplastic lymphoma kinase (ALK) fusion gene has been identified as a driver mutation in non-small-cell lung cancer (NSCLC). However, the role of EML4-ALK in malignant transformation is not entirely clear. Here, for the first time, we showed that H1299 NSCLC cells stably expressing EML4-ALK acquire EMT phenotype, associated with enhanced invasive migration and increased expression of EMT-inducing transcription factors. H1299-EML4-ALK cells also displayed cancer stem cell-like properties with a concomitant up-regulation of CD133 and enhanced ability of mammospheres formation. Moreover, we found that inhibition of ERK1/2 reversed EMT induced by EML4-ALK in H1299 cells. Taken together, these results suggested that EML4-ALK induced ERK activation is mechanistically associated with EMT phenotype. Thus, inhibition of ERK signaling pathway could be a potential strategy in treatment of NSCLC patients with EML4-ALK translocation.

  11. Critical roles of the TGF-beta type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development.

    PubMed

    Matsunobu, Tomoya; Torigoe, Kiyoyuki; Ishikawa, Masaki; de Vega, Susana; Kulkarni, Ashok B; Iwamoto, Yukihide; Yamada, Yoshihiko

    2009-08-15

    TGF-beta has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-beta in skeletal development is unclear. In this study, we investigated the role of TGF-beta signaling in growth plate development by creating mice with a conditional knockout of the TGF-beta type I receptor ALK5 (ALK5(CKO)) in skeletal progenitor cells using Dermo1-Cre mice. ALK5(CKO) mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5(CKO) growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5(CKO) growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-beta signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-beta signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.

  12. Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

    PubMed Central

    Matsunobu, Tomoya; Torigoe, Kiyoyuki; Ishikawa, Masaki; de Vega, Susana; Kulkarni, Ashok B.; Iwamoto, Yukihide; Yamada, Yoshihiko

    2009-01-01

    TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5CKOCKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development. PMID:19501582

  13. Deficient BIM Expression as a Mechanism of Intrinsic and Acquired Resistance to Targeted Therapies in EGFR-Mutant and ALK-Positive Lung Cancers

    DTIC Science & Technology

    2015-08-01

    EGFR- Mutant and ALK-Positive Lung Cancers PRINCIPAL INVESTIGATOR: Lecia Sequist MD. CONTRACTING ORGANIZATION: Massachusetts General Hospital Boston...and Acquired Resistance to Targeted Therapies in EGFR- Mutant and ALK-Positive Lung Cancers 5b. GRANT NUMBER W81XWH-13-1-0227 5c. PROGRAM ELEMENT...to a specific kinase, inhibition of that kinase often leads to cell growth arrest and apoptosis. For example, EGFR mutant and EML4-ALK lung cancers

  14. Deficient BIM Expression as a Mechanism of Intrinsic and Acquired Resistance to Targeted Therapies in EGFR-Mutant and ALK-Positive Lung Cancers

    DTIC Science & Technology

    2015-08-01

    EGFR- Mutant and ALK-Positive Lung Cancers PRINCIPAL INVESTIGATOR: Jeffrey Engelman MD PhD. CONTRACTING ORGANIZATION: Massachusetts General Hospital...Intrinsic and Acquired Resistance to Targeted Therapies in EGFR- Mutant and ALK-Positive Lung Cancers 5b. GRANT NUMBER W81XWH-13-1-0226 5c. PROGRAM...to a specific kinase, inhibition of that kinase often leads to cell growth arrest and apoptosis. For example, EGFR mutant and EML4-ALK lung cancers

  15. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    SciTech Connect

    Xu, Fei; Li, Hongling; Sun, Yong

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  16. Activated Alk triggers prolonged neurogenesis and Ret upregulation providing a therapeutic target in ALK-mutated neuroblastoma

    PubMed Central

    Cazes, Alex; Lopez-Delisle, Lucille; Tsarovina, Konstantina; Pierre-Eugène, Cécile; De Preter, Katleen; Peuchmaur, Michel; Nicolas, André; Provost, Claire; Louis-Brennetot, Caroline; Daveau, Romain; Kumps, Candy; Cascone, Ilaria; Schleiermacher, Gudrun; Prignon, Aurélie; Speleman, Frank; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

    2014-01-01

    Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma. PMID:24811913

  17. Reversal of microRNA-150 silencing disadvantages crizotinib-resistant NPM-ALK(+) cell growth

    PubMed Central

    Hoareau-Aveilla, Coralie; Valentin, Thibaud; Daugrois, Camille; Quelen, Cathy; Mitou, Géraldine; Quentin, Samuel; Jia, Jinsong; Spicuglia, Salvatore; Ferrier, Pierre; Ceccon, Monica; Giuriato, Sylvie; Gambacorti-Passerini, Carlo; Brousset, Pierre; Lamant, Laurence; Meggetto, Fabienne

    2015-01-01

    The regulatory microRNA miR-150 is involved in the development of hemopathies and is downregulated in T-lymphomas, such as anaplastic large-cell lymphoma (ALCL) tumors. ALCL is defined by the presence or absence of translocations that activate the anaplastic lymphoma kinase (ALK), with nucleophosmin-ALK (NPM-ALK) fusions being the most common. Here, we compared samples of primary NPM-ALK(+) and NPM-ALK(–) ALCL to investigate the role of miR-150 downstream of NPM-ALK. Methylation of the MIR150 gene was substantially elevated in NPM-ALK(+) biopsies and correlated with reduced miR-150 expression. In NPM-ALK(+) cell lines, DNA hypermethylation–mediated miR-150 repression required ALK-dependent pathways, as ALK inhibition restored miR-150 expression. Moreover, epigenetic silencing of miR-150 was due to the activation of STAT3, a major downstream substrate of NPM-ALK, in cooperation with DNA methyltransferase 1 (DNMT1). Accordingly, miR-150 repression was turned off following treatment with the DNMT inhibitor, decitabine. In murine NPM-ALK(+) xenograft models, miR-150 upregulation induced antineoplastic activity. Treatment of crizotinib-resistant NPM-ALK(+) KARPAS-299-CR06 cells with decitabine or ectopic miR-150 expression reduced viability and growth. Altogether, our results suggest that hypomethylating drugs, alone or in combination with other agents, may benefit ALK(+) patients harboring tumors resistant to crizotinib and other anti-ALK tyrosine kinase inhibitors (TKIs). Moreover, these results support further work on miR-150 in these and other ALK(+) malignancies. PMID:26258416

  18. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    PubMed Central

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  19. Protein expression-yeast.

    PubMed

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline.

  20. Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

    PubMed Central

    Kim, Bu-Yeo; Jeong, SangKyun; Lee, Seo-Young; Lee, So Min; Gweon, Eun Jeong; Ahn, Hyunjun; Kim, Janghwan; Chung, Sun-Ku

    2016-01-01

    Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1, encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs. PMID:27256111

  1. microRNA classifiers are powerful diagnostic/prognostic tools in ALK-, EGFR-, and KRAS-driven lung cancers.

    PubMed

    Gasparini, Pierluigi; Cascione, Luciano; Landi, Lorenza; Carasi, Stefania; Lovat, Francesca; Tibaldi, Carmelo; Alì, Greta; D'Incecco, Armida; Minuti, Gabriele; Chella, Antonio; Fontanini, Gabriella; Fassan, Matteo; Cappuzzo, Federico; Croce, Carlo M

    2015-12-01

    microRNAs (miRNAs) can act as oncosuppressors or oncogenes, induce chemoresistance or chemosensitivity, and are major posttranscriptional gene regulators. Anaplastic lymphoma kinase (ALK), EGF receptor (EGFR), and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) are major drivers of non-small cell lung cancer (NSCLC). The aim of this study was to assess the miRNA profiles of NSCLCs driven by translocated ALK, mutant EGFR, or mutant KRAS to find driver-specific diagnostic and prognostic miRNA signatures. A total of 85 formalin-fixed, paraffin-embedded samples were considered: 67 primary NSCLCs and 18 matched normal lung tissues. Of the 67 primary NSCLCs, 17 were echinoderm microtubule-associated protein-like 4-ALK translocated (ALK(+)) lung cancers; the remaining 50 were not (ALK(-)). Of the 50 ALK(-) primary NSCLCs, 24 were EGFR and KRAS mutation-negative (i.e., WT; triple negative); 11 were mutant EGFR (EGFR(+)), and 15 were mutant KRAS (KRAS(+)). We developed a diagnostic classifier that shows how miR-1253, miR-504, and miR-26a-5p expression levels can classify NSCLCs as ALK-translocated, mutant EGFR, or mutant KRAS versus mutation-free. We also generated a prognostic classifier based on miR-769-5p and Let-7d-5p expression levels that can predict overall survival. This classifier showed better performance than the commonly used classifiers based on mutational status. Although it has several limitations, this study shows that miRNA signatures and classifiers have great potential as powerful, cost-effective next-generation tools to improve and complement current genetic tests. Further studies of these miRNAs can help define their roles in NSCLC biology and in identifying best-performing chemotherapy regimens.

  2. Excess of NPM-ALK oncogenic signaling promotes cellular apoptosis and drug dependency

    PubMed Central

    Mologni, Luca; Poggio, Teresa; Varesio, Lydia M.; Menotti, Matteo; Bombelli, Silvia; Rigolio, Roberta; Manazza, Andrea D.; Di Giacomo, Filomena; Ambrogio, Chiara; Giudici, Giovanni; Casati, Cesare; Mastini, Cristina; Compagno, Mara; Turner, Suzanne D.; Gambacorti-Passerini, Carlo; Chiarle, Roberto; Voena, Claudia

    2016-01-01

    Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK. NPM-ALK deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive due to heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or re-localization of NPM-ALK to the cytoplasm by NPM genetic knock-out or knock-down caused ERK1/2 increased phosphorylation and cell death through the engagement of an ATM/Chk2 and γH2AX mediated DNA damage response. Remarkably, human NPM-ALK amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A “drug holiday” where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification. PMID:26657151

  3. BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

    PubMed

    Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

    2015-11-01

    Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

  4. Fluoride Regulate Osteoblastic Transforming Growth Factor-β1 Signaling by Mediating Recycling of the Type I Receptor ALK5

    PubMed Central

    Yang, Chen; Wang, Yan; Xu, Hui

    2017-01-01

    This study aimed to preliminary investigate the role of activin receptor-like kinase (ALK) 5 as one of TGF-βR1 subtypes in bone turnover and osteoblastic differentiation induced by fluoride. We analyzed bone mineral density and the expression of genes related with transforming growth factor-β1(TGF-β1) signaling and bone turnover in rats treated by different concentrations of fluoride with or without SB431542 in vivo. Moreover, MTT assay, alkaline phosphatase staining, RT-PCR, immunocytochemical analysis and western blot analysis were used to detect the influence on bone marrow stem cells (BMSC) after stimulating by varying concentration of fluoride with or without SB431542 in vitro. The in vivo study showed SB431542 treatment affected bone density and gene expression of rats, which indicated TGF-β1 and ALK5 might take part in fluoride-induced bone turnover and bone formation. The in vitro study showed low concentration of fluoride improved BMSC cells viability, alkaline phosphatase activity, and osteocalcin protein expression which were inhibited by high concentration of fluoride. The gene expression of Runx2 and ALK5 in cells increased after low concentration fluoride treatment which was also inhibited by high concentration of fluoride. Fluoride treatment inhibited gene and protein expression of Samd3 (except 1 mgF-/L). Compared with fluoride treatment alone, cells differentiation was inhibited with SB431542 treatment. Moreover, the expression of Runx2, ALK5 and Smad3 were influenced by SB431542 treatment. In conclusion, this preliminary study indicated that fluoride regulated osteoblastic TGFβ1 signaling in bone turnover and cells differentiation via ALK5. PMID:28125630

  5. Activation of the orphan receptor tyrosine kinase ALK by zinc.

    PubMed

    Bennasroune, Aline; Mazot, Pierre; Boutterin, Marie-Claude; Vigny, Marc

    2010-08-06

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.

  6. Efficiency of Crizotinib on an ALK-Positive Inflammatory Myofibroblastic Tumor of the Central Nervous System: A Case Report

    PubMed Central

    Chennouf, Anas; Arslanian, Elizabeth; Roberge, David; Berthelet, France; Bojanowski, Michel; Bahary, Jean-Paul; Masucci, Laura; Belanger, Karl; Florescu, Marie

    2017-01-01

    Inflammatory myofibroblastic tumors (IMT) of the central nervous system (CNS) are rare entities that have a predilection for local recurrences. Approximately half of the inflammatory myofibroblastic tumors contain translocations that result in the over-expression of the anaplastic lymphoma kinase (ALK) gene. We hereby present the case of a patient diagnosed with a left parieto-occipital IMT that recurred after multiple surgeries and radiotherapy. Immuno-histochemical examination of the tumor demonstrated ALK overexpression and the presence of an ALK rearrangement observed in lung cancers. The patient was subsequently started on an ALK inhibitor. A response evaluation criteria in solid tumors (RECIST) partial response was observed by the seventh month of ALK inhibition and the tumor remained in control for 14 months. The current case reiterates the activity of ALK inhibitors within the CNS and suggests that radiotherapy may potentiate the permeability of ALK inhibitors in CNS tumors addicted to ALK signalling.

  7. Alkane hydroxylase gene (alkB) phylotype composition and diversity in northern Gulf of Mexico bacterioplankton

    PubMed Central

    Smith, Conor B.; Tolar, Bradley B.; Hollibaugh, James T.; King, Gary M.

    2013-01-01

    Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM), a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU) indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with AlkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter). Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables. PMID:24376439

  8. RecA stimulates AlkB-mediated direct repair of DNA adducts

    PubMed Central

    Shivange, Gururaj; Monisha, Mohan; Nigam, Richa; Kodipelli, Naveena; Anindya, Roy

    2016-01-01

    The Escherichia coli AlkB protein is a 2-oxoglutarate/Fe(II)-dependent demethylase that repairs alkylated single stranded and double stranded DNA. Immunoaffinity chromatography coupled with mass spectrometry identified RecA, a key factor in homologous recombination, as an AlkB-associated protein. The interaction between AlkB and RecA was validated by yeast two-hybrid assay; size-exclusion chromatography and standard pull down experiment and was shown to be direct and mediated by the N-terminal domain of RecA. RecA binding results AlkB–RecA heterodimer formation and RecA–AlkB repairs alkylated DNA with higher efficiency than AlkB alone. PMID:27378775

  9. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

    PubMed

    Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara; Bosia, Amalia; D'Amico, Lucia; Riera, Ludovica; Cheng, Mangeng; Ruggeri, Bruce; Jensen, Ole N; Goss, Valerie L; Lee, Kimberly; Nardone, Julie; Rush, John; Polakiewicz, Roberto D; Comb, Michael J; Chiarle, Roberto; Inghirami, Giorgio

    2009-03-19

    Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

  10. Alk7 Depleted Mice Exhibit Prolonged Cardiac Repolarization and Are Predisposed to Ventricular Arrhythmia

    PubMed Central

    Ying, Shaozhen; Cao, Hong; Hu, He; Wang, Xin; Tang, Yanhong; Huang, Congxin

    2016-01-01

    We aimed to investigate the role of activin receptor-like kinase (ALK7) in regulating cardiac electrophysiology. Here, we showed that Alk7-/- mice exhibited prolonged QT intervals in telemetry ECG recordings. Furthermore, Langendorff-perfused Alk7-/- hearts had significantly longer action potential duration (APD) and greater incidence of ventricular arrhythmia (AV) induced by burst pacing. Using whole-cell patch clamp, we found that the densities of repolarizing K+ currents Ito and IK1 were profoundly reduced in Alk7-/- ventricular cardiomyocytes. Mechanistically, the expression of Kv4.2 (a major subunit of Ito carrying channel) and KCHIP2 (a key accessory subunit of Ito carrying channel), was markedly decreased in Alk7-/- hearts. These findings suggest that endogenous expression of ALK7 is necessary to maintain repolarizing K+ currents in ventricular cardiomyocytes, and finally prevent action potential prolongation and ventricular arrhythmia. PMID:26882027

  11. Screening for ALK abnormalities in central nervous system metastases of non-small-cell lung cancer: ALK abnormalities in CNS metastases of NSCLC.

    PubMed

    Nicoś, Marcin; Jarosz, Bożena; Krawczyk, Paweł; Wojas-Krawczyk, Kamila; Kucharczyk, Tomasz; Sawicki, Marek; Pankowski, Juliusz; Trojanowski, Tomasz; Milanowski, Janusz

    2016-11-23

    Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3-7% of primary non-small-cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non-smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (p=0.0002; χ(2) =16.783) in former-smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Our results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, we showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations. This article is protected by copyright. All rights reserved.

  12. Characterization of a CYP153 alkane hydroxylase gene in a Gram-positive Dietzia sp. DQ12-45-1b and its "team role" with alkW1 in alkane degradation.

    PubMed

    Nie, Yong; Liang, Jie-Liang; Fang, Hui; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-01-01

    CYP153 and AlkB-like hydroxylases were recently discovered in Gram-positive alkane-degrading bacteria. However, it is unclear whether they cooperate with each other in alkane degradation as they do in Gram-negative bacteria. In this paper, we cloned the CYP153 gene from a representative Gram-positive alkane-degrading bacterium, Dietzia sp. DQ12-45-1b. The CYP153 gene transcription in Dietzia sp. DQ12-45-1b and heterologous expression in alkB gene knockout mutant strain Pseudomonas fluorescens KOB2∆1 both confirmed the functions of CYP153 on C6-C10 n-alkanes degradation, but not on longer chain-length n-alkanes. In addition, substrate-binding analysis of the purified CYP153 protein revealed different substrate affinities to C6-C16 n-alkanes, confirming n-alkanes binding to CYP153 protein. Along with AlkW1, an AlkB-like alkane hydroxylase in Dietzia sp. DQ12-45-1b, a teamwork pattern was found in n-alkane degradation, i.e. CYP153 was responsible for hydroxylating n-alkanes shorter than C10 while AlkW1 was responsible for those longer than C14. Further sequence analysis suggested that the high horizontal gene transfer (HGT) potential of CYP153 genes may be universal in Gram-positive alkane-degrading actinomycetes that contain both alkB and CYP153 genes.

  13. A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

    DTIC Science & Technology

    2014-09-01

    AWARD NUMBER: W81XWH-13-1-0220 TITLE: A Genetically Engineered Mouse Model of Neuroblastoma ...CONTRACT NUMBER A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN 5b. GRANT NUMBER W81XWH-13-1-0220 5c...common ALK mutations in neuroblastoma , F1174L and R1275Q. We have determined that in tumors cells expressing mutated ALK, different downstream

  14. Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells

    PubMed Central

    Aveic, Sanja; Pantile, Marcella; Seydel, Anke; Esposito, Maria Rosaria; Zanon, Carlo; Li, Gary; Tonini, Gian Paolo

    2016-01-01

    Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03–5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib. PMID:26735175

  15. BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy.

    PubMed

    Shahid, Mohd; Spagnolli, Ester; Ernande, Laura; Thoonen, Robrecht; Kolodziej, Starsha A; Leyton, Patricio A; Cheng, Juan; Tainsh, Robert E T; Mayeur, Claire; Rhee, David K; Wu, Mei X; Scherrer-Crosbie, Marielle; Buys, Emmanuel S; Zapol, Warren M; Bloch, Kenneth D; Bloch, Donald B

    2016-04-15

    Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

  16. Detection of ALK rearrangements in lung cancer patients using a homebrew PCR assay.

    PubMed

    Yu, Hui; Chang, JianHua; Liu, Fang; Wang, Qifeng; Lu, YongMing; Zhang, ZhuanXu; Shen, Jiabing; Zhai, Qing; Meng, Xia; Wang, Jialei; Ye, Xun

    2017-01-31

    Lung cancer patients with anaplastic lymphoma kinase (ALK) rearrangements are candidates for targeted therapeutics. However, patients must be tested with a companion diagnostic assay to realize their ALK rearrangement status. We analyzed the publicly available E-GEOD-31210 microarray dataset and identified a non-coding RNA, sweyjawbu, which is strongly associated with ALK rearrangements. We validated these results using quantitative real-time PCR in an independent cohort consisting of 4 cell lines and 83 clinical samples. We could differentiate between ALK rearrangement-positive and -negative lung cancer samples by comparing sweyjawbu expression. Additionally, ALK rearrangement status was determined by comparing the expression of the 5' and 3' regions of the ALK transcript or by detecting known ALK hybrid subtypes. Thus, using our homebrew PCR assay, we were able to accurately detect ALK rearrangements, which could be used for diagnostic screening of lung cancer patients. The prototype could potentially be transferred to an automatic multiplex PCR platform (FilmArray) to differentiate between ALK rearrangement-positive and -negative patients in point-of-care settings.

  17. Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

    PubMed

    Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

    2012-10-01

    The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

  18. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism

    PubMed Central

    Nord, Christoffer; Ahlgren, Ulf; Eriksson, Maria; Vernersson-Lindahl, Emma; Helland, Åslaug; Alexeyev, Oleg A.; Hallberg, Bengt; Palmer, Ruth H.

    2015-01-01

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis. PMID:25955180

  19. Prospective and clinical validation of ALK immunohistochemistry: results from the phase I/II study of alectinib for ALK-positive lung cancer (AF-001JP study)

    PubMed Central

    Takeuchi, K.; Togashi, Y.; Kamihara, Y.; Fukuyama, T.; Yoshioka, H.; Inoue, A.; Katsuki, H.; Kiura, K.; Nakagawa, K.; Seto, T.; Maemondo, M.; Hida, T.; Harada, M.; Ohe, Y.; Nogami, N.; Yamamoto, N.; Nishio, M.; Tamura, T.

    2016-01-01

    Background Anaplastic lymphoma kinase (ALK) fusions need to be accurately and efficiently detected for ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) remains the reference test. Although increasing data are supporting that ALK immunohistochemistry (IHC) is highly concordant with FISH, IHC screening needed to be clinically and prospectively validated. Patients and methods In the AF-001JP trial for alectinib, 436 patients were screened for ALK fusions through IHC (n = 384) confirmed with FISH (n = 181), multiplex RT-PCR (n = 68), or both (n = 16). IHC results were scored with iScore. Result ALK fusion was positive in 137 patients and negative in 250 patients. Since the presence of cancer cells in the samples for RT-PCR was not confirmed, ALK fusion negativity could not be ascertained in 49 patients. IHC interpreted with iScore showed a 99.4% (173/174) concordance with FISH. All 41 patients who had iScore 3 and were enrolled in phase II showed at least 30% tumor reduction with 92.7% overall response rate. Two IHC-positive patients with an atypical FISH pattern responded to ALK inhibitor therapy. The reduction rate was not correlated with IHC staining intensity. Conclusions Our study showed (i) that when sufficiently sensitive and appropriately interpreted, IHC can be a stand-alone diagnostic for ALK inhibitor therapies; (ii) that when atypical FISH patterns are accompanied by IHC positivity, the patients should be considered as candidates for ALK inhibitor therapies, and (iii) that the expression level of ALK fusion is not related to the level of response to ALK inhibitors and is thus not required for patient selection. Registration number JapicCTI-101264 (This study is registered with the Japan Pharmaceutical Information Center). PMID:26487585

  20. ALK and ROS1 as a joint target for the treatment of lung cancer: a review.

    PubMed

    Puig de la Bellacasa, Raimon; Karachaliou, Niki; Estrada-Tejedor, Roger; Teixidó, Jordi; Costa, Carlota; Borrell, José I

    2013-04-01

    Rearrangements of the anaplastic lymphoma kinase (ALK) have been described in multiple malignancies, including non-small cell lung cancer (NSCLC). ALK fusions have gain of function properties while activating mutations in wild-type ALK can also occur within the tyrosine kinase domain. ALK rearrangements define a new molecular subtype of NSCLC that is exquisitely sensitive to ALK inhibition. Crizotinib, an orally available small molecule ATP-mimetic compound which was originally designed as a MET inhibitor, was recognized to have "off-target" anti-ALK activity and has been approved in the USA for the treatment of patients with ALK-positive NSCLC. Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase have also been recently described in NSCLC, while crizotinib is currently under clinical trial in this molecular subset of NSCLC patients. The basic approaches of any computer aided drug design work in terms of structure and ligand based drug design. Details of each of these approaches should be covered with an emphasis on utilizing both in order to develop multi-targeted small-molecule kinase inhibitors. Such multi-targeted tyrosine kinase inhibitors can have antiproliferative activity against both ROS1and ALK rearranged NSCLC. Herein, we highlight the importance of targeting these proteins and the advances in optimizing more potent and selective ALK and ROS1 kinase inhibitors.

  1. ALK and ROS1 as a joint target for the treatment of lung cancer: a review

    PubMed Central

    Puig de la Bellacasa, Raimon; Karachaliou, Niki; Estrada-Tejedor, Roger; Teixidó, Jordi; Costa, Carlota

    2013-01-01

    Rearrangements of the anaplastic lymphoma kinase (ALK) have been described in multiple malignancies, including non-small cell lung cancer (NSCLC). ALK fusions have gain of function properties while activating mutations in wild-type ALK can also occur within the tyrosine kinase domain. ALK rearrangements define a new molecular subtype of NSCLC that is exquisitely sensitive to ALK inhibition. Crizotinib, an orally available small molecule ATP-mimetic compound which was originally designed as a MET inhibitor, was recognized to have “off-target” anti-ALK activity and has been approved in the USA for the treatment of patients with ALK-positive NSCLC. Chromosomal rearrangements involving the ROS1 receptor tyrosine kinase have also been recently described in NSCLC, while crizotinib is currently under clinical trial in this molecular subset of NSCLC patients. The basic approaches of any computer aided drug design work in terms of structure and ligand based drug design. Details of each of these approaches should be covered with an emphasis on utilizing both in order to develop multi-targeted small-molecule kinase inhibitors. Such multi-targeted tyrosine kinase inhibitors can have antiproliferative activity against both ROS1and ALK rearranged NSCLC. Herein, we highlight the importance of targeting these proteins and the advances in optimizing more potent and selective ALK and ROS1 kinase inhibitors. PMID:25806218

  2. Conformational features and binding affinities to Cripto, ALK7 and ALK4 of Nodal synthetic fragments.

    PubMed

    Calvanese, Luisa; Sandomenico, Annamaria; Caporale, Andrea; Focà, Annalia; Focà, Giuseppina; D'Auria, Gabriella; Falcigno, Lucia; Ruvo, Menotti

    2015-04-01

    Nodal, a member of the TGF-β superfamily, is a potent embryonic morphogen also implicated in tumor progression. As for other TGF-βs, it triggers the signaling functions through the interaction with the extracellular domains of type I and type II serine/threonine kinase receptors and with the co-receptor Cripto. Recently, we reported the molecular models of Nodal in complex with its type I receptors (ALK4 and ALK7) as well as with Cripto, as obtained by homology modeling and docking simulations. From such models, potential binding epitopes have been identified. To validate such hypotheses, a series of mutated Nodal fragments have been synthesized. These peptide analogs encompass residues 44-67 of the Nodal protein, corresponding to the pre-helix loop and the H3 helix, and reproduce the wild-type sequence or bear some modifications to evaluate the hot-spot role of modified residues in the receptor binding. Here, we show the structural characterization in solution by CD and NMR of the Nodal peptides and the measurement of binding affinity toward Cripto by surface plasmon resonance. Data collected by both conformational analyses and binding measurements suggest a role for Y58 of Nodal in the recognition with Cripto and confirm that previously reported for E49 and E50. Surface plasmon resonance binding assays with recombinant proteins show that Nodal interacts in vitro also with ALK7 and ALK4 and preliminary data, generated using the Nodal synthetic fragments, suggest that Y58 of Nodal may also be involved in the recognition with these protein partners.

  3. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  4. Tackling ALK in non-small cell lung cancer: the role of novel inhibitors

    PubMed Central

    Facchinetti, Francesco; Di Maio, Massimo; Graziano, Paolo; Bria, Emilio; Rossi, Giulio; Novello, Silvia

    2016-01-01

    Crizotinib is an oral inhibitor of anaplastic lymphoma kinase (ALK) with remarkable clinical activity in patients suffering from ALK-rearranged non-small cell lung cancer (NSCLC), accounting to its superiority compared to chemotherapy. Unfortunately, virtually all ALK-rearranged tumors acquire resistance to crizotinib, frequently within one year since the treatment initiation. To date, therapeutic strategies to overcome crizotinib resistance have focused on the use of more potent and structurally different compounds. Second-generation ALK inhibitors such as ceritinib (LDK378), alectinib (CH5424802/RO5424802) and brigatinib (AP26113) have shown relevant clinical activity, consequently fostering their rapid clinical development and their approval by health agencies. The third-generation inhibitor lorlatinib (PF-06463922), selectively active against ALK and ROS1, harbors impressive biological potency; its efficacy in reversing resistance to crizotinib and to other ALK inhibitors is being proven by early clinical trials. The NTRK1-3 and ROS1 inhibitor entrectinib (RXDX-101) has been reported to act against NSCLC harboring ALK fusion proteins too. Despite the quick development of these novel agents, several issues remain to be discussed in the treatment of patients suffering from ALK-rearranged NSCLC. This position paper will discuss the development, the current evidence and approvals, as long as the future perspectives of new ALK inhibitors beyond crizotinib. Clinical behaviors of ALK-rearranged NSCLC vary significantly among patients and differential molecular events responsible of crizotinib resistance account for the most important quote of this heterogeneity. The precious availability of a wide range of active anti-ALK compounds should be approached in a critical and careful perspective, in order to develop treatment strategies tailored on the disease evolution of every single patient. PMID:27413712

  5. Uterine ALK3 is essential during the window of implantation

    PubMed Central

    Monsivais, Diana; Clementi, Caterina; Peng, Jia; Titus, Mary M.; Barrish, James P.; Creighton, Chad J.; Lydon, John P.; DeMayo, Francesco J.; Matzuk, Martin M.

    2016-01-01

    The window of implantation is defined by the inhibition of uterine epithelial proliferation, structural epithelial cell remodeling, and attenuated estrogen (E2) response. These changes occur via paracrine signaling between the uterine epithelium and stroma. Because implantation defects are a major cause of infertility in women, identifying these signaling pathways will improve infertility interventions. Bone morphogenetic proteins (BMPs) are TGF-β family members that regulate the postimplantation and midgestation stages of pregnancy. In this study, we discovered that signaling via activin-like kinase 3 (ALK3/BMPR1A), a BMP type 1 receptor, is necessary for blastocyst attachment. Conditional knockout (cKO) of ALK3 in the uterus was obtained by producing Alk3flox/flox-Pgr-cre–positive females. Alk3 cKO mice are sterile and have defects in the luminal uterine epithelium, including increased microvilli density and maintenance of apical cell polarity. Moreover, Alk3 cKO mice exhibit an elevated uterine E2 response and unopposed epithelial cell proliferation during the window of implantation. We determined that dual transcriptional regulation of Kruppel-like factor 15 (Klf15), by both the transforming growth factor β (TGF-β) transcription factor SMAD family member 4 (SMAD4) and progesterone receptor (PR), is necessary to inhibit uterine epithelial cell proliferation, a key step for embryo implantation. Our findings present a convergence of BMP and steroid hormone signaling pathways in the regulation of uterine receptivity. PMID:26721398

  6. Lumican Peptides: Rational Design Targeting ALK5/TGFBRI

    NASA Astrophysics Data System (ADS)

    Gesteira, Tarsis Ferreira; Coulson-Thomas, Vivien J.; Yuan, Yong; Zhang, Jianhua; Nader, Helena B.; Kao, Winston W.-Y.

    2017-02-01

    Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFβ) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.

  7. Lumican Peptides: Rational Design Targeting ALK5/TGFBRI

    PubMed Central

    Gesteira, Tarsis Ferreira; Coulson-Thomas, Vivien J.; Yuan, Yong; Zhang, Jianhua; Nader, Helena B.; Kao, Winston W.-Y.

    2017-01-01

    Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFβ) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand. PMID:28181591

  8. Deficient BIM Expression as a Mechanism of Intrinsic and Acquired Resistance to Targeted Therapies in EGFR-Mutant and ALK-Positive Lung Cancers

    DTIC Science & Technology

    2014-08-01

    depressed   in  patients’  specimens  following  acquired...second-­‐generation  ALK  inhibitors.   2C.  Current  objectives:  Determine  if  BIM  levels  are   depressed  (or  BIM  has...whether  resistant  lines  have  a   depressed  apoptotic  response  to  second-­‐line  targeted   therapies  by

  9. Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer

    PubMed Central

    Kelly, Lindsey M.; Barila, Guillermo; Liu, Pengyuan; Evdokimova, Viktoria N.; Trivedi, Sumita; Panebianco, Federica; Gandhi, Manoj; Carty, Sally E.; Hodak, Steven P.; Luo, Jianhua; Dacic, Sanja; Yu, Yan P.; Nikiforova, Marina N.; Ferris, Robert L.; Altschuler, Daniel L.; Nikiforov, Yuri E.

    2014-01-01

    Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone–independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well-differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients. PMID:24613930

  10. Variant translocation partners of the anaplastic lymphoma kinase (ALK) gene in two cases of anaplastic large cell lymphoma, identified by inverse cDNA polymerase chain reaction.

    PubMed

    Takeoka, Kayo; Okumura, Atsuko; Honjo, Gen; Ohno, Hitoshi

    2014-01-01

    In anaplastic large cell lymphoma (ALCL), the anaplastic lymphoma kinase (ALK) gene is rearranged with diverse partners due to variant translocations/inversions. Case 1 was a 39-year-old man who developed multiple tumors in the mediastinum, psoas muscle, lung, and lymph nodes. A biopsy specimen of the inguinal node was effaced by large tumor cells expressing CD30, epithelial membrane antigen, and cytoplasmic ALK, which led to a diagnosis of ALK(+) ALCL. Case 2 was a 51-year-old man who was initially diagnosed with undifferentiated carcinoma. He developed multiple skin tumors eight years after his initial presentation, and was finally diagnosed with ALK(+) ALCL. He died of therapy-related acute myeloid leukemia. G-banding and fluorescence in situ hybridization using an ALK break-apart probe revealed the rearrangement of ALK and suggested variant translocation in both cases. We applied an inverse cDNA polymerase chain reaction (PCR) strategy to identify the partner of ALK. Nucleotide sequencing of the PCR products and a database search revealed that the sequences of ATIC in case 1 and TRAF1 in case 2 appeared to follow those of ALK. We subsequently confirmed ATIC-ALK and TRAF1-ALK fusions by reverse transcriptase PCR and nucleotide sequencing. We successfully determined the partner gene of ALK in two cases of ALK(+) ALCL. ATIC is the second most common partner of variant ALK rearrangements, while the TRAF1-ALK fusion gene was first reported in 2013, and this is the second reported case of ALK(+) ALCL carrying TRAF1-ALK.

  11. RANBP2-ALK fusion combined with monosomy 7 in acute myelomonocytic leukemia.

    PubMed

    Lim, Ji-Hun; Jang, Seongsoo; Park, Chan-Jeoung; Cho, Young-Uk; Lee, Je-Hwan; Lee, Kyoo-Hyung; Lee, Jin-Ok; Shin, Jong-Yeon; Kim, Jong-Il; Huh, Jooryung; Seo, Eul-Ju

    2014-01-01

    Anaplastic lymphoma receptor tyrosine kinase (ALK) is located on chromosome 2p23; the chromosomal rearrangements of this gene are common genetic alterations, resulting in the creation of multiple fusion genes involved in tumorigenesis. However, the presence of an ALK fusion in myeloid malignancies is extremely rare. We report a case of acute myelomonocytic leukemia in a 31-year-old woman with an unusual rearrangement between RAN-binding protein 2 (RANBP2) and ALK and a karyotype of 45,XX,inv(2)(p23q21),-7[20]. We detected an ALK rearrangement using fluorescence in situ hybridization, identified the ALK fusion partner by using RNA transcriptome sequencing, and demonstrated the RANBP2-ALK fusion transcript by reverse transcriptase--PCR and Sanger sequencing. Immunohistochemistry for ALK showed strong staining of the nuclear membrane in leukemic cells. The patient had an unfavorable clinical course. Our results, together with a literature review, suggest the RANBP2-ALK fusion combined with monosomy 7 may be related to a unique clonal hematologic disorder of childhood and adolescence, characterized by myelomonocytic leukemia and a poor prognosis.

  12. Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations

    PubMed Central

    Chen, Hua-Jun; Zhou, Qing; Yan, Li-Xu; Xie, Zhi; Su, Jian; Chen, Zhi-Hong; Tu, Hai-Yan; Yan, Hong-Hong; Wang, Zhen; Xu, Chong-Rui; Jiang, Ben-Yuan; Wang, Bin-Chao; Bai, Xiao-Yan; Zhong, Wen-Zhao; Wu, Yi-Long; Yang, Jin-Ji

    2016-01-01

    The co-occurrence of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements constitutes a rare molecular subtype of non-small-cell lung cancer (NSCLC). Herein, we assessed the clinical outcomes and incidence of acquired resistance to tyrosine kinase inhibitors (TKIs) in this subtype. So we enrolled 118 advanced NSCLC treated with TKIs. EGFR mutations and ALK rearrangements were detected by DNA sequencing or Scorpion amplification refractory mutation system and fluorescence in situ hybridization respectively. Immunohistochemistry was used to evaluate the activation of associated proteins. We found that nine in ten patients with EGFR/ALK co-alterations had good response with first-line EGFR TKI, and the objective response rate (ORR) of EGFR TKIs was 80% (8/10) for EGFR/ALK co-altered and 65.5% (55/84) for EGFR-mutant (P = 0.57), with a median progression-free survival (PFS) of 11.2 and 13.2 months, (hazard ratio [HR]=0.95, 95% [CI], 0.49-1.84, P= 0.87). ORR of crizotinib was 40% (2/5) for EGFR/ALK co-altered and 73.9% (17/23) for ALK-rearranged (P= 0.29), with a median PFS of 1.9 and 6.9 months (hazard ratio [HR], 0.40; 95% [CI] 0.15-1.10, P = 0.08). The median overall survival (OS) was 21.3, 23.7, and 18.5 months in EGFR-mutant, ALK-rearranged, and EGFR/ALK co-altered (P= 0.06), and there existed a statistically significant difference in OS between ALK-rearranged and EGFR/ALK co-altered (P=0.03). Taken together, the first-line EGFR-TKI might be the reasonable care for advanced NSCLC harbouring EGFR/ALK co-alterations, whether or nor to use sequential crizotinib should be guided by the status of ALK rearrangement and the relative level of phospho-EGFR and phospho-ALK. PMID:27533086

  13. Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) oncoprotein induces the T regulatory cell phenotype by activating STAT3

    PubMed Central

    Kasprzycka, Monika; Marzec, Michal; Liu, Xiaobin; Zhang, Qian; Wasik, Mariusz A.

    2006-01-01

    The mechanisms of malignant cell transformation mediated by the oncogenic, chimeric nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) tyrosine kinase remain only partially understood. Here we report that the NPM/ALK-carrying T cell lymphoma (ALK+TCL) cells secrete IL-10 and TGF-β and express FoxP3, indicating their T regulatory (Treg) cell phenotype. The secreted IL-10 suppresses proliferation of normal immune, CD3/CD28-stimulated peripheral blood mononuclear cells and enhances viability of the ALK+TCL cells. The Treg phenotype of the affected cells is strictly dependent on NPM/ALK expression and function as demonstrated by transfection of the kinase into BaF3 cells and inhibition of its enzymatic activity and expression in ALK+TCL cells. NPM/ALK, in turn, induces the phenotype through activation of its key signal transmitter, signal transducer and activator of transcription 3 (STAT3). These findings identify a mechanism of NPM/ALK-mediated oncogenesis based on induction of the Treg phenotype of the transformed CD4+ T cells. These results also provide an additional rationale to therapeutically target the chimeric kinase and/or STAT3 in ALK+TCL. PMID:16766651

  14. Recombinant protein expression in Nicotiana.

    PubMed

    Matoba, Nobuyuki; Davis, Keith R; Palmer, Kenneth E

    2011-01-01

    Recombinant protein pharmaceuticals are now widely used in treatment of chronic diseases, and several recombinant protein subunit vaccines are approved for human and veterinary use. With growing demand for complex protein pharmaceuticals, such as monoclonal antibodies, manufacturing capacity is becoming limited. There is increasing need for safe, scalable, and economical alternatives to mammalian cell culture-based manufacturing systems, which require substantial capital investment for new manufacturing facilities. Since a seminal paper reporting immunoglobulin expression in transgenic plants was published in 1989, there have been many technological advances in plant expression systems to the present time where production of proteins in leaf tissues of nonfood crops such as Nicotiana species is considered a viable alternative. In particular, transient expression systems derived from recombinant plant viral vectors offer opportunities for rapid expression screening, construct optimization, and expression scale-up. Extraction of recombinant proteins from Nicotiana leaf tissues can be achieved by collection of secreted protein fractions, or from a total protein extract after grinding the leaves with buffer. After separation from solids, the major purification challenge is contamination with elements of the photosynthetic complex, which can be solved by application of a variety of facile and proven strategies. In conclusion, the technologies required for safe, efficient, scalable manufacture of recombinant proteins in Nicotiana leaf tissues have matured to the point where several products have already been tested in phase I clinical trials and will soon be followed by a rich pipeline of recombinant vaccines, microbicides, and therapeutic proteins.

  15. Dickkopf-3 Upregulates VEGF in Cultured Human Endothelial Cells by Activating Activin Receptor-Like Kinase 1 (ALK1) Pathway

    PubMed Central

    Busceti, Carla L.; Marchitti, Simona; Bianchi, Franca; Di Pietro, Paola; Riozzi, Barbara; Stanzione, Rosita; Cannella, Milena; Battaglia, Giuseppe; Bruno, Valeria; Volpe, Massimo; Fornai, Francesco; Nicoletti, Ferdinando; Rubattu, Speranza

    2017-01-01

    Dkk-3 is a member of the dickkopf protein family of secreted inhibitors of the Wnt pathway, which has been shown to enhance angiogenesis. The mechanism underlying this effect is currently unknown. Here, we used cultured HUVECs to study the involvement of the TGF-β and VEGF on the angiogenic effect of Dkk-3. Addition of hrDkk-3 peptide (1 or 10 ng/ml) to HUVECs for 6 or 12 h enhanced the intracellular and extracellular VEGF protein levels, as assessed by RTPCR, immunoblotting, immunocytochemistry and ELISA. The increase in the extracellular VEGF levels was associated to the VEGFR2 activation. Pharmacological blockade of VEGFR2 abrogated Dkk-3-induced endothelial cell tubes formation, indicating that VEGF is a molecular player of the angiogenic effects of Dkk-3. Moreover, Dkk-3 enhanced Smad1/5/8 phosphorylation and recruited Smad4 to the VEGF gene promoter, suggesting that Dkk-3 activated ALK1 receptor leading to a transcriptional activation of VEGF. This mechanism was instrumental to the increased VEGF expression and endothelial cell tubes formation mediated by Dkk-3, because both effects were abolished by siRNA-mediated ALK1 knockdown. In summary, we have found that Dkk-3 activates ALK1 to stimulate VEGF production and induce angiogenesis in HUVECs. PMID:28352232

  16. The bHLH transcription factor Hand is regulated by Alk in the Drosophila embryonic gut

    SciTech Connect

    Varshney, Gaurav K.; Palmer, Ruth H. . E-mail: Ruth.Palmer@ucmp.umu.se

    2006-12-29

    During embryonic development the midgut visceral muscle is formed by fusion of cells within the visceral mesoderm, a process initiated by the specification of a specialised cell type, the founder cell, within this tissue. Activation of the receptor tyrosine kinase Anaplastic lymphoma kinase (Alk) in the developing visceral muscle of Drosophila melanogaster initiates a signal transduction pathway required for muscle fusion. In this paper, we have investigated downstream components which are regulated by this novel signalling pathway. Here we show that Alk-mediated signal transduction drives the expression of the bHLH transcription factor Hand in vivo. Loss of Alk function results in a complete lack of Hand expression in this tissue, whereas Alk gain of function results in an expansion of Hand expression. Finally, we have investigated the process of muscle fusion in the gut of Hand mutant animals and can find no obvious defects in this process, suggesting that Hand is not critical for visceral muscle fusion per se.

  17. Growth Differentiation Factor-8 Decreases StAR Expression Through ALK5-Mediated Smad3 and ERK1/2 Signaling Pathways in Luteinized Human Granulosa Cells.

    PubMed

    Fang, Lanlan; Chang, Hsun-Ming; Cheng, Jung-Chien; Yu, Yiping; Leung, Peter C K; Sun, Ying-Pu

    2015-12-01

    Growth differentiation factor-8 (GDF-8) has been recently shown to be expressed in human granulosa cells, and the mature form of GDF-8 protein can be detected in the follicular fluid. However, the biological function and significance of this growth factor in the human ovary remains to be determined. Here, we investigated the effects of GDF-8 on steroidogenic enzyme expression and the potential mechanisms of action in luteinized human granulosa cells. We demonstrated that treatment with GDF-8 did not affect the mRNA levels of P450 side-chain cleavage enzyme and 3β-hydroxysteroid dehydrogenase, whereas it significantly down-regulated steroidogenic acute regulatory protein (StAR) expression and decreased progesterone production. The suppressive effect of GDF-8 on StAR expression was abolished by the inhibition of the TGF-β type I receptor. In addition, treatment with GDF-8 activated both Smad2/3 and ERK1/2 signaling pathways. Furthermore, knockdown of activin receptor-like kinase 5 reversed the effects of GDF-8 on Smad2/3 phosphorylation and StAR expression. The inhibition of Smad3 or ERK1/2 signaling pathways attenuated the GDF-8-induced down-regulation of StAR and production of progesterone. Interestingly, the concentrations of GDF-8 were negatively correlated with those of progesterone in human follicular fluid. These results indicate a novel autocrine function of GDF-8 to down-regulate StAR expression and decrease progesterone production in luteinized human granulosa cells, most likely through activin receptor-like kinase 5-mediated Smad3 and ERK1/2 signaling pathways. Our findings suggest that granulosa cells might play a critical role in the regulation of progesterone production to prevent premature luteinization during the final stage of folliculogenesis.

  18. The Utility of Thyroid Transcription Factor 1 (TTF-1), Napsin A, Excision Repair Cross-Complementing 1 (ERCC1), Anaplastic Lymphoma Kinase (ALK) and the Epidermal Growth Factor Receptor (EGFR) Expression in Small Biopsy in Prognosis of Patients with Lung Adenocarcinoma – A Retrograde Single-Center Study from Croatia

    PubMed Central

    Burazer, Marina Piljić; Mladinov, Suzana; Ćapkun, Vesna; Kuret, Sendi; Durdov, Merica Glavina

    2017-01-01

    Background The present study was carried out in order to evaluate our institutional experience with small biopsy in diagnosis and molecular testing of lung adenocarcinoma. Few specific and predictive markers have been evaluated and correlated with clinicopathologic characteristics and survival in patients with lung adenocarcinoma who received platinum-based chemotherapy. There have not been such reports from Croatia. Material/Methods A total of 142 cases of lung adenocarcinoma were retrospectively investigated in small biopsies for the immunohistochemical expression of TTF-1, napsin A, ERCC1, ALK, and the EGFR mutation by real-time polymerase chain reaction (rtPCR). Results TTF-1, napsin A, and ERCC1 expression was found in 81%, 78%, and 69% of patients, respectively, and the expressions were not significantly associated with subtype. Expression of ALK was found in 4% and EGFR mutation in 10% of patients. Exon 19 deletions were the most common. Longer survival was significantly associated with TTF-1 positivity (p=0.007) and napsin A positivity (p=0.026). Higher relative risk of death significantly correlated with positive expression of ERCC1 (p=0.041). Conclusions Positive TTF-1 and napsin A expressions in lung adenocarcinoma tissues were useful diagnostic and favorable prognostic parameters. Positive ERCC1 expression was identified as a negative prognostic marker in patients treated with platinum-based chemotherapy. The percentages of EGFR and ALK mutations corresponded to those in previously published reports for Caucasians. PMID:28128193

  19. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

    PubMed

    Fedeles, Bogdan I; Singh, Vipender; Delaney, James C; Li, Deyu; Essigmann, John M

    2015-08-21

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.

  20. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond*

    PubMed Central

    Fedeles, Bogdan I.; Singh, Vipender; Delaney, James C.; Li, Deyu; Essigmann, John M.

    2015-01-01

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. PMID:26152727

  1. Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

    PubMed Central

    Mazot, Pierre; Cazes, Alex; Dingli, Florent; Degoutin, Joffrey; Irinopoulou, Théano; Boutterin, Marie-Claude; Lombard, Bérangère; Loew, Damarys; Hallberg, Bengt; Palmer, Ruth Helen; Delattre, Olivier

    2012-01-01

    Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization. PMID:22479414

  2. Expression of TGF-β Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1β and TGF-β Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage

    PubMed Central

    Shanmugaapriya, S.; van Caam, A.; de Kroon, L.; Vitters, Elly L.; Walgreen, B; van Beuningen, H.; Davidson, E. Blaney; van der Kraan, Peter M.

    2016-01-01

    Objective RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-β (TGF-β)–mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1β (IL)-1β and TGF-β superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. Methods Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase–quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. Results RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-β inhibited RBPMS expression while BMP2 and IL-1β increased its expression. TGF-β-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. Conclusions Expression of RBPMS in chondrocytes is regulated by TGF-β superfamily members and IL-1β, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage. PMID:27688842

  3. Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

    PubMed

    Li, Qi; Huang, Yue; Liu, Xichun; Gan, Jianhua; Chen, Hao; Yang, Cai-Guang

    2016-05-20

    The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.

  4. Bone Morphogenetic Protein 2 and Transforming Growth Factor β1 Inhibit the Expression of the Proinflammatory Cytokine IL-34 in Rheumatoid Arthritis Synovial Fibroblasts.

    PubMed

    Chemel, Marguerite; Brion, Regis; Segaliny, Aude-Isabelle; Lamora, Audrey; Charrier, Celine; Brulin, Benedicte; Maugars, Yves; Le Goff, Benoit; Heymann, Dominique; Verrecchia, Franck

    2017-01-01

    IL-34 is a proinflammatory cytokine implicated in rheumatoid arthritis (RA). The current study aimed to assess the IL-34 expression in response to two members of the transforming growth factor (TGF)-β family, TGF-β1 and bone morphogenetic protein (BMP)-2, in synovial fibroblasts from RA patients. IL-34, TGF-β1, and BMP-2 productions were measured in patient synovial fluids by enzyme-linked immunosorbent assay. IL-34 mRNA levels were quantified by real-time quantitative PCR in human synovial fibroblasts and murine mesenchymal stem cells. Pharmacologic inhibitions were used to determine the involvement of activin receptor-like kinase 1 (ALK1) and ALK5 downstream TGF-β1 and BMP-2. IL-34, TGF-β1, and BMP-2 were expressed in synovial fluids from RA patients. We found a significant correlation between IL-34 and TGF-β1 expressions. Levels of both IL-34 and TGF-β1 were thus correlated with the total leukocyte counts in the synovial fluids. TGF-β1 and BMP-2 decreased IL-34 expression in the synovial fibroblasts or in murine mesenchymal stem cells in a dose- and time-dependent manner through ALK5 and ALK1 pathways, respectively. In addition, TGF-β1 and BMP-2 antagonized tumor necrosis factor α-induced IL-34 gene expression. This work identifies TGF-β1 and BMP-2 as potent inhibitors of IL-34 expression in RA synovial fibroblasts. These cytokines, as upstream inhibitors of IL-34, may thus contribute to antagonize inflammation and bone erosions in RA.

  5. Effects of Pharmacologic and Genetic Inhibition of Alk on Cognitive Impairments in NF1 Mutant Mice

    DTIC Science & Technology

    2014-06-01

    neuroblastoma 12-15. Orally active small molecule inhibitors have shown notable effectiveness in the treatment of lung cancer and are actively being...tested for the treatment of neuroblastoma 16-18. The normal function of Alk in humans is less clear though its expression in both the developing and...Y. P. et al. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature 455, 930-935 (2008). 13 Janoueix-Lerosey, I. et

  6. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

    PubMed

    Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-01-01

    Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer.

  7. Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

    PubMed Central

    Katayama, Ryohei; Khan, Tahsin M.; Benes, Cyril; Lifshits, Eugene; Ebi, Hiromichi; Rivera, Victor M.; Shakespeare, William C.; Iafrate, A. John; Engelman, Jeffrey A.; Shaw, Alice T.

    2011-01-01

    The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations. PMID:21502504

  8. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  9. In vivo imaging models of bone and brain metastases and pleural carcinomatosis with a novel human EML4-ALK lung cancer cell line.

    PubMed

    Nanjo, Shigeki; Nakagawa, Takayuki; Takeuchi, Shinji; Kita, Kenji; Fukuda, Koji; Nakada, Mitsutoshi; Uehara, Hisanori; Nishihara, Hiroshi; Hara, Eiji; Uramoto, Hidetaka; Tanaka, Fumihiro; Yano, Seiji

    2015-03-01

    EML4-ALK lung cancer accounts for approximately 3-7% of non-small-cell lung cancer cases. To investigate the molecular mechanism underlying tumor progression and targeted drug sensitivity/resistance in EML4-ALK lung cancer, clinically relevant animal models are indispensable. In this study, we found that the lung adenocarcinoma cell line A925L expresses an EML4-ALK gene fusion (variant 5a, E2:A20) and is sensitive to the ALK inhibitors crizotinib and alectinib. We further established highly tumorigenic A925LPE3 cells, which also have the EML4-ALK gene fusion (variant 5a) and are sensitive to ALK inhibitors. By using A925LPE3 cells with luciferase gene transfection, we established in vivo imaging models for pleural carcinomatosis, bone metastasis, and brain metastasis, all of which are significant clinical concerns of advanced EML4-ALK lung cancer. Interestingly, crizotinib caused tumors to shrink in the pleural carcinomatosis model, but not in bone and brain metastasis models, whereas alectinib showed remarkable efficacy in all three models, indicative of the clinical efficacy of these ALK inhibitors. Our in vivo imaging models of multiple organ sites may provide useful resources to analyze further the pathogenesis of EML4-ALK lung cancer and its response and resistance to ALK inhibitors in various organ microenvironments.

  10. ALK gene rearranged lung adenocarcinomas: molecular genetics and morphology in cohort of patients from North India.

    PubMed

    Bal, Amanjit; Singh, Navneet; Agarwal, Parimal; Das, Ashim; Behera, Digambar

    2016-10-01

    ALK gene rearrangement in the lung adenocarcinomas is the second most common (1.6-11.7% of NSCLC) targetable genomic change after EGFR mutations. However, the prevalence and clinicopathological features of ALK-rearranged lung adenocarcinomas from North India are lacking. A total of 240 cases of lung adenocarcinoma were screened for EGFR mutations and for ALK expression. Smoking status, TNM stage, and treatment response were recorded in all cases. Out of 240 cases screened, 37 cases were positive for EGFR mutations and 17 cases (7.08%) showed ALK positivity with immunohistochemistry and break-apart FISH. On excluding 37 EGFR mutation-positive cases, the incidence of ALK-positive adenocarcinoma appears to be higher (17/203 cases, 8.03%). Eight were men and nine were women with mean age of 51.7 years. Majority (62.5%) were non-smokers and had unresectable disease (70.6% stage IV, 17.6% IIIB). The morphological patterns noted were solid (12 cases), papillary (four cases), and micropapillary (one case). Signet ring (two cases) and clear cell change (one cases) were noted. Out of five patients who received crizotinib, three had partial response and two had stable disease. ALK-rearranged lung adenocarcinomas account for a minor proportion of NSCLC with prevalence similar to that reported in literature. However, as contrast to published data in our series, patients were in older age group and had solid and papillary pattern on morphology with an aggressive course.

  11. ALK: a tyrosine kinase target for cancer therapy

    PubMed Central

    Holla, Vijaykumar R.; Elamin, Yasir Y.; Bailey, Ann Marie; Johnson, Amber M.; Litzenburger, Beate C.; Khotskaya, Yekaterina B.; Sanchez, Nora S.; Zeng, Jia; Shufean, Md Abu; Shaw, Kenna R.; Mendelsohn, John; Mills, Gordon B.; Meric-Bernstam, Funda; Simon, George R.

    2017-01-01

    The anaplastic lymphoma kinase (ALK) gene plays an important physiologic role in the development of the brain and can be oncogenically altered in several malignancies, including non-small-cell lung cancer (NSCLC) and anaplastic large cell lymphomas (ALCL). Most prevalent ALK alterations are chromosomal rearrangements resulting in fusion genes, as seen in ALCL and NSCLC. In other tumors, ALK copy-number gains and activating ALK mutations have been described. Dramatic and often prolonged responses are seen in patients with ALK alterations when treated with ALK inhibitors. Three of these—crizotinib, ceritinib, and alectinib—are now FDA approved for the treatment of metastatic NSCLC positive for ALK fusions. However, the emergence of resistance is universal. Newer ALK inhibitors and other targeting strategies are being developed to counteract the newly emergent mechanism(s) of ALK inhibitor resistance. This review outlines the recent developments in our understanding and treatment of tumors with ALK alterations. PMID:28050598

  12. Expression of bioactive bone morphogenetic proteins in the subacromial bursa of patients with chronic degeneration of the rotator cuff

    PubMed Central

    Neuwirth, Jana; Fuhrmann, Renée AE; Veit, Amanda; Aurich, Matthias; Stonâns, Ilmars; Trommer, Tilo; Hortschansky, Peter; Chubinskaya, Susanna; Mollenhauer, Juergen A

    2006-01-01

    Degeneration of the rotator cuff is often associated with inflammation of the subacromial bursa and focal mineralization of the supraspinatus tendon. Portions of the supraspinatus tendon distant from the insertion site could transform into fibrous cartilage, causing rotator-cuff tears owing to mechanical instability. Indirect evidence is presented to link this pathology to ectopic production and secretion of bioactive bone morphogenetic proteins (BMPs) from sites within the subacromial bursa. Surgically removed specimens of subacromial bursa tissue from patients with chronic tears of the rotator cuff were analyzed by immunohistochemistry and reverse transcription-PCR. Bioactive BMP was detected in bursa extracts by a bioassay based on induction of alkaline phosphatase in the osteogenic/myogenic cell line C2C12. Topical and differential expression of BMP-2/4 and BMP-7 mRNA and protein was found in bursa tissue. The bioassay of C2C12 cells revealed amounts of active BMP high enough to induce osteogenic cell types, and blocking BMP with specific antibodies or soluble BMP receptors Alk-3 and Alk-6 abolished the inductive properties of the extract. Sufficient information was gathered to explain how ectopic expression of BMP might induce tissue transformation into ectopic bone/cartilage and, therefore, promote structural degeneration of the rotator cuff. Early surgical removal of the subacromial bursa might present an option to interrupt disease progression. PMID:16719933

  13. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer

    PubMed Central

    Koivunen, Jussi P.; Mermel, Craig; Zejnullahu, Kreshnik; Murphy, Carly; Lifshits, Eugene; Holmes, Alison J.; Choi, Hwan Geun; Kim, Jhingook; Chiang, Derek; Thomas, Roman; Lee, Jinseon; Richards, William G.; Sugarbaker, David J.; Ducko, Christopher; Lindeman, Neal; Marcoux, J. Paul; Engelman, Jeffrey A.; Gray, Nathanael S.; Lee, Charles; Meyerson, Matthew; Jänne, Pasi A.

    2011-01-01

    Purpose The EML4-ALK fusion gene has been detected in ~7% of Japanese non-small cell lung cancers (NSCLC). We determined the frequency of EML4-ALK in Caucasian NSCLCs and in NSCLC cell lines. We also determined whether TAE684, a specific ALK kinase inhibitor, would inhibit the growth of EML4-ALK containing cell lines in vitro and in vivo. Experimental Design We screened 305 primary NSCLCs (both US (n=138) and Korean (n=167) patients) and 83 NSCLC cell lines using RT-PCR and by exon array analyses. We evaluated the efficacy of TAE684 against NSCLC cell lines in vitro and in vivo. Results We detected 4 different variants, including two novel variants, of EML4-ALK using RT-PCR in 8/305 tumors (3%) and in 3/83 (3.6%) NSCLC cell lines. All EML4-ALK containing tumors and cell lines were adenocarcinomas. EML4-ALK was detected more frequently in NSCLC patients who were never or light (< 10 pack years) cigarette smokers compared to current/former smokers (6% vs. 1%; p=0.049). TAE684 inhibited the growth of 1 of 3 (H3122) EML4-ALK containing cell lines in vitro and in vivo, inhibited Akt phosphorylation and caused apoptosis. In another EML4-ALK cell line, DFCI032, TAE684 was ineffective due to co-activation of EGFR and ERBB2. The combination of TAE684 and CL-387,785 (EGFR/ERBB2 kinase inhibitor), inhibited growth and Akt phosphorylation and led to apoptosis in the DFCI032 cell line. Conclusions EML4-ALK is found in the minority of NSCLCs. ALK kinase inhibitors alone or in combination may nevertheless be clinically effective treatments for NSCLC patients whose tumors contain EML4-ALK. PMID:18594010

  14. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway

    PubMed Central

    Zhu, Dongxing; Mackenzie, Neil Charles Wallace; Shanahan, Catherine M; Shroff, Rukshana C; Farquharson, Colin; MacRae, Vicky Elizabeth

    2015-01-01

    The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention. PMID:25297851

  15. Activin A Stimulates Aromatase via the ALK4-Smad Pathway in Endometriosis

    PubMed Central

    Zheng, Juan; Qu, Juan; Lu, Pinhong; Mao, Yundong; Qi, Xiaochen; Ji, Hui; Liu, Jiayin

    2016-01-01

    Endometriosis is an estrogen-dependent disease. We previously found that the expression of Activin A was upregulated in the peritoneal fluid of patients with endometriosis. The results of the present study indicated that Activin A induced estradiol secretion and P450arom expression in endometrial stromal cells (ESCs) derived from endometriosis patients. The mechanism of estrogenic synthesis was regulated by the Activin-Smad pathway in endometrial lesions. The data showed that the effect of Activin A on ESCs was partially abrogated by pretreatment with an inhibitor of ALK4 (the type I receptor, ActRIB) and Smad4-siRNA. Cumulatively, these data suggest that Activin A promotes the secretion of estradiol from ESCs by increasing the expression of P450arom via the ALK4-Smad pathway. These findings indicate the ALK4-Smad pathway may promote ectopic lesion survival and development. PMID:27833918

  16. Kinetic mechanism for the excision of hypoxanthine by Escherichia coli AlkA and evidence for binding to DNA ends.

    PubMed

    Zhao, Boyang; O'Brien, Patrick J

    2011-05-24

    The Escherichia coli 3-methyladenine DNA glycosylase II protein (AlkA) recognizes a broad range of oxidized and alkylated base lesions and catalyzes the hydrolysis of the N-glycosidic bond to initiate the base excision repair pathway. Although the enzyme was one of the first DNA repair glycosylases to be discovered more than 25 years ago and there are multiple crystal structures, the mechanism is poorly understood. Therefore, we have characterized the kinetic mechanism for the AlkA-catalyzed excision of the deaminated purine, hypoxanthine. The multiple-turnover glycosylase assays are consistent with Michaelis-Menten kinetics. However, under single-turnover conditions that are commonly employed for studying other DNA glycosylases, we observe an unusual biphasic protein saturation curve. Initially, the observed rate constant for excision increases with an increasing level of AlkA protein, but at higher protein concentrations, the rate constant decreases. This behavior can be most easily explained by tight binding to DNA ends and by crowding of multiple AlkA protamers on the DNA. Consistent with this model, crystal structures have shown the preferential binding of AlkA to DNA ends. By varying the position of the lesion, we identified an asymmetric substrate that does not show inhibition at higher concentrations of AlkA, and we performed pre-steady state and steady state kinetic analysis. Unlike the situation in other glycosylases, release of the abasic product is faster than N-glycosidic bond cleavage. Nevertheless, AlkA exhibits significant product inhibition under multiple-turnover conditions, and it binds approximately 10-fold more tightly to an abasic site than to a hypoxanthine lesion site. This tight binding could help protect abasic sites when the adaptive response to DNA alkylation is activated and very high levels of AlkA protein are present.

  17. Expression of multiple proteins in transgenic plants

    DOEpatents

    Vierstra, Richard D.; Walker, Joseph M.

    2002-01-01

    A method is disclosed for the production of multiple proteins in transgenic plants. A DNA construct for introduction into plants includes a provision to express a fusion protein of two proteins of interest joined by a linking domain including plant ubiquitin. When the fusion protein is produced in the cells of a transgenic plant transformed with the DNA construction, native enzymes present in plant cells cleave the fusion protein to release both proteins of interest into the cells of the transgenic plant. Since the proteins are produced from the same fusion protein, the initial quantities of the proteins in the cells of the plant are approximately equal.

  18. Novel alkane hydroxylase gene (alkB) diversity in sediments associated with hydrocarbon seeps in the Timor Sea, Australia.

    PubMed

    Wasmund, Kenneth; Burns, Kathryn A; Kurtböke, D Ipek; Bourne, David G

    2009-12-01

    Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem's response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation.

  19. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-07

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  20. Atypical Carcinoid Tumor with Anaplastic Lymphoma Kinase (ALK) Rearrangement Successfully Treated by an ALK Inhibitor.

    PubMed

    Nakajima, Masayuki; Uchiyama, Naoki; Shigemasa, Rie; Matsumura, Takeshi; Matsuoka, Ryota; Nomura, Akihiro

    This is the first report in which crizotinib, an anaplastic lymphoma kinase (ALK) inhibitor, reduced an atypical carcinoid tumor with ALK rearrangement. A 70-year-old man developed a tumor in the left lung and multiple metastases to the lung and brain. The pathology of transbronchial biopsied specimens demonstrated an atypical carcinoid pattern. Combined with immunohistochemical findings, we diagnosed the tumor as atypical carcinoid. ALK gene rearrangement was observed by both immunohistochemical (IHC) and fluorescence in situ hybridization. He was treated with chemotherapy as first-line therapy, however, the tumor did not respond to chemotherapy. Thereafter, he was treated with crizotinib, which successfully reduced the tumors.

  1. Coevolution of gene expression among interacting proteins

    SciTech Connect

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  2. Efficacy of ALK5 inhibition in myelofibrosis

    PubMed Central

    Zhao, Wanke; Ho, Wanting Tina; Han, Ying; Murdun, Cem; Mailloux, Adam W.; Zhang, Ling; Wang, Xuefeng; Budhathoki, Anjali; Pradhan, Kith; Rapaport, Franck; Wang, Huaquan; Shao, Zonghong; Ren, Xiubao; Steidl, Ulrich; Levine, Ross L.; Zhao, Zhizhuang Joe; Verma, Amit; Epling-Burnette, Pearlie K.

    2017-01-01

    Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF.

  3. Detection of anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer and related issues in ALK inhibitor therapy: a literature review.

    PubMed

    Yi, Eunhee S; Chung, Jin-Haeng; Kulig, Kimary; Kerr, Keith M

    2012-06-01

    Anaplastic lymphoma kinase (ALK) encodes a receptor tyrosine kinase, and ALK gene rearrangement (ALK+) is implicated in the oncogenesis of non-small cell lung carcinomas (NSCLCs), especially adenocarcinomas. The ALK inhibitor crizotinib was approved in August 2011 by the US Food and Drug Administration (FDA) for treating late-stage NSCLCs that are ALK+, with a companion fluorescent in situ hybridization (FISH) test using the Vysis ALK Break Apart FISH Probe Kit. This review covers pertinent issues in ALK testing, including approaches to select target patients for the test, pros and cons of different detection methods, and mechanisms as well as monitoring of acquired crizotinib resistance in ALK+ NSCLCs.

  4. A malignant inflammatory myofibroblastic tumor of the hypopharynx harboring the 3a/b variants of the EML4-ALK fusion gene

    PubMed Central

    Muscarella, Lucia Anna; Rossi, Giulio; Trombetta, Domenico; La Torre, Annamaria; Di Candia, Leonarda; Mengoli, Maria Cecilia; Sparaneo, Angelo; Fazio, Vito Michele; Graziano, Paolo

    2017-01-01

    Inflammatory myofibroblastic tumors (IMT) in the head and neck region are rare neoplasms that generally mimic benign/low-grade neoplasms. Overexpression of anaplastic lymphoma kinase (ALK) has been reported in 50% of IMT cases, secondary to ALK activation by structural rearrangements in the ALK gene, which results in a fusion protein with echinoderm microtubule associated protein like 4 (EML4) in ~20% of cases. The present study describes a case of a 74-year-old woman with a malignant IMT in the right posterior hypopharynx harboring a previously unreported chromosomal rearrangement resulting in EML4 and ALK gene fusion. Strong ALK immunoreactivity was observed in neoplastic cells, while fluorescent in situ hybridization combined with fluorescent fragment analysis and direct sequencing identified the first case of the 3a/b variants of the EML4-ALK fusion gene in IMT. The results of the current study highlight the uncommon occurrence of ALK-positive IMT in the head/neck region and demonstrate the importance of integrating different molecular methodologies to identify unequivocal gene fusion characterization. PMID:28356934

  5. Sensitivity Analysis of the NPM-ALK Signalling Network Reveals Important Pathways for Anaplastic Large Cell Lymphoma Combination Therapy

    PubMed Central

    Buetti-Dinh, Antoine; O’Hare, Thomas

    2016-01-01

    A large subset of anaplastic large cell lymphoma (ALCL) patients harbour a somatic aberration in which anaplastic lymphoma kinase (ALK) is fused to nucleophosmin (NPM) resulting in a constitutively active signalling fusion protein, NPM-ALK. We computationally simulated the signalling network which mediates pathological cell survival and proliferation through NPM-ALK to identify therapeutically targetable nodes through which it may be possible to regain control of the tumourigenic process. The simulations reveal the predominant role of the VAV1-CDC42 (cell division control protein 42) pathway in NPM-ALK-driven cellular proliferation and of the Ras / mitogen-activated ERK kinase (MEK) / extracellular signal-regulated kinase (ERK) cascade in controlling cell survival. Our results also highlight the importance of a group of interleukins together with the Janus kinase 3 (JAK3) / signal transducer and activator of transcription 3 (STAT3) signalling in the development of NPM-ALK derived ALCL. Depending on the activity of JAK3 and STAT3, the system may also be sensitive to activation of protein tyrosine phosphatase-1 (SHP1), which has an inhibitory effect on cell survival and proliferation. The identification of signalling pathways active in tumourigenic processes is of fundamental importance for effective therapies. The prediction of alternative pathways that circumvent classical therapeutic targets opens the way to preventive approaches for countering the emergence of cancer resistance. PMID:27669408

  6. A novel immunohistochemical classifier to distinguish Hodgkin lymphoma from ALK anaplastic large cell lymphoma.

    PubMed

    Döring, Claudia; Hansmann, Martin-Leo; Agostinelli, Claudio; Piccaluga, Pier P; Facchetti, Fabio; Pileri, Stefano; Küppers, Ralf; Newrzela, Sebastian; Hartmann, Sylvia

    2014-10-01

    Classical Hodgkin lymphoma and ALK(-) anaplastic large cell lymphoma share many features like strong CD30 expression and usually loss of B- and T-cell markers. However, their clinical course is dramatically different with curability rates of >90% for classical Hodgkin lymphoma and an unfavorable prognosis for anaplastic large cell lymphoma. Classical Hodgkin lymphoma and ALK(-) anaplastic large cell lymphoma can usually be distinguished by PAX5 expression in the Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma and expression of cytotoxic molecules in tumor cells of anaplastic large cell lymphoma. However, in some cases the differential diagnosis is difficult owing to absence of established markers. To be able to better classify these cases, we reevaluated gene expression data of microdissected tumor cells of both lymphomas for differentially expressed genes. A classifier was established, comprising four genes strongly expressed in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma (MDC/CCL22, CD83, STAT3, and TUBB2B). Applying this classifier to a test cohort, Hodgkin lymphoma was successfully distinguished from ALK(-) anaplastic large cell lymphoma with an accuracy of 97% (43/44). MDC/CCL22, CD83, and STAT3 have also been found to be expressed in antigen-presenting cells. Therefore, based on our established classifier, Hodgkin and Reed-Sternberg cells differ from tumor cells of anaplastic large cell lymphoma, which can successfully be applied for practical purposes in histopathologic diagnostics.

  7. Stimulation of the midkine/ALK axis renders glioma cells resistant to cannabinoid antitumoral action

    PubMed Central

    Lorente, M; Torres, S; Salazar, M; Carracedo, A; Hernández-Tiedra, S; Rodríguez-Fornés, F; García-Taboada, E; Meléndez, B; Mollejo, M; Campos-Martín, Y; Lakatosh, S A; Barcia, J; Guzmán, M; Velasco, G

    2011-01-01

    Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas. PMID:21233844

  8. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

    Chen, Qiang; Dent, Matthew; Hurtado, Jonathan; Stahnke, Jake; McNulty, Alyssa; Leuzinger, Kahlin; Lai, Huafang

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

  9. Integral Membrane Protein Expression in Saccharomyces cerevisiae.

    PubMed

    Boswell-Casteel, Rebba C; Johnson, Jennifer M; Stroud, Robert M; Hays, Franklin A

    2016-01-01

    Eukaryotic integral membrane proteins are challenging targets for crystallography or functional characterization in a purified state. Since expression is often a limiting factor when studying this difficult class of biological macromolecules, the intent of this chapter is to focus on the expression of eukaryotic integral membrane proteins (IMPs) using the model organism Saccharomyces cerevisiae. S. cerevisiae is a prime candidate for the expression of eukaryotic IMPs because it offers the convenience of using episomal expression plasmids, selection of positive transformants, posttranslational modifications, and it can properly fold and target IMPs. Here we present a generalized protocol and insights based on our collective knowledge as an aid to overcoming the challenges faced when expressing eukaryotic IMPs in S. cerevisiae.

  10. Reduced mural cell coverage and impaired vessel integrity after angiogenic stimulation in the Alk1-deficient brain

    PubMed Central

    Chen, Wanqiu; Guo, Yi; Walker, Espen J.; Shen, Fanxia; Jun, Kristine; Oh, S. Paul; Degos, Vincent; Lawton, Michael T.; Tihan, Tarik; Davalos, Dimitrios; Akassoglou, Katerina; Nelson, Jeffrey; Pile-Spellman, John; Su, Hua; Young, William L.

    2013-01-01

    Objective Vessels in brain arteriovenous malformations (bAVM) are prone to rupture. The underlying pathogenesis is not clear. Hereditary hemorrhagic telangiectasia type 2 (HHT2) patients with activin receptor-like kinase 1 (Alk1) mutation have a higher incidence of bAVM than the general population. We tested the hypothesis that vascular endothelial growth factor (VEGF) impairs vascular integrity in the Alk1-deficient brain through reduction of mural cell-coverage. Methods and Results Adult Alk11f/2f mice (loxP sites flanking exons 4-6) and wild-type (WT) mice were injected with 2×107 PFU Ad-Cre and 2×109 genome copies of AAV-VEGF to induce focal homozygous Alk1 deletion (in Alk11f/2f mice) and angiogenesis. Brain vessels were analyzed eight weeks later. Compared to WT mice, the Alk1-deficient brain had more fibrin (99±30×103 pixels/mm2 vs. 40±13×103, P=0.001), iron deposition (508±506 pixels/mm2 vs. 6 ±49, P=0.04), and Iba1+ microglia/macrophage infiltration (888±420 Iba1+ cells/mm2 vs. 240±104 Iba1+, P=0.001) after VEGF stimulation. In the angiogenic foci, the Alk1-deficient brain had more α-SMA- vessels (52±9% vs. 12±7%, P<0.001), fewer vascular associated pericytes (503±179/mm2 vs. 931±115, P<0.001), and reduced PDGFR-β expression (26±9%, P<0.001). Conclusion Reduction of mural cell coverage in response to VEGF stimulation is a potential mechanism for the impairment of vessel wall integrity in HHT2-associated bAVM. PMID:23241407

  11. Biotechnology Protein Expression and Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The purpose of the Project Scientist Core Facility is to provide purified proteins, both recombinant and natural, to the Biotechnology Science Team Project Scientists and the NRA-Structural Biology Test Investigators. Having a core facility for this purpose obviates the need for each scientist to develop the necessary expertise and equipment for molecular biology, protein expression, and protein purification. Because of this, they are able to focus their energies as well as their funding on the crystallization and structure determination of their target proteins.

  12. In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

    PubMed

    Mathivet, Thomas; Mazot, Pierre; Vigny, Marc

    2007-12-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.

  13. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

    PubMed

    Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

    2016-01-01

    Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations.

  14. Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

    PubMed Central

    Fennewald, M; Benson, S; Oppici, M; Shapiro, J

    1979-01-01

    We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes. Images PMID:479111

  15. Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch

    PubMed Central

    Aspalter, Irene Maria; Gordon, Emma; Dubrac, Alexandre; Ragab, Anan; Narloch, Jarek; Vizán, Pedro; Geudens, Ilse; Collins, Russell Thomas; Franco, Claudio Areias; Abrahams, Cristina Luna; Thurston, Gavin; Fruttiger, Marcus; Rosewell, Ian; Eichmann, Anne; Gerhardt, Holger

    2015-01-01

    Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-β/BMP signalling. PMID:26081042

  16. Streamlined expressed protein ligation using split inteins.

    PubMed

    Vila-Perelló, Miquel; Liu, Zhihua; Shah, Neel H; Willis, John A; Idoyaga, Juliana; Muir, Tom W

    2013-01-09

    Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  17. Achievements and future developments of ALK-TKIs in the management of CNS metastases from ALK-positive NSCLC

    PubMed Central

    Cappuzzo, Federico

    2016-01-01

    Non-small cell lung cancer (NSCLC) represents the paradigm of personalized treatment of human cancer. Several oncogenic druggable alterations have been so far identified, with anaplastic lymphoma kinase (ALK) gene rearrangements being one of the newest and most appealing. Presence of ALK fusions is associated with some particular clinical and pathological features, including a preferential seeding into the central nervous system (CNS). In addition, ALK rearrangements are recognized as the strongest predictor for benefit of anti-ALK therapy. Crizotinib, the first ALK inhibitor (ALK-I) licensed in clinical practice, is the standard of care for newly diagnosed patients. Unfortunately, within the first year of treatment the majority of patients become insensitive to crizotinib, with approximately one third of them developing brain metastases (BMs). Optimal management of BMs is one of the major challenges in treating ALK positive NSCLC. Several novel and highly CNS penetrant ALK-Is are currently under investigation and available data clearly indicated their ability in controlling intracranial disease. PMID:28149753

  18. Expression and purification of membrane proteins.

    PubMed

    Kubicek, Jan; Block, Helena; Maertens, Barbara; Spriestersbach, Anne; Labahn, Jörg

    2014-01-01

    Approximately 30% of a genome encodes for membrane proteins. They are one of the most important classes of proteins in that they can receive, differentiate, and transmit intra- and intercellular signals. Some examples of classes of membrane proteins include cell-adhesion molecules, translocases, and receptors in signaling pathways. Defects in membrane proteins may be involved in a number of serious disorders such as neurodegenerative diseases (e.g., Alzheimer's) and diabetes. Furthermore, membrane proteins provide natural entry and anchoring points for the molecular agents of infectious diseases. Thus, membrane proteins constitute ~50% of known and novel drug targets. Progress in this area is slowed by the requirement to develop methods and procedures for expression and isolation that are tailored to characteristic properties of membrane proteins. A set of standard protocols for the isolation of the targets in quantities that allow for the characterization of their individual properties for further optimization is required. The standard protocols given below represent a workable starting point. If optimization of yields is desired, a variation of conditions as outlined in the theory section is recommended.

  19. Engineering Genes for Predictable Protein Expression

    PubMed Central

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2013-01-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering. PMID:22425659

  20. Engineering genes for predictable protein expression.

    PubMed

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  1. Synergistic activity of ALK and mTOR inhibitors for the treatment of NPM-ALK positive lymphoma

    PubMed Central

    Redaelli, Sara; Ceccon, Monica; Antolini, Laura; Rigolio, Roberta; Pirola, Alessandra; Peronaci, Marco; Gambacorti-Passerini, Carlo; Mologni, Luca

    2016-01-01

    ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/mTOR, indispensable for survival of ALK-driven tumors. Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while long-term exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients. PMID:27662658

  2. Clinicopathological features and relation between anaplastic lymphoma kinase (ALK) mutation and histological subtype of lung adenocarcinoma in Eastern European Caucasian population

    PubMed Central

    Zaric, Bojan; Stojsic, Vladimir; Panjkovic, Milana; Tegeltija, Dragana; Stepanov, Vanesa; Kovacevic, Tomi; Sarcev, Tatjana; Radosavljevic, Davorin; Milovancev, Aleksandar; Adamidis, Vasilis; Zarogoulidis, Paul; Hohenforst-Schmidt, Wolfgang; Trakada, Georgia; Rapti, Aggeliki; Perin, Branislav

    2016-01-01

    Introduction: The incidence of echinoderm microtubule-associated protein-like4-anaplastic lymphoma kinase (EML4-ALK) mutation among surgically treated patients with adenocarcinoma of the lung of the Eastern European ethnicity is underreported. The aim of this trial was the determination of EML4-ALK mutation frequency in investigated population, and the evaluation of correlations between lung adenocarcinoma subtype and clinical characteristics with mutation status. Patients and methods: This was a prospective trial which included 195 patients with adenocarcinoma of the lung who underwent surgical treatment. ALK mutation screening was performed by immunohistochemistry (IHC). IHC scores of 2+ and 3+ were regarded as positive. Confirmatory FISH was performed in all IHC positive and in 2:1 ratio in negative patients. Results: Overall ALK mutation rate established by IHC was 6.2%, while FISH confirmed rate of 5.1%. The FISH confirmed ALK positivity in 7.6% Hungarians, 5.5% Serbians, and 6.6% Slovakians. Acinar subtype of adenocarcinoma of the lung was significantly (p=0.02) related to EML4-ALK positive mutation status. Most of the patients were males (56.9%), smokers (50.8%), or former smokers (28.7%) with acinar (55.4%) or solid (35.9%) adenocarcinoma of the lung. Sensitivity and specificity of IHC were 100% and 98.9% respectively. Conclusions: ALK mutation rate in surgically treated patients with adenocarcinoma of the lung was found to be 6.2% by IHC and 5.1% by FISH. Acinar subtype of the adenocarcinoma of the lung was significantly related to ALK positive mutation. PMID:27994656

  3. Responses to crizotinib in patients with ALK-positive lung adenocarcinoma who tested immunohistochemistry (IHC)-positive and fluorescence in situ hybridization (FISH)-negative

    PubMed Central

    Yang, Lin; Mu, Xinlin; Wang, Yan; Zhao, Xinming; Li, Junling; Lin, Dongmei

    2016-01-01

    Although the Ventana immunohistochemistry (IHC) platform for detecting anaplastic lymphoma kinase gene (ALK) (D5F3) expression was recently approved by the US Food and Drugs Administration (FDA), fluorescence in situ hybridization (FISH) is still the “gold-standard” method recommended by the US National Comprehensive Cancer Network (NCCN) guideline for NSCLC. We evaluated 6 ALK-positive lung adenocarcinoma patients who tested Ventana IHC-positive and FISH-negative and assessed their clinical responses to the ALK tyrosine kinase inhibitor (TKI) crizotinib. Histologic and cytologic specimens from the 6 patients were stained with Ventana anti-ALK(D5F3) rabbit monoclonal primary antibody using the OptiView™ DAB IHC detection kit and OptiView™ amplification kit on a Ventana BenchMark XT processor. In addition, they were also tested by FISH, qRT-PCR, next-generation sequencing (NGS), and RNAscope ISH analysis. All patients received crizotinib treatment and their follow-up clinical data were recorded. The objective response rate achieved with crizotinib therapy was 66.7% (4/6 partial responses and 2/6 stable disease). One patient in whom a new fusion type (EML4->EXOC6B->ALK fusion) was identified obtained a partial response. These findings indicate that patients with ALK-positive lung adenocarcinoma who test Ventana IHC-positive and FISH-negative may still respond to crizotinib therapy. PMID:27418132

  4. Comparison of small biopsy specimens and surgical specimens for the detection of EGFR mutations and EML4-ALK in non-small-cell lung cancer

    PubMed Central

    Xiao, DeSheng; Lu, Can; Zhu, Wei; He, QiuYan; Li, Yong; Fu, ChunYan; Zhou, JianHua; Liu, Shuang; Tao, YongGuang

    2016-01-01

    Epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) fusion genes represent novel oncogenes that are associated with non–small-cell lung cancers (NSCLC). The feasibility of detecting EGFR mutations and ALK fusion genes in small biopsy specimens or surgical specimens was determined. Of the 721 NSCLC patients, a total of 305 cases were positive for EGFR mutations (42.3%). The rate of EGFR mutations in women was significantly higher than that in men. Histologically, the EGFR mutation rate in adenocarcinomas was significantly higher than that in squamous cell carcinomas. No difference in the EGFR mutation rate was observed between surgical specimens (42.1%) and small biopsy specimens (42.4%), which indicated that the EGFR mutation ratios in surgical specimens and small biopsy specimens were not different. In 385 NSCLC patients, 26 cases were positive for EML4-ALK (6.8%). However, 11.7% of the surgical specimens were EML4-ALK-positive, whereas the positive proportion in the small biopsy specimens was only 4.7%, which indicated that EML4-ALK-positive rate in the surgical specimens was significantly higher than that in the small biopsy specimens. Detection of EGFR gene mutations was feasible in small biopsy specimens, and screening for EML4-ALK expression in small biopsy specimens can be used to guide clinical treatments. PMID:27322143

  5. Structure of Escherichia coli AlkA in Complex with Undamaged DNA*

    PubMed Central

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; Verdine, Gregory L.

    2010-01-01

    Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures with that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions. PMID:20843803

  6. Structure of Escherichia coli AlkA in Complex with Undamaged DNA

    DOE PAGES

    Bowman, Brian R.; Lee, Seongmin; Wang, Shuyu; ...

    2010-11-22

    Because DNA damage is so rare, DNA glycosylases interact for the most part with undamaged DNA. Whereas the structural basis for recognition of DNA lesions by glycosylases has been studied extensively, less is known about the nature of the interaction between these proteins and undamaged DNA. Here we report the crystal structures of the DNA glycosylase AlkA in complex with undamaged DNA. The structures revealed a recognition mode in which the DNA is nearly straight, with no amino acid side chains inserted into the duplex, and the target base pair is fully intrahelical. A comparison of the present structures withmore » that of AlkA recognizing an extrahelical lesion revealed conformational changes in both the DNA and protein as the glycosylase transitions from the interrogation of undamaged DNA to catalysis of nucleobase excision. Modeling studies with the cytotoxic lesion 3-methyladenine and accompanying biochemical experiments suggested that AlkA actively interrogates the minor groove of the DNA while probing for the presence of lesions.« less

  7. Expression Differentiation Is Constrained to Low-Expression Proteins over Ecological Timescales.

    PubMed

    Margres, Mark J; Wray, Kenneth P; Seavy, Margaret; McGivern, James J; Herrera, Nathanael D; Rokyta, Darin R

    2016-01-01

    Protein expression level is one of the strongest predictors of protein sequence evolutionary rate, with high-expression protein sequences evolving at slower rates than low-expression protein sequences largely because of constraints on protein folding and function. Expression evolutionary rates also have been shown to be negatively correlated with expression level across human and mouse orthologs over relatively long divergence times (i.e., ∼100 million years). Long-term evolutionary patterns, however, often cannot be extrapolated to microevolutionary processes (and vice versa), and whether this relationship holds for traits evolving under directional selection within a single species over ecological timescales (i.e., <5000 years) is unknown and not necessarily expected. Expression is a metabolically costly process, and the expression level of a particular protein is predicted to be a tradeoff between the benefit of its function and the costs of its expression. Selection should drive the expression level of all proteins close to values that maximize fitness, particularly for high-expression proteins because of the increased energetic cost of production. Therefore, stabilizing selection may reduce the amount of standing expression variation for high-expression proteins, and in combination with physiological constraints that may place an upper bound on the range of beneficial expression variation, these constraints could severely limit the availability of beneficial expression variants. To determine whether rapid-expression evolution was restricted to low-expression proteins owing to these constraints on highly expressed proteins over ecological timescales, we compared venom protein expression levels across mainland and island populations for three species of pit vipers. We detected significant differentiation in protein expression levels in two of the three species and found that rapid-expression differentiation was restricted to low-expression proteins. Our

  8. Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation.

    PubMed

    Yasmin, Nighat; Bauer, Thomas; Modak, Madhura; Wagner, Karin; Schuster, Christopher; Köffel, Rene; Seyerl, Maria; Stöckl, Johannes; Elbe-Bürger, Adelheid; Graf, Daniel; Strobl, Herbert

    2013-11-18

    Human Langerhans cell (LC) precursors populate the epidermis early during prenatal development and thereafter undergo massive proliferation. The prototypic antiproliferative cytokine TGF-β1 is required for LC differentiation from human CD34(+) hematopoietic progenitor cells and blood monocytes in vitro. Similarly, TGF-β1 deficiency results in LC loss in vivo. However, immunohistology studies revealed that human LC niches in early prenatal epidermis and adult basal (germinal) keratinocyte layers lack detectable TGF-β1. Here we demonstrated that these LC niches express high levels of bone morphogenetic protein 7 (BMP7) and that Bmp7-deficient mice exhibit substantially diminished LC numbers, with the remaining cells appearing less dendritic. BMP7 induces LC differentiation and proliferation by activating the BMP type-I receptor ALK3 in the absence of canonical TGF-β1-ALK5 signaling. Conversely, TGF-β1-induced in vitro LC differentiation is mediated via ALK3; however, co-induction of ALK5 diminished TGF-β1-driven LC generation. Therefore, selective ALK3 signaling by BMP7 promotes high LC yields. Within epidermis, BMP7 shows an inverse expression pattern relative to TGF-β1, the latter induced in suprabasal layers and up-regulated in outer layers. We observed that TGF-β1 inhibits microbial activation of BMP7-generated LCs. Therefore, TGF-β1 in suprabasal/outer epidermal layers might inhibit LC activation, resulting in LC network maintenance.

  9. PARP-1 protein expression in glioblastoma multiforme

    PubMed Central

    Galia, A.; Calogero, A.E.; Condorelli, R.A.; Fraggetta, F.; La Corte, C.; Ridolfo, F.; Bosco, P.; Castiglione, R.; Salemi, M.

    2012-01-01

    One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker. PMID:22472897

  10. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  11. A novel lead compound CM-118: antitumor activity and new insight into the molecular mechanism and combination therapy strategy in c-Met- and ALK-dependent cancers.

    PubMed

    Meng, Lanfang; Shu, Mengjun; Chen, Yaqing; Yang, Dexiao; He, Qun; Zhao, Hui; Feng, Zhiyong; Liang, Chris; Yu, Ker

    2014-06-01

    The anaplastic lymphoma kinase (ALK) and the c-Met receptor tyrosine kinase play essential roles in the pathogenesis in multiple human cancers and present emerging targets for cancer treatment. Here, we describe CM-118, a novel lead compound displaying low nanomolar biochemical potency against both ALK and c-Met with selectivity over>90 human kinases. CM-118 potently abrogated hepatocyte growth factor (HGF)-induced c-Met phosphorylation and cell migration, phosphorylation of ALK, EML4-ALK, and ALK resistance mutants in transfected cells. CM-118 inhibited proliferation and/or induced apoptosis in multiple c-Met- and ALK-addicted cancer lines with dose response profile correlating target blockade. We show that the CM-118-induced apoptosis in c-Met-amplified H1993 NSCLC cells involved a rapid suppression of c-Met activity and c-Met-to-EGFR cross-talk, and was profoundly potentiated by EGFR inhibitors as shown by the increased levels of apoptotic proteins cleaved-PARP and Bim as well as reduction of the survival protein Mcl-1. Bim-knockdown or Mcl-1 overexpression each significantly attenuated apoptosis. We also revealed a key role by mTOR in mediating CM-118 action against the EML4-ALK-dependent NSCLC cells. Abrogation of EML4-ALK in H2228 cells profoundly reduced signaling capacity of the rapamycin-sensitive mTOR pathway leading to G 1 cell cycle arrest and mitochondrial hyperpolarization, a metabolic perturbation linked to mTOR inhibition. Depletion of mTOR or mTORC1 inhibited H2228 cell growth, and mTOR inhibitors potentiated CM-118's antitumor activity in vitro and in vivo. Oral administration of CM-118 at a wide range of well tolerated dosages diminished c-Met- and ALK phosphorylation in vivo, and caused tumor regression or growth inhibition in multiple c-Met- and ALK-dependent tumor xenografts in mice. CM-118 exhibits favorable pharmacokinetic and drug metabolism properties hence presents a candidate for clinical evaluation.

  12. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  13. Structural and mechanistic insight into alkane hydroxylation by Pseudomonas putida AlkB.

    PubMed

    Alonso, Hernan; Kleifeld, Oded; Yeheskel, Adva; Ong, Poh C; Liu, Yu C; Stok, Jeanette E; De Voss, James J; Roujeinikova, Anna

    2014-06-01

    Pseudomonas putida GPo1 alkane hydroxylase (AlkB) is an integral membrane protein that catalyses the hydroxylation of medium-chain alkanes (C3-C12). 1-Octyne irreversibly inhibits this non-haem di-iron mono-oxygenase under turnover conditions, suggesting that it acts as a mechanism-based inactivator. Upon binding to the active site, 1-octyne is postulated to be oxidized to an oxirene that rapidly rearranges to a reactive ketene which covalently acylates nearby residues, resulting in enzyme inactivation. In analysis of inactivated AlkB by LC-MS/MS, several residues exhibited a mass increase of 126.1 Da, corresponding to the octanoyl moiety derived from oxidative activation of 1-octyne. Mutagenesis studies of conserved acylated residues showed that Lys18 plays a critical role in enzyme function, as a single-point mutation of Lys18 to alanine (K18A) completely abolished enzymatic activity. Finally, we present a computational 3D model structure of the transmembrane domain of AlkB, which revealed the overall packing arrangement of the transmembrane helices within the lipid bilayer and the location of the active site mapped by the 1-octyne modifications.

  14. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  15. Alk1 controls arterial endothelial cell migration in lumenized vessels.

    PubMed

    Rochon, Elizabeth R; Menon, Prahlad G; Roman, Beth L

    2016-07-15

    Heterozygous loss of the arterial-specific TGFβ type I receptor, activin receptor-like kinase 1 (ALK1; ACVRL1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular mis-steps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild-type embryos, alk1-positive cranial arterial endothelial cells generally migrate towards the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.

  16. Wheat germ systems for cell-free protein expression.

    PubMed

    Harbers, Matthias

    2014-08-25

    Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.

  17. Trypanosoma cruzi expresses diverse repetitive protein antigens.

    PubMed Central

    Hoft, D F; Kim, K S; Otsu, K; Moser, D R; Yost, W J; Blumin, J H; Donelson, J E; Kirchhoff, L V

    1989-01-01

    We screened a Trypanosoma cruzi cDNA expression library with human and rabbit anti-T. cruzi sera and identified cDNA clones that encode polypeptides containing tandemly arranged repeats which are 6 to 34 amino acids in length. The peptide repeats encoded by these cDNAs varied markedly in sequence, copy number, and location relative to the polyadenylation site of the mRNAs from which they were derived. The repeats were specific for T. cruzi, but in each case the sizes of the corresponding mRNAs and the total number of repeat copies encoded varied considerably among different isolates of the parasite. Expression of the peptide repeats was not stage specific. One of the peptide repeats occurred in a protein with an Mr of greater than 200,000 and one was in a protein of Mr 75,000 to 105,000. The frequent occurrence and diversity of these peptide repeats suggested that they may play a role in the ability of the parasite to evade immune destruction in its invertebrate and mammalian hosts, but the primary roles of these macromolecules may be unrelated to the host-parasite relationship. Images PMID:2659529

  18. Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.

    PubMed

    Roy, Todd W; Bhagwat, A S

    2007-01-01

    The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) alpha-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.

  19. Late Alk4/5/7 signaling is required for anterior skeletal patterning in sea urchin embryos.

    PubMed

    Piacentino, Michael L; Ramachandran, Janani; Bradham, Cynthia A

    2015-03-01

    Skeletal patterning in the sea urchin embryo requires a conversation between the skeletogenic primary mesenchyme cells (PMCs) and the overlying pattern-dictating ectoderm; however, our understanding of the molecular basis for this process remains incomplete. Here, we show that TGF-β-receptor signaling is required during gastrulation to pattern the anterior skeleton. To block TGF-β signaling, we used SB431542 (SB43), a specific inhibitor of the TGF-β type I receptor Alk4/5/7. Treatment with SB43 during gastrulation blocks anterior PMC positioning and the formation of the anterior skeleton, but does not perturb general ectoderm specification or development. This is the first example of a signaling event required for patterning of a specific part of the skeleton. Alk4/5/7 inhibition does not prevent the formation of a mouth, although SB43-treated plutei display reduced feeding ability, presumably due to the loss of the structural support for the mouth conferred by the anterior skeleton. Both Univin and Nodal are potential ligands for Alk4/5/7; however, Nodal is unilaterally expressed on only the right side, whereas Univin is bilaterally expressed in the ectoderm adjacent to the anterior skeleton during the relevant time period. Our results demonstrate that Univin is both necessary and sufficient for secondary skeletal development in a control background, consistent with the hypothesis that Univin is a relevant Alk4/5/7 ligand for anterior skeletal patterning. Taken together, our data demonstrate that Alk4/5/7 signaling during gastrulation is required to direct PMCs to the oral hood, and suggest that Univin is a relevant ligand for this signaling event.

  20. Therapeutic efficacy of the bromodomain inhibitor OTX015/MK-8628 in ALK-positive anaplastic large cell lymphoma: an alternative modality to overcome resistant phenotypes

    PubMed Central

    Vurchio, Valentina; Yang, Shao Ning; Moon, John; Kwee, Ivo; Rinaldi, Andrea; Pan, Heng; Crescenzo, Ramona; Cheng, Mangeng; Cerchietti, Leandro; Elemento, Olivier; Riveiro, Maria E.; Cvitkovic, Esteban; Bertoni, Francesco; Inghirami, Giorgio

    2016-01-01

    Anaplastic large cell lymphomas (ALCL) represent a peripheral T-cell lymphoma subgroup, stratified based on the presence or absence of anaplastic lymphoma kinase (ALK) chimeras. Although ALK-positive ALCLs have a more favorable outcome than ALK-negative ALCL, refractory and/or relapsed forms are common and novel treatments are needed. Here we investigated the therapeutic potential of a novel bromodomain inhibitor, OTX015/MK-8628 in ALK-positive ALCLs. The effects of OTX015 on a panel of ALK+ ALCL cell lines was evaluated in terms of proliferation, cell cycle and downstream signaling, including gene expression profiling analyses. Synergy was tested with combination targeted therapies. Bromodomain inhibition with OTX015 led primarily to ALCL cell cycle arrest in a dose-dependent manner, along with downregulation of MYC and its downstream regulated genes. MYC overexpression did not compensate this OTX015-mediated phenotype. Transcriptomic analysis of OTX015-treated ALCL cells identified a gene signature common to various hematologic malignancies treated with bromodomain inhibitors, notably large cell lymphoma. OTX015-modulated genes included transcription factors (E2F2, NFKBIZ, FOS, JUNB, ID1, HOXA5 and HOXC6), members of multiple signaling pathways (ITK, PRKCH, and MKNK2), and histones (clusters 1-3). Combination of OTX015 with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib led to cell cycle arrest then cell death, and combination with suboptimal doses of the ALK inhibitor CEP28122 caused cell cycle arrest. When OTX015 was associated with GANT61, a selective GLI1/2 inhibitor, C1156Y-resistant ALK ALCL growth was impaired. These findings support OTX015 clinical trials in refractory ALCL in combination with inhibitors of interleukin-2-inducible kinase or SHH/GLI1. PMID:27793034

  1. PF-06463922, an ALK/ROS1 inhibitor, overcomes resistance to 1st and 2nd generation ALK inhibitors in pre-clinical models

    PubMed Central

    Zou, Helen Y.; Friboulet, Luc; Kodack, David P.; Engstrom, Lars D.; Li, Qiuhua; West, Melissa; Tang, Ruth W.; Wang, Hui; Tsaparikos, Konstantinos; Wang, Jinwei; Timofeevski, Sergei; Katayama, Ryohei; Dinh, Dac M.; Lam, Hieu; Lam, Justine L.; Yamazaki, Shinji; Hu, Wenyue; Patel, Bhushankumar; Bezwada, Divya; Frias, Rosa L.; Lifshits, Eugene; Mahmood, Sidra; Gainor, Justin F.; Affolter, Timothy; Lappin, Patrick B.; Gukasyan, Hovhannes; Lee, Nathan; Deng, Shibing; Jain, Rakesh K; Johnson, Ted W.; Shaw, Alice T.; Fantin, Valeria R.; Smeal, Tod

    2015-01-01

    SUMMARY We report the preclinical evaluation of PF-06463922, a potent and brain penetrant ALK/ROS1 inhibitor. Compared to other clinically available ALK inhibitors, PF-06463922 displayed superior potency against all known clinically acquired ALK mutations, including the highly resistant G1202R mutant. Furthermore, PF-06463922 treatment led to regression of EML4-ALK driven brain metastases, leading to prolonged mouse survival, in a superior manner. Finally, PF-06463922 demonstrated high selectivity and safety margins in a variety of preclinical studies. These results suggest that PF-06463922 will be highly effective for the treatment of patients with ALK-driven lung cancers, including those who relapsed on clinically available ALK inhibitors due to secondary ALK kinase domain mutations and/or due to the failed control of brain metastases. PMID:26144315

  2. ALKBH3, a human AlkB homologue, contributes to cell survival in human non-small-cell lung cancer

    PubMed Central

    Tasaki, M; Shimada, K; Kimura, H; Tsujikawa, K; Konishi, N

    2011-01-01

    Background: We have demonstrated for the first time that a novel human AlkB homologue, ALKBH3, contributes to prostate cancer development, but its clinical and biological roles in lung cancer remain unclear. Methods: Expression of both mRNA and protein of PCA-1 was examined by RT–PCR and western blotting. We also assessed association with senescence and in vivo ALKBH3 treatment on orthotopic tumour cell inoculation, and analysed it clinicopathologically. Results: We have since found novel biological roles for ALKBH3 in human lung cancers, particularly in adenocarcinoma. Our immunohistochemical analysis of human adenocarcinomas and squamous cell carcinomas of the lung not only showed overexpression of ALKBH3 in these tumours but the percentage of cells positive for ALKBH3 also correlated statistically to recurrence-free survival in adenocarcinoma. Knockdown of ALKBH3 by siRNA transfection induced expression of p21WAF1/Cip1 and p27Kip1 in the human lung adenocarcinoma cell line A549, resulting in cell cycle arrest, senescence and strong suppression of cell growth in vitro. In vivo, peritoneal tumour growth and dissemination was inhibited in nude mice, previously inoculated with the A549 cell line, by intraperitoneal injection of ALKBH3 siRNA + atelocollagen, as demonstrated by the reduction in both number and diameter of tumours developing in the peritoneum. Conclusion: We suggest that ALKBH3 contributes significantly to cancer cell survival and may be a therapeutic target for human adenocarcinoma of the lung. PMID:21285982

  3. Green Fluorescent Protein as a Marker for Gene Expression

    NASA Astrophysics Data System (ADS)

    Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

    1994-02-01

    A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.

  4. Compound genetically engineered mouse models of cancer reveal dual targeting of ALK1 and endoglin as a synergistic opportunity to impinge on angiogenic TGF-β signaling

    PubMed Central

    Eleftheriou, Nikolas M.; Sjölund, Jonas; Bocci, Matteo; Cortez, Eliane; Lee, Se-Jin

    2016-01-01

    Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-β family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET. PMID:27741515

  5. Effects of immunosuppressive treatment on protein expression in rat kidney

    PubMed Central

    Kędzierska, Karolina; Sporniak-Tutak, Katarzyna; Sindrewicz, Krzysztof; Bober, Joanna; Domański, Leszek; Parafiniuk, Mirosław; Urasińska, Elżbieta; Ciechanowicz, Andrzej; Domański, Maciej; Smektała, Tomasz; Masiuk, Marek; Skrzypczak, Wiesław; Ożgo, Małgorzata; Kabat-Koperska, Joanna; Ciechanowski, Kazimierz

    2014-01-01

    The structural proteins of renal tubular epithelial cells may become a target for the toxic metabolites of immunosuppressants. These metabolites can modify the properties of the proteins, thereby affecting cell function, which is a possible explanation for the mechanism of immunosuppressive agents’ toxicity. In our study, we evaluated the effect of two immunosuppressive strategies on protein expression in the kidneys of Wistar rats. Fragments of the rat kidneys were homogenized after cooling in liquid nitrogen and then dissolved in lysis buffer. The protein concentration in the samples was determined using a protein assay kit, and the proteins were separated by two-dimensional electrophoresis. The obtained gels were then stained with Coomassie Brilliant Blue, and their images were analyzed to evaluate differences in protein expression. Identification of selected proteins was then performed using mass spectrometry. We found that the immunosuppressive drugs used in popular regimens induce a series of changes in protein expression in target organs. The expression of proteins involved in drug, glucose, amino acid, and lipid metabolism was pronounced. However, to a lesser extent, we also observed changes in nuclear, structural, and transport proteins’ synthesis. Very slight differences were observed between the group receiving cyclosporine, mycophenolate mofetil, and glucocorticoids (CMG) and the control group. In contrast, compared to the control group, animals receiving tacrolimus, mycophenolate mofetil, and glucocorticoids (TMG) exhibited higher expression of proteins responsible for renal drug metabolism and lower expression levels of cytoplasmic actin and the major urinary protein. In the TMG group, we observed higher expression of proteins responsible for drug metabolism and a decrease in the expression of respiratory chain enzymes (thioredoxin-2) and markers of distal renal tubular damage (heart fatty acid-binding protein) compared to expression in the CMG

  6. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  7. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P

    2015-01-13

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

  8. Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

    DOEpatents

    Mayfield, Stephen P.

    2010-03-16

    Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

  9. A Catalytic Role for C-H/π Interactions in Base Excision Repair by Bacillus cereus DNA Glycosylase AlkD.

    PubMed

    Parsons, Zachary D; Bland, Joshua M; Mullins, Elwood A; Eichman, Brandt F

    2016-09-14

    DNA glycosylases protect genomic integrity by locating and excising aberrant nucleobases. Substrate recognition and excision usually take place in an extrahelical conformation, which is often stabilized by π-stacking interactions between the lesion nucleobase and aromatic side chains in the glycosylase active site. Bacillus cereus AlkD is the only DNA glycosylase known to catalyze base excision without extruding the damaged nucleotide from the DNA helix. Instead of contacting the nucleobase itself, the AlkD active site interacts with the lesion deoxyribose through a series of C-H/π interactions. These interactions are ubiquitous in protein structures, but evidence for their catalytic significance in enzymology is lacking. Here, we show that the C-H/π interactions between AlkD and the lesion deoxyribose participate in catalysis of glycosidic bond cleavage. This is the first demonstration of a catalytic role for C-H/π interactions as intermolecular forces important to DNA repair.

  10. Analysis of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK)-reactive CD8(+) T cell responses in children with NPM-ALK(+) anaplastic large cell lymphoma.

    PubMed

    K Singh, V; Werner, S; Hackstein, H; Lennerz, V; Reiter, A; Wölfel, T; Damm-Welk, C; Woessmann, W

    2016-10-01

    Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

  11. Phosphoproteomics reveals ALK promote cell progress via RAS/JNK pathway in neuroblastoma

    PubMed Central

    Xu, Guofeng; Zhang, Min; Wu, Yeming; Wu, Zhixiang

    2016-01-01

    Emerging evidence suggests receptor tyrosine kinase ALK as a promising therapeutic target in neuroblastoma. However, clinical trials reveal that a limited proportion of ALK-positive neuroblastoma patients experience clinical benefits from Crizotinib, a clinically approved specific inhibitor of ALK. The precise molecular mechanisms of aberrant ALK activity in neuroblastoma remain elusive, limiting the clinical application of ALK as a therapeutic target in neuroblastoma. Here, we describe a deep quantitative phosphoproteomic approach in which Crizotinib-treated neuroblastoma cell lines bearing aberrant ALK are used to investigate downstream regulated phosphoproteins. We identified more than 19,500—and quantitatively analyzed approximately 10,000—phosphorylation sites from each cell line, ultimately detecting 450–790 significantly-regulated phosphorylation sites. Multiple layers of bioinformatic analysis of the significantly-regulated phosphoproteins identified RAS/JNK as a downstream signaling pathway of ALK, independent of the ALK variant present. Further experiments demonstrated that ALK/JNK signaling could be inactivated by either ALK- or JNK-specific inhibitors, resulting in cell growth inhibition by induction of cell cycle arrest and cell apoptosis. Our study broadly defines the phosphoproteome in response to ALK inhibition and provides a resource for further clinical investigation of ALK as therapeutic target for the treatment of neuroblastoma. PMID:27732954

  12. Calreticulin: Roles in Cell-Surface Protein Expression

    PubMed Central

    Jiang, Yue; Dey, Sandeepa; Matsunami, Hiroaki

    2014-01-01

    In order to perform their designated functions, proteins require precise subcellular localizations. For cell-surface proteins, such as receptors and channels, they are able to transduce signals only when properly targeted to the cell membrane. Calreticulin is a multi-functional chaperone protein involved in protein folding, maturation, and trafficking. However, evidence has been accumulating that calreticulin can also negatively regulate the surface expression of certain receptors and channels. In these instances, depletion of calreticulin enhances cell-surface expression and function. In this review, we discuss the role of calreticulin with a focus on its negative effects on the expression of cell-surface proteins. PMID:25230046

  13. Human SUMO fusion systems enhance protein expression and solubility.

    PubMed

    Wang, Zhongyuan; Li, Haolong; Guan, Wei; Ling, Haili; Wang, Zhiyong; Mu, Tianyang; Shuler, Franklin D; Fang, Xuexun

    2010-10-01

    A major challenge associated with recombinant protein production in Escherichia coli is generation of large quantities of soluble, functional protein. Yeast SUMO (small ubiquitin-related modifier), has been shown to enhance heterologous protein expression and solubility as fusion tag, however, the effects of human SUMOs on protein expression have not been investigated. Here we describe the use of human SUMO1 and SUMO2 as a useful gene fusion technology. Human SUMO1 and SUMO2 fusion expression vectors were constructed and tested in His-tag and ubiquitin fusion expression systems. Two difficult-to-express model proteins, matrix metalloprotease-13 (MMP13) and enhanced green fluorescence protein (eGFP) were fused to the C-terminus of the human SUMO1 and SUMO2 expression vectors. These constructs were expressed in E. coli and evaluation of MMP13 and eGFP expression and solubility was conducted. We found that both SUMO1 and SUMO2 had the ability to enhance the solubility of MMP13 and eGFP, with the SUMO2 tag having a more significant effect. Since fusion tags produce varying quantities of soluble proteins, we assessed the effect of SUMO2 coupled with ubiquitin (Ub). SUMO2-ubiquitin and ubiquitin-SUMO2 fusion expression plasmids were constructed with eGFP as a passenger protein. Following expression in E. coli, both plasmids could improve eGFP expression and solubility similar to the SUMO2 fusion and better than the ubiquitin fusion. The sequential order of SUMO2 and ubiquitin had little effect on expression and solubility of eGFP. Purification of eGFP from the gene fusion product, SUMO2-ubiquitin-eGFP, involved cleavage by a deubiquitinase (Usp2-cc) and Ni-Sepharose column chromatography. The eGFP protein was purified to high homogeneity. In summary, human SUMO1 and SUMO2 are useful gene fusion technologies enhancing the expression, solubility and purification of model heterologous proteins.

  14. Efficient protein production method for NMR using soluble protein tags with cold shock expression vector.

    PubMed

    Hayashi, Kokoro; Kojima, Chojiro

    2010-11-01

    The E. coli protein expression system is one of the most useful methods employed for NMR sample preparation. However, the production of some recombinant proteins in E. coli is often hampered by difficulties such as low expression level and low solubility. To address these problems, a modified cold-shock expression system containing a glutathione S-transferase (GST) tag, the pCold-GST system, was investigated. The pCold-GST system successfully expressed 9 out of 10 proteins that otherwise could not be expressed using a conventional E. coli expression system. Here, we applied the pCold-GST system to 84 proteins and 78 proteins were successfully expressed in the soluble fraction. Three other cold-shock expression systems containing a maltose binding protein tag (pCold-MBP), protein G B1 domain tag (pCold-GB1) or thioredoxin tag (pCold-Trx) were also developed to improve the yield. Additionally, we show that a C-terminal proline tag, which is invisible in ¹H-¹⁵N HSQC spectra, inhibits protein degradation and increases the final yield of unstable proteins. The purified proteins were amenable to NMR analyses. These data suggest that pCold expression systems combined with soluble protein tags can be utilized to improve the expression and purification of various proteins for NMR analysis.

  15. A patient previously treated with ALK inhibitors for central nervous system lesions from ALK rearranged lung cancer: a case report

    PubMed Central

    Kashima, Jumpei; Okuma, Yusuke; Hishima, Tsunekazu

    2016-01-01

    Background Patients with anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC) are now preferentially treated with tyrosine kinase inhibitors (TKIs). However, patients treated with ALK inhibitors end up with acquired resistance. Case presentation We present a patient with recurrent ALK-rearranged NSCLC that developed multiple brain metastases and meningitis carcinomatosa after sequential treatment with several lines of cytotoxic chemotherapy, crizotinib, and alectinib. After the patient underwent retreatment with crizotinib as salvage therapy because of poor performance status, the intracranial metastatic foci and meningeal thickening were shrank within 1 week. Conclusion Our experience with this case suggests that alectinib may restore sensitivity to crizotinib or amplified pathway such as MET which bestowed alectinib resistance was inhibited with crizotinib. PMID:27785052

  16. Expression, Solubilization, and Purification of Bacterial Membrane Proteins.

    PubMed

    Jeffery, Constance J

    2016-02-02

    Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.

  17. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  18. Cell-free protein synthesis as a promising expression system for recombinant proteins.

    PubMed

    Ge, Xumeng; Xu, Jianfeng

    2012-01-01

    Cell-free protein synthesis (CFPS) has major advantages over traditional cell-based methods in the capability of high-throughput protein synthesis and special protein production. During recent decades, CFPS has become an alternative protein production platform for both fundamental and applied purposes. Using Renilla luciferase as model protein, we describe a typical process of CFPS in wheat germ extract system, including wheat germ extract preparation, expression vector construction, in vitro protein synthesis (transcription/translation), and target protein assay.

  19. Maltose-Binding Protein (MBP), a Secretion-Enhancing Tag for Mammalian Protein Expression Systems.

    PubMed

    Reuten, Raphael; Nikodemus, Denise; Oliveira, Maria B; Patel, Trushar R; Brachvogel, Bent; Breloy, Isabelle; Stetefeld, Jörg; Koch, Manuel

    2016-01-01

    Recombinant proteins are commonly expressed in eukaryotic expression systems to ensure the formation of disulfide bridges and proper glycosylation. Although many proteins can be expressed easily, some proteins, sub-domains, and mutant protein versions can cause problems. Here, we investigated expression levels of recombinant extracellular, intracellular as well as transmembrane proteins tethered to different polypeptides in mammalian cell lines. Strikingly, fusion of proteins to the prokaryotic maltose-binding protein (MBP) generally enhanced protein production. MBP fusion proteins consistently exhibited the most robust increase in protein production in comparison to commonly used tags, e.g., the Fc, Glutathione S-transferase (GST), SlyD, and serum albumin (ser alb) tag. Moreover, proteins tethered to MBP revealed reduced numbers of dying cells upon transient transfection. In contrast to the Fc tag, MBP is a stable monomer and does not promote protein aggregation. Therefore, the MBP tag does not induce artificial dimerization of tethered proteins and provides a beneficial fusion tag for binding as well as cell adhesion studies. Using MBP we were able to secret a disease causing laminin β2 mutant protein (congenital nephrotic syndrome), which is normally retained in the endoplasmic reticulum. In summary, this study establishes MBP as a versatile expression tag for protein production in eukaryotic expression systems.

  20. Cell-free expression of G-protein-coupled receptors.

    PubMed

    Orbán, Erika; Proverbio, Davide; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank

    2015-01-01

    Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.

  1. Canadian consensus: inhibition of ALK-positive tumours in advanced non-small-cell lung cancer

    PubMed Central

    Melosky, B.; Agulnik, J.; Albadine, R.; Banerji, S.; Bebb, D.G.; Bethune, D.; Blais, N.; Butts, C.; Cheema, P.; Cheung, P.; Cohen, V.; Deschenes, J.; Ionescu, D.N.; Juergens, R.; Kamel-Reid, S.; Laurie, S.A.; Liu, G.; Morzycki, W.; Tsao, M.S.; Xu, Z.; Hirsh, V.

    2016-01-01

    Anaplastic lymphoma kinase (alk) is an oncogenic driver in non-small-cell lung cancer (nsclc). Chromosomal rearrangements involving the ALK gene occur in up to 4% of nonsquamous nsclc patients and lead to constitutive activation of the alk signalling pathway. ALK-positive nsclc is found in relatively young patients, with a median age of 50 years. Patients frequently have brain metastasis. Targeted inhibition of the alk pathway prolongs progression-free survival in patients with ALK-positive advanced nsclc. The results of several recent clinical trials confirm the efficacy and safety benefit of crizotinib and ceritinib in this population. Canadian oncologists support the following consensus statement: All patients with advanced nonsquamous nsclc (excluding pure neuroendocrine carcinoma) should be tested for the presence of an ALK rearrangement. If an ALK rearrangement is present, treatment with a targeted alk inhibitor in the first-line setting is recommended. As patients become resistant to first-generation alk inhibitors, other treatments, including second-generation alk inhibitors can be considered. PMID:27330348

  2. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

    PubMed

    Nalaskowski, Marcus M; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W

    2012-09-01

    Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

  3. An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

    PubMed

    Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

    2015-05-01

    Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket.

  4. New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration

    PubMed Central

    Pasteuning-Vuhman, Svitlana; Boertje-van der Meulen, Johanna W.; van Putten, Maaike; Overzier, Maurice; ten Dijke, Peter; Kiełbasa, Szymon M.; Arindrarto, Wibowo; Wolterbeek, Ron; Lezhnina, Ksenia V.; Ozerov, Ivan V.; Aliper, Aleksandr M.; Hoogaars, Willem M.; Aartsma-Rus, Annemieke; Loomans, Cindy J. M.

    2017-01-01

    Skeletal muscle fibrosis and impaired muscle regeneration are major contributors to muscle wasting in Duchenne muscular dystrophy (DMD). Muscle growth is negatively regulated by myostatin (MSTN) and activins. Blockage of these pathways may improve muscle quality and function in DMD. Antisense oligonucleotides (AONs) were designed specifically to block the function of ALK4, a key receptor for the MSTN/activin pathway in skeletal muscle. AON-induced exon skipping resulted in specific Alk4 down-regulation, inhibition of MSTN activity, and increased myoblast differentiation in vitro. Unexpectedly, a marked decrease in muscle mass (10%) was found after Alk4 AON treatment in mdx mice. In line with in vitro results, muscle regeneration was stimulated, and muscle fiber size decreased markedly. Notably, when Alk4 was down-regulated in adult wild-type mice, muscle mass decreased even more. RNAseq analysis revealed dysregulated metabolic functions and signs of muscle atrophy. We conclude that ALK4 inhibition increases myogenesis but also regulates the tight balance of protein synthesis and degradation. Therefore, caution must be used when developing therapies that interfere with MSTN/activin pathways.—Pasteuning-Vuhman, S., Boertje-van der Meulen, J. W., van Putten, M., Overzier, M., ten Dijke, P., Kiełbasa, S. M., Arindrarto, W., Wolterbeek, R., Lezhnina, K. V., Ozerov, I. V., Aliper, A. M., Hoogaars, W. M., Aartsma-Rus, A., Loomans, C. J. M. New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration. PMID:27733450

  5. Durable brain response with pulse-dose crizotinib and ceritinib in ALK-positive non-small cell lung cancer compared with brain radiotherapy.

    PubMed

    Dudnik, Elizabeth; Siegal, Tali; Zach, Leor; Allen, Aaron M; Flex, Dov; Yust-Katz, Shlomit; Limon, Dror; Hirsch, Fred R; Peled, Nir

    2016-04-01

    Crizotinib achieves excellent systemic control in anaplastic lymphoma kinase-rearranged (ALK+) non-small cell lung cancer (NSCLC); however, central nervous system (CNS) metastases frequently occur as an early event. Whole brain irradiation, the standard treatment, results in neurocognitive impairment. We present a case series of three ALK+ NSCLC patients with progressing CNS metastases who were treated with pulse-dose crizotinib followed by ceritinib. Three ALK+ NSCLC patients treated between 2011 and 2014 (two males, two never smokers, age range 20-54years, all echinoderm microtubule-associated protein-like 4/ALK rearrangement), were diagnosed with progressing cerebral disease while receiving crizotinib. Clinico-pathological characteristics, treatments, and outcomes were analyzed. In two patients the progression was limited to the CNS, and radiological evidence of leptomeningeal spread was present in one patient. Sequential use of crizotinib 500mg administered once daily (pulse-dose) followed by ceritinib on progression achieved control of the disease in the CNS for over 18 months and over 7 months in Patient 1 and Patient 2, respectively. This strategy provided durable CNS control after whole-brain radiotherapy failure in Patient 1, and allowed the whole-brain radiotherapy to be deferred in Patient 2. Limited CNS progression was documented in Patient 3 while he was on standard-dose/pulse-dose crizotinib for 15months; durable (over 7 months) complete remission was achieved with stereotactic radiotherapy and ceritinib. Manipulating the crizotinib schedule in ALK+ NSCLC patients with CNS metastases and using a novel ALK-inhibitor at the time of further progression may provide durable CNS control and allow brain radiotherapy to be deferred.

  6. Nucleic Acid Programmable Protein Array: A Just-In-Time Multiplexed Protein Expression and Purification Platform

    PubMed Central

    Qiu, Ji; LaBaer, Joshua

    2012-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally. PMID:21943897

  7. Nucleic acid programmable protein array a just-in-time multiplexed protein expression and purification platform.

    PubMed

    Qiu, Ji; LaBaer, Joshua

    2011-01-01

    Systematic study of proteins requires the availability of thousands of proteins in functional format. However, traditional recombinant protein expression and purification methods have many drawbacks for such study at the proteome level. We have developed an innovative in situ protein expression and capture system, namely NAPPA (nucleic acid programmable protein array), where C-terminal tagged proteins are expressed using an in vitro expression system and efficiently captured/purified by antitag antibodies coprinted at each spot. The NAPPA technology presented in this chapter enable researchers to produce and display fresh proteins just in time in a multiplexed high-throughput fashion and utilize them for various downstream biochemical researches of interest. This platform could revolutionize the field of functional proteomics with it ability to produce thousands of spatially separated proteins in high density with narrow dynamic rand of protein concentrations, reproducibly and functionally.

  8. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  9. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  10. Expression of heat shock protein genes in insect stress responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The heat shock proteins (HSPs) that are abundantly expressed in insects are important modulators of insect survival. Expression of HSP genes in insects is not only developmentally regulated, but also induced by various stressors in order to confer protection against such stressors. The expression o...

  11. Evolution, diversification, and expression of KNOX proteins in plants

    PubMed Central

    Gao, Jie; Yang, Xue; Zhao, Wei; Lang, Tiange; Samuelsson, Tore

    2015-01-01

    The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification. PMID:26557129

  12. Major cancer protein amplifies global gene expression

    Cancer.gov

    Scientists may have discovered why a protein called MYC can provoke a variety of cancers. Like many proteins associated with cancer, MYC helps regulate cell growth. A new study carried out by researchers at the National Institutes of Health and colleagues

  13. Transient protein expression in three Pisum sativum (green pea) varieties.

    PubMed

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  14. Mobile phone radiation might alter protein expression in human skin

    PubMed Central

    Karinen, Anu; Heinävaara, Sirpa; Nylund, Reetta; Leszczynski, Dariusz

    2008-01-01

    Background Earlier we have shown that the mobile phone radiation (radiofrequency modulated electromagnetic fields; RF-EMF) alters protein expression in human endothelial cell line. This does not mean that similar response will take place in human body exposed to this radiation. Therefore, in this pilot human volunteer study, using proteomics approach, we have examined whether a local exposure of human skin to RF-EMF will cause changes in protein expression in living people. Results Small area of forearm's skin in 10 female volunteers was exposed to RF-EMF (specific absorption rate SAR = 1.3 W/kg) and punch biopsies were collected from exposed and non-exposed areas of skin. Proteins extracted from biopsies were separated using 2-DE and protein expression changes were analyzed using PDQuest software. Analysis has identified 8 proteins that were statistically significantly affected (Anova and Wilcoxon tests). Two of the proteins were present in all 10 volunteers. This suggests that protein expression in human skin might be affected by the exposure to RF-EMF. The number of affected proteins was similar to the number of affected proteins observed in our earlier in vitro studies. Conclusion This is the first study showing that molecular level changes might take place in human volunteers in response to exposure to RF-EMF. Our study confirms that proteomics screening approach can identify protein targets of RF-EMF in human volunteers. PMID:18267023

  15. Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

    PubMed

    Ardini, Elena; Menichincheri, Maria; Banfi, Patrizia; Bosotti, Roberta; De Ponti, Cristina; Pulci, Romana; Ballinari, Dario; Ciomei, Marina; Texido, Gemma; Degrassi, Anna; Avanzi, Nilla; Amboldi, Nadia; Saccardo, Maria Beatrice; Casero, Daniele; Orsini, Paolo; Bandiera, Tiziano; Mologni, Luca; Anderson, David; Wei, Ge; Harris, Jason; Vernier, Jean-Michel; Li, Gang; Felder, Eduard; Donati, Daniele; Isacchi, Antonella; Pesenti, Enrico; Magnaghi, Paola; Galvani, Arturo

    2016-04-01

    Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

  16. Galangin inhibits hypertrophic scar formation via ALK5/Smad2/3 signaling pathway.

    PubMed

    Zhang, Yifan; Shan, Shengzhou; Wang, Jing; Cheng, Xinyu; Yi, Bo; Zhou, Jia; Li, Qingfeng

    2016-02-01

    Hypertrophic scar (HS) is characterized by excessive fibrosis associated with aberrant function of fibroblasts. Currently, no satisfactory drug has been developed to treat the disease. Here we found that a flavonoid natural product, galangin, could significantly attenuate hypertrophic scar formation in a mechanical load-induced mouse model. Both in vivo and in vitro studies demonstrated that galangin remarkably inhibited collagen production, proliferation, and activation of fibroblasts. Besides, galangin suppressed the contractile ability of hypertrophic scar fibroblasts. Further Western blot analysis revealed that galangin dose-dependently down-regulated Smad2 and Smad3 phosphorylation. Such bioactivity of galangin resulted from its selective targeting to the activin receptor-like kinase 5 (ALK5) was demonstrated by ALK5 knockdown and over-expression experiments. Taken together, this compound could simultaneously inhibit both the accumulation of collagen and abnormal activation/proliferation of fibroblasts, which were the two pivotal factors for hypertrophic scar formation, thus suggesting that galangin serves as a potential agent for treatment of HS or other fibroproliferative disorders.

  17. Reducing progoitrin and enriching glucoraphanin in Brassica napus seeds through silencing of the GSL-ALK gene family.

    PubMed

    Liu, Zheng; Hirani, Arvind H; McVetty, Peter B E; Daayf, Fouad; Quiros, Carlos F; Li, Genyi

    2012-05-01

    The hydrolytic products of glucosinolates in brassica crops are bioactive compounds. Some glucosinolate derivatives such as oxazolidine-2-thione from progoitrin in brassica oilseed meal are toxic and detrimental to animals, but some isothiocyanates such as sulforaphane are potent anti-carcinogens that have preventive effects on several human cancers. In most B. rapa, B. napus and B. juncea vegetables and oilseeds, there is no or only trace amount of glucoraphanin that is the precursor to sulforaphane. In this paper, RNA interference (RNAi) of the GSL-ALK gene family was used to down-regulate the expression of GSL-ALK genes in B. napus. The detrimental glucosinolate progoitrin was reduced by 65 %, and the beneficial glucosinolate glucoraphanin was increased to a relatively high concentration (42.6 μmol g(-1) seed) in seeds of B. napus transgenic plants through silencing of the GSL-ALK gene family. Therefore, there is potential application of the new germplasm with reduced detrimental glucosinolates and increased beneficial glucosinolates for producing improved brassica vegetables.

  18. Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB

    PubMed Central

    Yu, Bomina; Hunt, John F.

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis–Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this “kcat/Km compensation,” which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding. PMID:19706517

  19. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  20. Protein Production for Structural Genomics Using E. coli Expression

    PubMed Central

    Makowska-Grzyska, Magdalena; Kim, Youngchang; Maltseva, Natalia; Li, Hui; Zhou, Min; Joachimiak, Grazyna; Babnigg, Gyorgy; Joachimiak, Andrzej

    2014-01-01

    The goal of structural biology is to reveal details of the molecular structure of proteins in order to understand their function and mechanism. X-ray crystallography and NMR are the two best methods for atomic level structure determination. However, these methods require milligram quantities of proteins. In this chapter a reproducible methodology for large-scale protein production applicable to a diverse set of proteins is described. The approach is based on protein expression in E. coli as a fusion with a cleavable affinity tag that was tested on over 20,000 proteins. Specifically, a protocol for fermentation of large quantities of native proteins in disposable culture vessels is presented. A modified protocol that allows for the production of selenium-labeled proteins in defined media is also offered. Finally, a method for the purification of His6-tagged proteins on immobilized metal affinity chromatography columns that generates high-purity material is described in detail. PMID:24590711

  1. Protein Expression Dynamics During Postnatal Mouse Brain Development

    PubMed Central

    Laeremans, Annelies; Van de Plas, Babs; Clerens, Stefan; Van den Bergh, Gert; Arckens, Lutgarde; Hu, Tjing-Tjing

    2013-01-01

    We explored differential protein expression profiles in the mouse forebrain at different stages of postnatal development, including 10-day (P10), 30-day (P30), and adult (Ad) mice, by large-scale screening of proteome maps using two-dimensional difference gel electrophoresis. Mass spectrometry analysis resulted in the identification of 251 differentially expressed proteins. Most molecular changes were observed between P10 compared to both P30 and Ad. Computational ingenuity pathway analysis (IPA) confirmed these proteins as crucial molecules in the biological function of nervous system development. Moreover, IPA revealed Semaphorin signaling in neurons and the protein ubiquitination pathway as essential canonical pathways in the mouse forebrain during postnatal development. For these main biological pathways, the transcriptional regulation of the age-dependent expression of selected proteins was validated by means of in situ hybridization. In conclusion, we suggest that proteolysis and neurite outgrowth guidance are key biological processes, particularly during early brain maturation. PMID:25157209

  2. 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

  3. Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signaling.

    PubMed

    Chang, Mei-Chi; Chang, Hsiao-Hua; Lin, Po-Shuan; Huang, Yu-An; Chan, Chiu-Po; Tsai, Yi-Ling; Lee, Shen-Yang; Jeng, Po-Yuan; Kuo, Han-Yueh; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-10-09

    Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signaling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analyzed by Pathscan ELISA or western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor), U0126 (MEK/ERK inhibitor) for examining the related signaling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 up-regulated both RNA and protein expression of PAI-1 and uPAR, whereas down-regulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA nd uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signaling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. This article is protected by copyright. All rights reserved.

  4. Expression of Yes-associated protein modulates Survivin expression in primary liver malignancies.

    PubMed

    Bai, Haibo; Gayyed, Mariana F; Lam-Himlin, Dora M; Klein, Alison P; Nayar, Suresh K; Xu, Yang; Khan, Mehtab; Argani, Pedram; Pan, Duojia; Anders, Robert A

    2012-09-01

    Hepatocellular carcinoma and intrahepatic cholangiocarcinoma account for 95% of primary liver cancer. For each of these malignancies, the outcome is dismal; incidence is rapidly increasing, and mechanistic understanding is limited. We observed abnormal proliferation of both biliary epithelium and hepatocytes in mice after genetic manipulation of Yes-associated protein, a transcription coactivator. Here, we comprehensively documented Yes-associated protein expression in the human liver and primary liver cancers. We showed that nuclear Yes-associated protein expression is significantly increased in human intrahepatic cholangiocarcinoma and hepatocellular carcinoma. We found that increased Yes-associated protein levels in hepatocellular carcinoma are due to multiple mechanisms including gene amplification and transcriptional and posttranscriptional regulation. Survivin, a member of the inhibitors-of-apoptosis protein family, has been reported as an independent prognostic factor for poor survival in both hepatocellular carcinoma and intrahepatic cholangiocarcinoma. We found that nuclear Yes-associated protein expression correlates significantly with nuclear Survivin expression for both intrahepatic cholangiocarcinoma and hepatocellular carcinoma. Furthermore, using mice engineered to conditionally overexpress Yes-associated protein in the liver, we found that Survivin messenger RNA expression depends upon Yes-associated protein levels. Our findings suggested that Yes-associated protein contributes to primary liver tumorigenesis and likely mediates its oncogenic effects through modulating Survivin expression.

  5. Performance benchmarking of four cell-free protein expression systems.

    PubMed

    Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill

    2016-02-01

    Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins.

  6. GTP cyclohydrolase I expression, protein, and activity determine intracellular tetrahydrobiopterin levels, independent of GTP cyclohydrolase feedback regulatory protein expression.

    PubMed

    Tatham, Amy L; Crabtree, Mark J; Warrick, Nicholas; Cai, Shijie; Alp, Nicholas J; Channon, Keith M

    2009-05-15

    GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

  7. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  8. Recombinant Brucella abortus gene expressing immunogenic protein

    SciTech Connect

    Mayfield, J.E.; Tabatabai, L.B.

    1991-06-11

    This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

  9. Treatment modalities for advanced ALK-rearranged non-small-cell lung cancer.

    PubMed

    Sullivan, Ivana; Planchard, David

    2016-04-01

    The ALK gene plays a key role in the pathogenesis of non-small-cell lung cancer (NSCLC). Patients with NSCLC harboring an ALK-rearrangement represent the second oncogene addiction to be identified in this disease. Crizotinib was the first ALK inhibitor showing pronounced clinical activity, and is now a reference treatment for ALK-positive NSCLC disease. However, despite initial impressive responses to crizotinib, acquired resistance almost invariably develops within 12 months. The pressing need for effective second-line agents has prompted the rapid development of next-generation ALK inhibitors. These agents, notably ceritinib and alectinib as the most developed, have a higher potency against ALK than crizotinib, along with activity against tumors harboring crizotinib-resistant mutations and potentially improved CNS penetration.

  10. Targeting brain metastases in ALK-rearranged non-small-cell lung cancer.

    PubMed

    Zhang, Isabella; Zaorsky, Nicholas G; Palmer, Joshua D; Mehra, Ranee; Lu, Bo

    2015-10-01

    The incidence of brain metastases has increased as a result of improved systemic control and advances in imaging. However, development of novel therapeutics with CNS activity has not advanced at the same rate. Research on molecular markers has revealed many potential targets for antineoplastic agents, and a particularly important aberration is translocation in the ALK gene, identified in non-small-cell lung cancer (NSCLC). ALK inhibitors have shown systemic efficacy against ALK-rearranged NSCLC in many clinical trials, but the effectiveness of crizotinib in CNS disease is limited by poor blood-brain barrier penetration and acquired drug resistance. In this Review, we discuss potential pathways to target ALK-rearranged brain metastases, including next generation ALK inhibitors with greater CNS penetration and mechanisms to overcome resistance. Other important mechanisms to control CNS disease include targeting pathways downstream of ALK phosphorylation, increasing the permeability of the blood-brain barrier, modifying the tumour microenvironment, and adding concurrent radiotherapy.

  11. Enhanced membrane protein expression by engineering increased intracellular membrane production

    PubMed Central

    2013-01-01

    Background Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of

  12. A Statistical Study on Oscillatory Protein Expression

    NASA Astrophysics Data System (ADS)

    Yan, Shiwei

    Motivated by the experiments on the dynamics of a common network motif, p53 and Mdm2 feedback loop, by Lahav et al. [Nat. Genet 36, 147(2004)] in individual cells and Lev Bar-or et al. [Proc. Natl. Acad. Sci. USA 97, 11250(2000)] at the population of cells, we propose a statistical signal-response model with aiming to describe the different oscillatory behaviors for the activities of p53 and Mdm2 proteins both in individual and in population of cells in a unified way. At the cellular level, the activities of p53 and Mdm2 proteins are described by a group of nonlinear dynamical equations where the damage-derived signal is assumed to have the form with abrupt transition (”on” leftrightarrow ”off”) as soon as signal strength passes forth and back across a threshold. Each cell responses to the damage with different time duration within which the oscillations persist. For the case of population of cells, the activities of p53 and Mdm2 proteins will be the population average of the individual cells, which results damped oscillations, due to the averaging over the cell population with the different response time.

  13. ALK Signaling and Target Therapy in Anaplastic Large Cell Lymphoma

    PubMed Central

    Tabbó, Fabrizio; Barreca, Antonella; Piva, Roberto; Inghirami, Giorgio

    2012-01-01

    The discovery by Morris et al. (1994) of the genes contributing to the t(2;5)(p23;q35) translocation has laid the foundation for a molecular based recognition of anaplastic large cell lymphoma and highlighted the need for a further stratification of T-cell neoplasia. Likewise the detection of anaplastic lymphoma kinase (ALK) genetic lesions among many human cancers has defined unique subsets of cancer patients, providing new opportunities for innovative therapeutic interventions. The objective of this review is to appraise the molecular mechanisms driving ALK-mediated transformation, and to maintain the neoplastic phenotype. The understanding of these events will allow the design and implementation of novel tailored strategies for a well-defined subset of cancer patients. PMID:22649787

  14. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  15. Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary?

    PubMed

    Toyokawa, Gouji; Seto, Takashi; Takenoyama, Mitsuhiro; Ichinose, Yukito

    2015-12-01

    Anaplastic lymphoma kinase (ALK) has been identified to exert a potent transforming activity through its rearrangement in non-small cell lung cancer (NSCLC), and patients (pts) with ALK rearrangement can be treated more successfully with ALK inhibitors, such as crizotinib, alectinib, and ceritinib, than with chemotherapy. Despite the excellent efficacy of ALK inhibitors, resistance to these drugs is inevitably encountered in most ALK-rearranged pts. Cases of resistance are subtyped into three groups, i.e., systemic, oligo, and central nervous system (CNS) types, with the CNS being used to be considered a sanctuary. With regard to the management of CNS lesions in pts with ALK+ NSCLC, a growing body of evidence has gradually demonstrated the intracranial (IC) efficacy of ALK inhibitor (ALKi) in ALK+ NSCLC pts with brain metastases (BMs). Although the efficacy of crizotinib for the CNS lesions remains controversial, a recent retrospective investigation of ALK+ pts with BM enrolled in PROFILE 1005 and PROFILE 1007 demonstrated that crizotinib is associated with a high disease control rate for BM. However, BM comprises the most common site of progressive disease in pts with or without baseline BMs, which is a serious problem for crizotinib. Furthermore, alectinib can be used to achieve strong and long-lasting inhibitory effects on BM. In addition to alectinib, the IC efficacy of other next-generation ALK inhibitors, such as ceritinib, AP26113 and PF-06463922, has been demonstrated. In this article, we review the latest evidence regarding the BM and IC efficacy of ALK inhibitors in pts with ALK+ NSCLC.

  16. A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-13-1-0220 TITLE: A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN PRINCIPAL...4. TITLE AND SUBTITLE A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN 5a. CONTRACT NUMBER 5b. GRANT NUMBER... genetic and epigenetic changes that occur during tumorigenesis. 15. SUBJECT TERMS Anaplastic lymphoma kinase, neuroblastoma, ALK, ALKF1174L, MYCN, CDK7

  17. Expression of rabies virus G protein in carrots (Daucus carota).

    PubMed

    Rojas-Anaya, Edith; Loza-Rubio, Elizabeth; Olivera-Flores, Maria Teresa; Gomez-Lim, Miguel

    2009-12-01

    Antigens derived from various pathogens can readily be synthesized at high levels in plants in their authentic forms. Such antigens administered orally can induce an immune response and, in some cases, result in protection against a subsequent challenge. We here report the expression of rabies virus G protein into carrots. The G gene was subcloned into the pUCpSSrabG vector and then used to transform carrot embryogenic cells by particle bombardment. The carrot cells were selected in liquid medium, a method previously unreported. The presence of the transgene was verified by PCR, and by RT-PCR. By western blot, G protein transgene was identified in 93.3% of adult carrot roots. The G protein was quantified by densitometric analysis (range 0.4-1.2%). The expressed protein was antigenic in mice. This confirms that the carrot is an adequate system for antigen expression.

  18. Vectors for the expression of tagged proteins in Drosophila.

    PubMed

    Parker, L; Gross, S; Alphey, L

    2001-12-01

    Regulated expression systems have been extremely useful in developmental studies, allowing the expression of specific proteins in defined spatial and temporal patterns. If these proteins are fused to an appropriate molecular tag, then they can be purified or visualized without the need to raise specific antibodies. If the tag is inherently fluorescent, then the proteins can even be visualized directly, in living tissue. We have constructed a series of P element-based transformation vectors for the most widely used expression system in Drosophila, GAL4/UAS. These vectors provide a series of useful tags for antibody detection, protein purification, and/or direct visualization, together with a convenient multiple cloning site into which the cDNA of interest can be inserted.

  19. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  20. Differential protein expression in Phalaenopsis under low temperature.

    PubMed

    Yuan, Xiu-Yun; Liang, Fang; Jiang, Su-Hua; Wan, Mo-Fei; Ma, Jie; Zhang, Xian-Yun; Cui, Bo

    2015-01-01

    A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

  1. Global Analysis of Protein Expression of Inner Ear Hair Cells.

    PubMed

    Hickox, Ann E; Wong, Ann C Y; Pak, Kwang; Strojny, Chelsee; Ramirez, Miguel; Yates, John R; Ryan, Allen F; Savas, Jeffrey N

    2017-02-01

    The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.

  2. Prolonged morphine administration alters protein expression in the rat myocardium

    PubMed Central

    2011-01-01

    Background Morphine is used in clinical practice as a highly effective painkiller as well as the drug of choice for treatment of certain heart diseases. However, there is lack of information about its effect on protein expression in the heart. Therefore, here we aimed to identify the presumed alterations in rat myocardial protein levels after prolonged morphine treatment. Methods Morphine was administered to adult male Wistar rats in high doses (10 mg/kg per day) for 10 days. Proteins from the plasma membrane- and mitochondria-enriched fractions or cytosolic proteins isolated from left ventricles were run on 2D gel electrophoresis, scanned and quantified with specific software to reveal differentially expressed proteins. Results Nine proteins were found to show markedly altered expression levels in samples from morphine-treaded rats and these proteins were identified by mass spectrometric analysis. They belong to different cell pathways including signaling, cytoprotective, and structural elements. Conclusions The present identification of several important myocardial proteins altered by prolonged morphine treatment points to global effects of this drug on heart tissue. These findings represent an initial step toward a more complex view on the action of morphine on the heart. PMID:22129148

  3. The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria.

    PubMed

    Jurelevicius, Diogo; Alvarez, Vanessa Marques; Peixoto, Raquel; Rosado, Alexandre S; Seldin, Lucy

    2013-01-01

    The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.

  4. The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

    PubMed Central

    Jurelevicius, Diogo; Alvarez, Vanessa Marques; Peixoto, Raquel; Rosado, Alexandre S.; Seldin, Lucy

    2013-01-01

    The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments. PMID:23825163

  5. Inhibition of TGF-beta signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis.

    PubMed

    de Gouville, Anne-Charlotte; Boullay, Valerie; Krysa, Gael; Pilot, Julia; Brusq, Jean-Marie; Loriolle, Florence; Gauthier, Jean-Michel; Papworth, Stephen A; Laroze, Alain; Gellibert, Françoise; Huet, Stephane

    2005-05-01

    1 Chronic liver disease is characterized by an exacerbated accumulation of matrix, causing progressive fibrosis, which may lead to cirrhosis. Transforming growth factor beta (TGF-beta), a well-known profibrotic cytokine, transduces its signal through the ALK5 ser/thr kinase receptor, and increases transcription of different genes including PAI-1 and collagens. The identification of GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), an ALK5 inhibitor, allowed us to evaluate the therapeutic potential of inhibiting TGF-beta pathway in different models of liver disease. 2 A cellular assay was used to identify GW6604 as a TGF-beta signaling pathway inhibitor. This ALK5 inhibitor was then tested in a model of liver hepatectomy in TGF-beta-overexpressing transgenic mice, in an acute model of liver disease and in a chronic model of dimethylnitrosamine (DMN)-induced liver fibrosis. 3 In vitro, GW6604 inhibited autophosphorylation of ALK5 with an IC(50) of 140 nM and in a cellular assay inhibited TGF-beta-induced transcription of PAI-1 (IC(50): 500 nM). In vivo, GW6604 (40 mg kg(-1) p.o.) increased liver regeneration in TGF-beta-overexpressing mice, which had undergone partial hepatectomy. In an acute model of liver disease, GW6604 reduced by 80% the expression of collagen IA1. In a chronic model of DMN-induced fibrosis where DMN was administered for 6 weeks and GW6604 dosed for the last 3 weeks (80 mg kg(-1) p.o., b.i.d.), mortality was prevented and DMN-induced elevations of mRNA encoding for collagen IA1, IA2, III, TIMP-1 and TGF-beta were reduced by 50-75%. Inhibition of matrix genes overexpression was accompanied by reduced matrix deposition and reduction in liver function deterioration, as assessed by bilirubin and liver enzyme levels. 4 Our results suggest that inhibition of ALK5 could be an attractive new approach to treatment of liver fibrotic diseases by both preventing matrix deposition and promoting hepatocyte regeneration.

  6. Recent patents on alphavirus protein expression and vector production.

    PubMed

    Aranda, Alejandro; Ruiz-Guillen, Marta; Quetglas, Jose I; Bezunartea, Jaione; Casales, Erkuden; Smerdou, Cristian

    2011-12-01

    Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.

  7. Cell Cycle Programs of Gene Expression Control Morphogenetic Protein Localization

    PubMed Central

    Lord, Matthew; Yang, Melody C.; Mischke, Michelle; Chant, John

    2000-01-01

    Genomic studies in yeast have revealed that one eighth of genes are cell cycle regulated in their expression. Almost without exception, the significance of cell cycle periodic gene expression has not been tested. Given that many such genes are critical to cellular morphogenesis, we wanted to examine the importance of periodic gene expression to this process. The expression profiles of two genes required for the axial pattern of cell division, BUD3 and BUD10/AXL2/SRO4, are strongly cell cycle regulated. BUD3 is expressed close to the onset of mitosis. BUD10 is expressed in late G1. Through promotor-swap experiments, the expression profile of each gene was altered and the consequences examined. We found that an S/G2 pulse of BUD3 expression controls the timing of Bud3p localization, but that this timing is not critical to Bud3p function. In contrast, a G1 pulse of BUD10 expression plays a direct role in Bud10p localization and function. Bud10p, a membrane protein, relies on the polarized secretory machinery specific to G1 to be delivered to its proper location. Such a secretion-based targeting mechanism for membrane proteins provides cells with flexibility in remodeling their architecture or evolving new forms. PMID:11134078

  8. Expression and biochemical characterization of recombinant human epididymis protein 4.

    PubMed

    Hua, Ling; Liu, Yunhui; Zhen, Shuai; Wan, Deyou; Cao, Jiyue; Gao, Xin

    2014-10-01

    Whey acidic proteins (WAP) belong to a large gene family of antibacterial peptides that perform critical immune system functions. The function of human epididymis protein 4 (HE4), a 124-amino acid long polypeptide that has two whey acidic protein four-disulfide core (WFDC) domains, is not well studied. Here, a fusion gene encoding the HE4 protein fused to an IgG1 Fc domain was constructed. The recombinant HE4 protein was expressed as a secretory protein in Pichia pastoris and mammalian HEK293-F cells and was subsequently purified. Our data suggested that the HE4 protein produced by these two expression systems bound to both gram-negative and gram-positive bacteria, but demonstrated slightly inhibitory activity towards the growth of Staphylococcus aureus. Moreover, HE4 exhibited proteinase inhibitory activity towards trypsin, elastase, matrix metallopeptidase 9, and the secretory proteinases from Bacillus subtilis. The effects of glycosylation on the biochemical characterization of HE4 were also investigated. LC-ESI-MS glycosylation analysis showed that the high-mannose glycosylated form of HE4 expressed by P. pastoris has lower biological activity when compared to its complex-glycosylated form produced from HEK293-F cells. The implications of this are discussed, which may be provide theoretical basis for its important role in the development of cancer and innate immune system.

  9. Protein Co-Expression Network Analysis (ProCoNA)

    SciTech Connect

    Gibbs, David L.; Baratt, Arie; Baric, Ralph; Kawaoka, Yoshihiro; Smith, Richard D.; Orwoll, Eric S.; Katze, Michael G.; Mcweeney, Shannon K.

    2013-06-01

    Biological networks are important for elucidating disease etiology due to their ability to model complex high dimensional data and biological systems. Proteomics provides a critical data source for such models, but currently lacks robust de novo methods for network construction, which could bring important insights in systems biology. We have evaluated the construction of network models using methods derived from weighted gene co-expression network analysis (WGCNA). We show that approximately scale-free peptide networks, composed of statistically significant modules, are feasible and biologically meaningful using two mouse lung experiments and one human plasma experiment. Within each network, peptides derived from the same protein are shown to have a statistically higher topological overlap and concordance in abundance, which is potentially important for inferring protein abundance. The module representatives, called eigenpeptides, correlate significantly with biological phenotypes. Furthermore, within modules, we find significant enrichment for biological function and known interactions (gene ontology and protein-protein interactions). Biological networks are important tools in the analysis of complex systems. In this paper we evaluate the application of weighted co-expression network analysis to quantitative proteomics data. Protein co-expression networks allow novel approaches for biological interpretation, quality control, inference of protein abundance, a framework for potentially resolving degenerate peptide-protein mappings, and a biomarker signature discovery.

  10. Thyroid-Related Protein Expression in the Human Thymus

    PubMed Central

    Park, Do Joon; Jung, Kyeong Cheon

    2017-01-01

    Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall's corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS. PMID:28386277

  11. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    PubMed Central

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2013-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT’s reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT’s reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease. PMID:23012103

  12. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.

    PubMed

    Bird, Louise E; Rada, Heather; Verma, Anil; Gasper, Raphael; Birch, James; Jennions, Matthew; Lӧwe, Jan; Moraes, Isabel; Owens, Raymond J

    2015-01-06

    The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

  13. Using ion exchange chromatography to purify a recombinantly expressed protein.

    PubMed

    Duong-Ly, Krisna C; Gabelli, Sandra B

    2014-01-01

    Ion exchange chromatography (IEX) separates molecules by their surface charge, a property that can vary vastly between different proteins. There are two types of IEX, cation exhange and anion exchange chromatography. The protocol that follows was designed by the authors for anion exchange chromatography of a recombinantly expressed protein having a pI of 4.9 and containing two cysteine residues and one tryptophan residue, using an FPLC system. Prior to anion exchange, the protein had been salted out using ammonium sulfate precipitation and partially purified via hydrophobic interaction chromatography (see Salting out of proteins using ammonium sulfate precipitation and Use and Application of Hydrophobic Interaction Chromatography for Protein Purification). Slight modifications to this protocol may be made to accommodate both the protein of interest and the availability of equipment.

  14. Increased functional protein expression using nucleotide sequence features enriched in highly expressed genes in zebrafish.

    PubMed

    Horstick, Eric J; Jordan, Diana C; Bergeron, Sadie A; Tabor, Kathryn M; Serpe, Mihaela; Feldman, Benjamin; Burgess, Harold A

    2015-04-20

    Many genetic manipulations are limited by difficulty in obtaining adequate levels of protein expression. Bioinformatic and experimental studies have identified nucleotide sequence features that may increase expression, however it is difficult to assess the relative influence of these features. Zebrafish embryos are rapidly injected with calibrated doses of mRNA, enabling the effects of multiple sequence changes to be compared in vivo. Using RNAseq and microarray data, we identified a set of genes that are highly expressed in zebrafish embryos and systematically analyzed for enrichment of sequence features correlated with levels of protein expression. We then tested enriched features by embryo microinjection and functional tests of multiple protein reporters. Codon selection, releasing factor recognition sequence and specific introns and 3' untranslated regions each increased protein expression between 1.5- and 3-fold. These results suggested principles for increasing protein yield in zebrafish through biomolecular engineering. We implemented these principles for rational gene design in software for codon selection (CodonZ) and plasmid vectors incorporating the most active non-coding elements. Rational gene design thus significantly boosts expression in zebrafish, and a similar approach will likely elevate expression in other animal models.

  15. SPINK 1 Protein Expression and Prostate Cancer Progression

    PubMed Central

    Flavin, Richard; Pettersson, Andreas; Hendrickson, Whitney K.; Fiorentino, Michelangelo; Finn, Stephen; Kunz, Lauren; Judson, Gregory L.; Lis, Rosina; Bailey, Dyane; Fiore, Christopher; Nuttall, Elizabeth; Martin, Neil E.; Stack, Edward; Penney, Kathryn L.; Rider, Jennifer R.; Sinnott, Jennifer; Sweeney, Christopher; Sesso, Howard D.; Fall, Katja; Giovannucci, Edward; Kantoff, Philip; Stampfer, Meir; Loda, Massimo; Mucci, Lorelei A.

    2014-01-01

    Purpose SPINK1 over-expression has been described in prostate cancer and is linked with poor prognosis in many cancers. The objective of this study was to characterize the association between SPINK1 over-expression and prostate cancer specific survival. Experimental Design The study included 879 participants in the US Physicians’ Health Study and Health Professionals Follow–Up Study, diagnosed with prostate cancer (1983 – 2004) and treated by radical prostatectomy. Protein tumor expression of SPINK1 was evaluated by immunohistochemistry on tumor tissue microarrays. Results 74/879 (8%) prostate cancer tumors were SPINK1 positive. Immunohistochemical data was available for PTEN, p-Akt, pS6, stathmin, androgen receptor (AR) and ERG (as a measure of the TMPRSS2:ERG translocation). Compared to SPINK1 negative tumors, SPINK1 positive tumors showed higher PTEN and stathmin expression, and lower expression of AR (p<0.01). SPINK1 over-expression was seen in 47 of 427 (11%) ERG negative samples and in 19 of 427 (4%) ERG positive cases (p=0.0003). We found no significant associations between SPINK1 status and Gleason grade or tumor stage. There was no association between SPINK1 expression and biochemical recurrence (p=0.56). Moreover, there was no association between SPINK1 expression and prostate cancer mortality (there were 75 lethal cases of prostate cancer during a mean of 13.5 years follow-up [HR 0.71 (95% confidence interval 0.29–1.76)]). Conclusions Our results suggest that SPINK1 protein expression may not be a predictor of recurrence or lethal prostate cancer amongst men treated by radical prostatectomy. SPINK1 and ERG protein expression do not appear to be entirely mutually exclusive, as some previous studies have suggested. PMID:24687926

  16. Protein Expression of Proteasome Subunits in Elderly Patients with Schizophrenia

    PubMed Central

    Scott, Madeline R; Rubio, Maria D; Haroutunian, Vahram; Meador-Woodruff, James H

    2016-01-01

    The ubiquitin proteasome system (UPS) is a major regulator of protein processing, trafficking, and degradation. While protein ubiquitination is utilized for many cellular processes, one major function of this system is to target proteins to the proteasome for degradation. In schizophrenia, studies have found UPS transcript abnormalities in both blood and brain, and we have previously reported decreased protein expression of ubiquitin-associated proteins in brain. To test whether the proteasome is similarly dysregulated, we measured the protein expression of proteasome catalytic subunits as well as essential subunits from proteasome regulatory complexes in 14 pair-matched schizophrenia and comparison subjects in superior temporal cortex. We found decreased expression of Rpt1, Rpt3, and Rpt6, subunits of the 19S regulatory particle essential for ubiquitin-dependent degradation by the proteasome. Additionally, the α subunit of the 11S αβ regulatory particle, which enhances proteasomal degradation of small peptides and unfolded proteins, was also decreased. Haloperidol-treated rats did not have altered expression of these subunits, suggesting the changes we observed in schizophrenia are likely not due to chronic antipsychotic treatment. Interestingly, expression of the catalytic subunits of both the standard and immunoproteasome were unchanged, suggesting the abnormalities we observed may be specific to the complexed state of the proteasome. Aging has significant effects on the proteasome, and several subunits (20S β2, Rpn10, Rpn13, 11Sβ, and 11Sγ) were significantly correlated with subject age. These data provide further evidence of dysfunction of the ubiquitin-proteasome system in schizophrenia, and suggest that altered proteasome activity may be associated with the pathophysiology of this illness. PMID:26202105

  17. Enhancement of G Protein-Coupled Receptor Surface Expression

    PubMed Central

    Dunham, Jill H.; Hall, Randy A.

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate physiological responses to a diverse array of stimuli and are the molecular targets for numerous therapeutic drugs. GPCRs primarily signal from the plasma membrane, but when expressed in heterologous cells many GPCRs exhibit poor trafficking to the cell surface. Multiple approaches have been taken to enhance GPCR surface expression in heterologous cells, including addition/deletion of receptor sequences, co-expression with interacting proteins, and treatment with pharmacological chaperones. In addition to allowing for enhanced surface expression of certain GPCRs in heterologous cells, these approaches have also shed light on the control of GPCR trafficking in vivo and in some cases have led to new therapeutic approaches for treating human diseases that result from defects in GPCR trafficking. PMID:19679364

  18. Expression, delivery and function of insecticidal proteins expressed by recombinant baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal po...

  19. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  20. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  1. p53 and MDM2 protein expression in actinic cheilitis.

    PubMed

    de Freitas, Maria da Conceição Andrade; Ramalho, Luciana Maria Pedreira; Xavier, Flávia Caló Aquino; Moreira, André Luis Gomes; Reis, Sílvia Regina Almeida

    2008-01-01

    Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

  2. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  3. AB223. Expression of tight junction proteins in rat vagina

    PubMed Central

    Oh, Kyung Jin; Lee, Hyun-Suk; Chung, Ho Suck; Ahn, Kyu Youn; Park, Kwangsung

    2014-01-01

    Aim Tight junction plays a role in apical cell-to-cell adhesion and epithelial polarity. In this study, we investigated the expression of tight junction proteins, such as Claudin-1, zonula occludens (ZO)-1, junction adhesion molecule (JAM)-A, and occludin in rat vagina. Methods Female Sprague-dawley rats (230-240 g, n=20) were divided into two groups: control (n=10) and bilateral ovariectomy (n=10). The expression and cellular localization of claudin-1, ZO-1, JAM-A, and occludin were determined in each group by immunohistochemistry and Western blot. Results Immunolabeling of ZO-1 was mainly expressed in the capillaries and venules of the vagina. Claudin-1, JAM-A, and occludin were expressed in the epithelium of the vagina. The immunoreactivity and protein expression of claudin-1 was significantly decreased in the ovariectomy group compared with the control group. Conclusions Our results suggest that tight junction proteins may have an important role in the vagina. Further studies are needed to clarify the role of each tight junction protein on vaginal lubrication.

  4. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  5. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  6. Metachronous neuroblastoma in an infant with germline translocation resulting in partial trisomy 2p: a role for ALK?

    PubMed

    Morgenstern, Daniel A; Soh, Shui Yen; Stavropoulos, Dimitri J; Bowdin, Sarah; Baruchel, Sylvain; Malkin, David; Meyn, M Stephen; Irwin, Meredith S

    2014-04-01

    A male infant with dysmorphic features, intestinal malrotation, and developmental delay was found to have a germline translocation resulting in partial trisomy 2p and monosomy 16p. At 3 and 9 months of age, he developed localized neuroblastoma in each adrenal, which was managed with surgical resection. Tumors were MYCN non-amplified, with 2p copy gain consistent with the germline translocation. The potential increased risk of neuroblastoma associated with partial trisomy 2p is discussed in the context of this and previously published cases, and may be due to increased constitutional expression of MYCN and ALK genes, both located within the duplicated 2p region.

  7. Fe-S Proteins that Regulate Gene Expression

    PubMed Central

    Mettert, Erin L.; Kiley, Patricia J.

    2014-01-01

    Iron-sulfur (Fe-S) cluster containing proteins that regulate gene expression are present in most organisms. The innate chemistry of their Fe-S cofactors makes these regulatory proteins ideal for sensing environmental signals, such as gases (e.g. O2 and NO), levels of Fe and Fe-S clusters, reactive oxygen species, and redox cycling compounds, to subsequently mediate an adaptive response. Here we review the recent findings that have provided invaluable insight into the mechanism and function of these highly significant Fe-S regulatory proteins. PMID:25450978

  8. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  9. Expression of Aequorea green fluorescent protein in plant cells.

    PubMed

    Hu, W; Cheng, C L

    1995-08-07

    The coding region of the green fluorescent protein (GFP) from Aequorea victoria has been fused to the cauliflower mosaic virus 35S promoter and introduced into maize leaf protoplasts. Transient expression of GFP was observed. In addition, the coding region of GFP was fused to an Arabidopsis heat shock promoter and co-transformed with another construct in which GFP has been replaced with chloramphenicol acetyltransferase (CAT). The heat-induced expression of GFP in maize protoplasts parallels that of CAT. While GFP was expressed in both dark-grown and green maize leaf protoplasts, no green fluorescence was observed in similarly transformed Arabidopsis protoplasts.

  10. Shear induced collateral artery growth modulated by endoglin but not by ALK1

    PubMed Central

    Seghers, Leonard; de Vries, Margreet R; Pardali, Evangelia; Hoefer, Imo E; Hierck, Beerend P; ten Dijke, Peter ten; Goumans, Marie Jose; Quax, Paul HA

    2012-01-01

    Transforming growth factor-beta (TGF-β) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-β receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis. PMID:22436015

  11. Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

    PubMed Central

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-01-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

  12. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  13. New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK

    PubMed Central

    Di Paolo, Daniela; Yang, D.; Pastorino, Fabio; Emionite, Laura; Cilli, Michele; Daga, Antonio; Destefanis, Elisa; Di Fiore, Annarita; Piaggio, Francesca; Brignole, Chiara; Xu, Xiaobao; Liang, Chris; Gibbons, James

    2015-01-01

    Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors. PMID:26299615

  14. New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK.

    PubMed

    Di Paolo, Daniela; Yang, D; Pastorino, Fabio; Emionite, Laura; Cilli, Michele; Daga, Antonio; Destafanis, Elisa; Di Fiore, Annarita; Piaggio, Francesca; Brignole, Chiara; Xu, Xiaobao; Liang, Chris; Gibbons, James; Ponzoni, Mirco; Perri, Patrizia

    2015-10-06

    Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.

  15. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  16. Salicylic acid enhances Staphylococcus aureus extracellular adhesin protein expression.

    PubMed

    Alvarez, Lucía P; Barbagelata, María S; Cheung, Ambrose L; Sordelli, Daniel O; Buzzola, Fernanda R

    2011-11-01

    One of the virulence factors required by Staphylococcus aureus at the early stages of infection is Eap, a secreted adhesin that binds many host proteins and is upregulated by the two-component regulatory system saeRS. The S. aureus Newman strain harbors a mutation in saeS that is thought to be responsible for the high level of Eap expression in this strain. This study was designed to ascertain whether salicylic acid (SAL) affects the expression of Eap and the internalization of S. aureus into epithelial cells. The strain Newman treated with SAL exhibited increased levels of eap transcription and protein expression. Furthermore, SAL treatment increased the eap promoter activity. SAL treatment enhanced Eap expression in the Newman and in other S. aureus strains that do not carry the mutation in saeS. Internalization of S. aureus eap and sae mutants into the MAC-T epithelial cells was significantly decreased compared with the wild-type counterparts. In conclusion, we demonstrated that a low concentration of SAL increased S. aureus Eap expression possibly due to enhancement of sae. SAL may create the conditions for S. aureus persistence in the host, not only by decreasing the capsular polysaccharide expression as shown before, but also by enhancing Eap expression.

  17. Cementum attachment protein/protein-tyrosine phosphotase-like member A is not expressed in teeth.

    PubMed

    Schild, Christof; Beyeler, Michael; Lang, Niklaus P; Trueb, Beat

    2009-02-01

    Cementum is a highly specialized connective tissue that covers tooth roots. The only cementum-specific protein described to date is the cementum attachment protein (CAP). A putative sequence for CAP was established from a cDNA clone isolated from a human cementifying fibroma cDNA library. This sequence overlaps with a phosphatase-like protein in muscle termed the protein-tyrosine phosphatase-like member A (PTPLA). To clarify the nature of CAP/PTPLA, we cloned the homologous rat protein and determined its sequence. The rat protein shared 94% sequence identity with the human protein. On Northern blots containing RNA from various rat tissues of different developmental stages, the cDNA hybridized to an mRNA expressed in heart and skeletal muscle but not in teeth. These results were confirmed by real-time PCR. Thus, the sequence deposited in public databanks under the name 'cementum attachment protein' does not represent genuine CAP.

  18. Selection of soluble protein expression constructs: the experimental determination of protein domain boundaries.

    PubMed

    Dyson, Michael R

    2010-08-01

    Proteins can contain multiple domains each of which is capable of possessing a separate independent function and three-dimensional structure. It is often useful to clone and express individual protein domains to study their biochemical properties and for structure determination. However, the annotated domain boundaries in databases such as Pfam or SMART are not always accurate. The present review summarizes various strategies for the experimental determination of protein domain boundaries.

  19. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  20. Computational codon optimization of synthetic gene for protein expression

    PubMed Central

    2012-01-01

    Background The construction of customized nucleic acid sequences allows us to have greater flexibility in gene design for recombinant protein expression. Among the various parameters considered for such DNA sequence design, individual codon usage (ICU) has been implicated as one of the most crucial factors affecting mRNA translational efficiency. However, previous works have also reported the significant influence of codon pair usage, also known as codon context (CC), on the level of protein expression. Results In this study, we have developed novel computational procedures for evaluating the relative importance of optimizing ICU and CC for enhancing protein expression. By formulating appropriate mathematical expressions to quantify the ICU and CC fitness of a coding sequence, optimization procedures based on genetic algorithm were employed to maximize its ICU and/or CC fitness. Surprisingly, the in silico validation of the resultant optimized DNA sequences for Escherichia coli, Lactococcus lactis, Pichia pastoris and Saccharomyces cerevisiae suggests that CC is a more relevant design criterion than the commonly considered ICU. Conclusions The proposed CC optimization framework can complement and enhance the capabilities of current gene design tools, with potential applications to heterologous protein production and even vaccine development in synthetic biotechnology. PMID:23083100

  1. Expression Trend of Selected Ribosomal Protein Genes in Nasopharyngeal Carcinoma

    PubMed Central

    Ma, Xiang-Ru; Sim, Edmund Ui-Hang; Ling, Teck-Yee; Tiong, Thung-Sing; Subramaniam, Selva Kumar; Khoo, Alan Soo-Beng

    2012-01-01

    Background: Ribosomal proteins are traditionally associated with protein biosynthesis until recent studies that implicated their extraribosomal functions in human diseases and cancers. Our previous studies using GeneFishing™ DEG method and microarray revealed underexpression of three ribosomal protein genes, RPS26, RPS27, and RPL32 in cancer of the nasopharynx. Herein, we investigated the expression pattern and nucleotide sequence integrity of these genes in nasopharyngeal carcinoma to further delineate their involvement in tumourigenesis. The relationship of expression level with clinicopathologic factors was also statistically studied. Methods: Quantitative Polymerase Chain Reaction was performed on nasopharyngeal carcinoma and their paired normal tissues. Expression and sequence of these three genes were analysed. Results: All three ribosomal protein genes showed no significant difference in transcript expressions and no association could be established with clinicopathologic factors studied. No nucleotide aberrancy was detected in the coding regions of these genes. Conclusion: There is no early evidence to substantiate possible involvement of RPS26, RPS27, and RPL32 genes in NPC tumourigenesis. PMID:23613646

  2. Expression and detection of LINE-1 ORF-encoded proteins.

    PubMed

    Dai, Lixin; LaCava, John; Taylor, Martin S; Boeke, Jef D

    2014-01-01

    LINE-1 (L1) elements are endogenous retrotransposons active in mammalian genomes. The L1 RNA is bicistronic, encoding two non-overlapping open reading frames, ORF1 and ORF2, whose protein products (ORF1p and ORF2p) bind the L1 RNA to form a ribonucleoprotein (RNP) complex that is presumed to be a critical retrotransposition intermediate. However, ORF2p is expressed at a significantly lower level than ORF1p; these differences are thought to be controlled at the level of translation, due to a low frequency ribosome reinitiation mechanism controlling ORF2 expression. As a result, while ORF1p is readily detectable, ORF2p has previously been very challenging to detect in vitro and in vivo. To address this, we recently tested several epitope tags fused to the N- or C-termini of the ORF proteins in an effort to enable robust detection and affinity purification from native (L1RP) and synthetic (ORFeus-Hs) L1 constructs. An analysis of tagged RNPs from both L1RP and ORFeus-Hs showed similar host-cell-derived protein interactors. Our observations also revealed that the tag sequences affected the retrotransposition competency of native and synthetic L1s differently although they encode identical ORF proteins. Unexpectedly, we observed apparently stochastic expression of ORF2p within seemingly homogenous L1-expressing cell populations.

  3. Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

    PubMed Central

    Pochon, Nathalie; Dementin, Sébastien; Hivin, Patrick; Boutigny, Sylvain; Rioux, Jean-Baptiste; Salvi, Daniel; Seigneurin-Berny, Daphné; Richaud, Pierre; Joyard, Jacques; Pignol, David; Sabaty, Monique; Desnos, Thierry; Pebay-Peyroula, Eva; Darrouzet, Elisabeth; Vernet, Thierry; Rolland, Norbert

    2011-01-01

    Background Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. Methodology/Principal Findings The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. Conclusions/Significance Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein. PMID:22216205

  4. ALK and ROS1 as targeted therapy paradigms and clinical implications to overcome crizotinib resistance.

    PubMed

    Ye, Mingxiang; Zhang, Xinxin; Li, Nan; Zhang, Yong; Jing, Pengyu; Chang, Ning; Wu, Jianxiong; Ren, Xinling; Zhang, Jian

    2016-03-15

    During the past decade, more than 10 targetable oncogenic driver genes have been validated in non-small cell lung cancer (NSCLC). Anaplastic lymphoma kinase (ALK) and ROS1 kinase are two new driver genes implicated in ALK- and ROS1-rearranged NSCLC. Inhibition of ALK and ROS1 by crizotinib has been reported to be highly effective and well tolerated in these patients. However, resistance to crizotinib emerges years after treatment, and increasing efforts have been made to overcome this issue. Here, we review the biology of ALK and ROS1 and their roles in cancer progression. We also summarize the ongoing and completed clinical trials validating ALK and ROS1 as targets for cancer treatment. In the last section of the review, we will discuss the molecular mechanisms of crizotinib resistance and focus approaches to overcome it. This review describes an exciting new area of research and may provide new insights for targeted cancer therapies.

  5. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    SciTech Connect

    Raymond, Amy; Lovell, Scott; Lorimer, Don; Walchli, John; Mixon, Mark; Wallace, Ellen; Thompkins, Kaitlin; Archer, Kimberly; Burgin, Alex; Stewart, Lance

    2009-12-01

    With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38{alpha}), viral polymerase (HCV NS5B), and bacterial structural protein (FtsZ) were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  6. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  7. Optimization of translation profiles enhances protein expression and solubility.

    PubMed

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  8. A characterization of structural proteins expressed by Bombyx mori bidensovirus.

    PubMed

    Lü, Peng; Xing, Yali; Hu, Zhaoyang; Yang, Yanhua; Pan, Ye; Chen, Kangmin; Zhu, Feifei; Zhou, Yajing; Chen, Keping; Yao, Qin

    2017-03-01

    Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5kb (viral DNA 1, VD1) and 6.0kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22-24nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism.

  9. Raman microscopy of bladder cancer cells expressing green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Mandair, Gurjit S.; Han, Amy L.; Keller, Evan T.; Morris, Michael D.

    2016-11-01

    Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

  10. Expression of glutamine metabolism-related proteins in thyroid cancer

    PubMed Central

    Kim, Hye Min; Lee, Yu Kyung; Koo, Ja Seung

    2016-01-01

    Purpose This study aimed to investigate the expression of glutamine metabolism-related protein in tumor and stromal compartments among the histologic subtypes of thyroid cancer. Results GLS1 and GDH expression in tumor and stromal compartments were the highest in AC than in other subtypes. Tumoral ASCT2 expression was higher in MC but lower in FC (p < 0.001). In PTC, tumoral GLS1 and tumoral GDH expression was higher in the conventional type than in the follicular variant (p = 0.043 and 0.001, respectively), and in PTC with BRAF V600E mutation than in PTC without BRAF V600E mutation (p<0.001). Stromal GDH positivity was the independent factor associated with short overall survival (hazard ratio: 21.48, 95% confidence interval: 2.178-211.8, p = 0.009). Methods We performed tissue microarrays with 557 thyroid cancer cases (papillary thyroid carcinoma [PTC]: 344, follicular carcinoma [FC]: 112, medullary carcinoma [MC]: 70, poorly differentiated carcinoma [PDC]: 23, and anaplastic carcinoma [AC]: 8) and 152 follicular adenoma (FA) cases. We performed immunohistochemical staining of glutaminolysis-related proteins (glutaminase 1 [GLS1], glutamate dehydrogenase [GDH], and amino acid transporter-2 [ASCT-2]). Conclusion Glutamine metabolism-related protein expression differed among the histologic subtypes of thyroid cancer. PMID:27447554

  11. Methods and constructs for expression of foreign proteins in photosynthetic organisms

    DOEpatents

    Laible, Philip D.; Hanson, Deborah K.

    2002-01-01

    A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

  12. Transient expression and cellular localization of recombinant proteins in cultured insect cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heterologous protein expression systems are used for production of recombinant proteins, interpretation of cellular trafficking/localization, and for the determination of biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for ...

  13. Dark proteins: effect of inclusion body formation on quantification of protein expression.

    PubMed

    Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R

    2008-09-01

    Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used

  14. Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins

    PubMed Central

    Bokhove, Marcel; Sadat Al Hosseini, Hamed; Saito, Takako; Dioguardi, Elisa; Gegenschatz-Schmid, Katharina; Nishimura, Kaoru; Raj, Isha; de Sanctis, Daniele; Han, Ling; Jovine, Luca

    2016-01-01

    We present a strategy to obtain milligrams of highly post-translationally modified eukaryotic proteins, transiently expressed in mammalian cells as rigid or cleavable fusions with a mammalianized version of bacterial maltose-binding protein (mMBP). This variant was engineered to combine mutations that enhance MBP solubility and affinity purification, as well as provide crystal-packing interactions for increased crystallizability. Using this cell type-independent approach, we could increase the expression of secreted and intracellular human proteins up to 200-fold. By molecular replacement with MBP, we readily determined five novel high-resolution structures of rigid fusions of targets that otherwise defied crystallization. PMID:26850170

  15. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup

  16. Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly.

    PubMed

    Sawatsky, Bevan; Bente, Dennis A; Czub, Markus; von Messling, Veronika

    2016-05-01

    The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

  17. Precision medicine in NSCLC and pathology: how does ALK fit in the pathway?

    PubMed

    Kerr, K M; López-Ríos, F

    2016-09-01

    The evolution of personalised medicine in lung cancer has dramatically impacted diagnostic pathology. Current challenges centre on the growing demands placed on small tissue samples by molecular diagnostic techniques. In this review, expert recommendations are provided regarding successful identification of anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung cancer (NSCLC). Steps to correctly process and conserve tumour tissue during diagnostic testing are essential to ensure tissue availability. For example, storing extra tissue sections ready for molecular diagnostic steps allows faster testing and preserves tissue. Fluorescence in situ hybridisation (FISH) is commonly used to detect ALK rearrangements, with most laboratories favouring screening by immunohistochemistry followed by a confirmatory FISH assay. Reverse transcription-polymerase chain reaction can also identify ALK fusion gene mRNA transcripts but can be limited by the quality of RNA and the risk that rare fusion variants may not be captured. Next-generation sequencing (NGS) technology has recently provided an alternative method for detecting ALK rearrangements. While current experience is limited, NGS is set to become the most efficient approach as an increasing number of genetic abnormalities is required to be tested. Upfront, reflex testing for ALK gene rearrangement should become routine as ALK tyrosine kinase inhibitor therapy moves into the first-line setting. Guidelines recommend that EGFR and ALK tests are carried out in parallel on all confirmed and potential adenocarcinomas, and this is more efficient in terms of tissue usage and testing turnaround time for both of these actionable gene alterations. The practice of sequential testing is not recommended. Identification of ALK rearrangements is now essential for the diagnosis of NSCLC, underpinned by the benefits of ALK inhibitors. As scientific understanding and diagnostic technology develops, ALK testing will continue to be an

  18. Grape seed extract inhibits VEGF expression via reducing HIF-1α protein expression

    PubMed Central

    Lu, Jianming; Zhang, Keqiang; Chen, Shiuan; Wen, Wei

    2009-01-01

    Grape seed extract (GSE) is a widely consumed dietary supplement that has antitumor activity. Here, we have investigated the inhibitory effect of GSE on the expression of vascular endothelial growth factor (VEGF) and the mechanism underlying this action. We found that GSE inhibited VEGF messenger RNA (mRNA) and protein expression in U251 human glioma cells and MDA-MB-231 human breast cancer cells. GSE inhibited transcriptional activation of the VEGF gene through reducing protein but not mRNA expression of hypoxia-inducible factor (HIF) 1α. The inhibitory effect of GSE on HIF-1α expression was mainly through inhibiting HIF-1α protein synthesis rather than promoting protein degradation. Consistent with this result, GSE-suppressed phosphorylation of several important components involved in HIF-1α protein synthesis, such as Akt, S6 kinase and S6 protein. Furthermore, in the MDA-MB-231 tumor, we found that GSE treatment inhibited the expression of VEGF and HIF-1α and the phosphorylation of S6 kinase without altering the subcellular localization of HIF-1α, correlating with reduced vessel density and tumor size. Depletion of polyphenol with polyvinylpyrrolidone abolished the inhibitory activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the inhibitory activity. Taken together, our results indicate that GSE inhibits VEGF expression by reducing HIF-1α protein synthesis through blocking Akt activation. This finding provides new insight into the mechanisms of anticancer activity of GSE and reveals a novel molecular mechanism underlying the antiangiogenic action of GSE. PMID:19131542

  19. Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

    PubMed Central

    Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

    2015-01-01

    Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

  20. Morphine Withdrawal Modifies Prion Protein Expression in Rat Hippocampus

    PubMed Central

    Mattei, Vincenzo; Martellucci, Stefano; Santilli, Francesca; Manganelli, Valeria; Garofalo, Tina; Candelise, Niccolò; Caruso, Alessandra; Sorice, Maurizio; Scaccianoce, Sergio

    2017-01-01

    The hippocampus is a vulnerable brain structure susceptible to damage during aging and chronic stress. Repeated exposure to opioids may alter the brain so that it functions normally when the drugs are present, thus, a prolonged withdrawal might lead to homeostatic changes headed for the restoration of the physiological state. Abuse of morphine may lead to Reacting Oxygen Species-induced neurodegeneration and apoptosis. It has been proposed that during morphine withdrawal, stress responses might be responsible, at least in part, for long-term changes of hippocampal plasticity. Since prion protein is involved in both, Reacting Oxygen Species mediated stress responses and synaptic plasticity, in this work we investigate the effect of opiate withdrawal in rats after morphine treatment. We hypothesize that stressful stimuli induced by opiate withdrawal, and the subsequent long-term homeostatic changes in hippocampal plasticity, might modulate the Prion protein expression. Our results indicate that abstinence from the opiate induced a time-dependent and region-specific modification in Prion protein content, indeed during morphine withdrawal a selective unbalance of hippocampal Prion Protein is observable. Moreover, Prion protein overexpression in hippocampal tissue seems to generate a dimeric structure of Prion protein and α-cleavage at the hydrophobic domain. Stress factors or toxic insults can induce cytosolic dimerization of Prion Protein through the hydrophobic domain, which in turn, it stimulates the α-cleavage and the production of neuroprotective Prion protein fragments. We speculate that this might be the mechanism by which stressful stimuli induced by opiate withdrawal and the subsequent long-term homeostatic changes in hippocampal plasticity, modulate the expression and the dynamics of Prion protein. PMID:28081197

  1. Expression and Localization of Plant Protein Disulfide Isomerase.

    PubMed Central

    Shorrosh, B. S.; Subramaniam, J.; Schubert, K. R.; Dixon, R. A.

    1993-01-01

    A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules. PMID:12231974

  2. The Bright Fluorescent Protein mNeonGreen Facilitates Protein Expression Analysis In Vivo

    PubMed Central

    Hostettler, Lola; Grundy, Laura; Käser-Pébernard, Stéphanie; Wicky, Chantal; Schafer, William R.; Glauser, Dominique A.

    2017-01-01

    The Green Fluorescent Protein (GFP) has been tremendously useful in investigating cell architecture, protein localization, and protein function. Recent developments in transgenesis and genome editing methods now enable working with fewer transgene copies and, consequently, with physiological expression levels. However, lower signal intensity might become a limiting factor. The recently developed mNeonGreen protein is a brighter alternative to GFP in vitro. The goal of the present study was to determine how mNeonGreen performs in vivo in Caenorhabditis elegans—a model used extensively for fluorescence imaging in intact animals. We started with a side-by-side comparison between cytoplasmic forms of mNeonGreen and GFP expressed in the intestine, and in different neurons, of adult animals. While both proteins had similar photostability, mNeonGreen was systematically 3–5 times brighter than GFP. mNeonGreen was also used successfully to trace endogenous proteins, and label specific subcellular compartments such as the nucleus or the plasma membrane. To further demonstrate the utility of mNeonGreen, we tested transcriptional reporters for nine genes with unknown expression patterns. While mNeonGreen and GFP reporters gave overall similar expression patterns, low expression tissues were detected only with mNeonGreen. As a whole, our work establishes mNeonGreen as a brighter alternative to GFP for in vivo imaging in a multicellular organism. Furthermore, the present research illustrates the utility of mNeonGreen to tag proteins, mark subcellular regions, and describe new expression patterns, particularly in tissues with low expression. PMID:28108553

  3. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  4. Tools to cope with difficult-to-express proteins.

    PubMed

    Saccardo, Paolo; Corchero, José Luís; Ferrer-Miralles, Neus

    2016-05-01

    The identification of DNA coding sequences contained in the genome of many organisms coupled to the use of high throughput approaches has fueled the field of recombinant protein production. Apart from basic research interests, the growing relevance of this field is highlighted by the global sales of the top ten biopharmaceuticals on the market, which exceeds the trillion USD in a steady increasing tendency. Therefore, the demand of biological compounds seems to have a long run on the market. One of the most popular expression systems is based on Escherichia coli cells which apart from being cost-effective counts with a large selection of resources. However, a significant percentage of the genes of interest are not efficiently expressed in this system, or the expressed proteins are accumulated within aggregates, degraded or lacking the desired biological activity, being finally discarded. In some instances, expressing the gene in a homologous expression system might alleviate those drawbacks but then the process usually increases in complexity and is not as cost-effective as the prokaryotic systems. An increasing toolbox is available to approach the production and purification of those difficult-to-express proteins, including different expression systems, promoters with different strengths, cultivation media and conditions, solubilization tags and chaperone coexpression, among others. However, in most cases, the process follows a non-integrative trial and error strategy with discrete success. This review is focused on the design of the whole process by using an integrative approach, taken into account the accumulated knowledge of the pivotal factors that affect any of the key processes, in an attempt to rationalize the efforts made in this appealing field.

  5. Expression of low molecular weight proteins in patients with leukaemia.

    PubMed

    Sheikh, N; Abid, R; Qureshi, A W; Basheer, T

    2012-06-01

    The current study is conducted to observe the differences in the level of low molecular weight proteins in the sera of patients with leukaemia in comparison to healthy subjects (control group). The sera of patients with leukaemia showed 15 peaks in the densitometric curve in comparison to the seven peaks of the controls. The peaks in the experimental samples that coincide with those in the control were of 134.14, 113.15, 76.06, 63.25, 48.07, 22.85 and 16.47 kDa molecular weights, respectively. Most of the new peaks appeared between the proteins of molecular weight 36-29 kDa in the experimental groups. Mean density of the 134.14 kDa protein band showed an increase in the protein in experimental groups I and II only whereas 113.15 and 22.85 kDa protein were increased in all experimental groups of patients with leukaemia. The expression of 76.06 and 63.25 kDa protein fraction was downregulated in the patients with leukaemia. A decline in the level of the protein of 48.07 kDa was observed in patients with leukaemia except in group I. Unlike the other protein fractions, the level of the protein of 16.47 kDa was significantly (p < 0.05) increased with a maximum density in group II. Intergroup experimental) comparison revealed an increasing pattern of 95.44 and 89.21 kDa with maximum level in group III sera. However the protein fractions of 38.07 and 34.94 kDa varied in the serum with maximum density in Group IV Protein fractions of 32.92 and 31.24 kDa were expressed in all age groups of patients with leukaemia with a maximum density in group III whereas the percentage densities of 14.42 and 13.56 kDa protein were quite different. This preliminary study will provide a basis to study the role of different proteins in patients with leukaemia.

  6. Profile of Ventana ALK (D5F3) companion diagnostic assay for non-small-cell lung carcinomas.

    PubMed

    Conde, Esther; Hernandez, Susana; Prieto, Mario; Martinez, Rebeca; Lopez-Rios, Fernando

    2016-06-01

    The development of several ALK inhibitors means that the importance of accurately identifying ALK-positive lung cancer has never been greater. Therefore, it is crucial that ALK testing assays become more standardized. The aim of this review is to comment on the recently FDA-approved VENTANA ALK (D5F3) Companion Diagnostic (CDx) Assay. This kit provides high sensitivity and specificity for the detection of ALK rearrangements and seamless integration into the laboratory workflow, with a fully automated analytical phase and fast interpretation. The use of controls increases the sensitivity and specificity and a dichotomous scoring approach enhances reproducibility.

  7. Expression and serological reactivity of hemorrhagic enteritis virus hexon protein.

    PubMed

    Lobová, Dana; Celer, Vladimír

    2016-05-01

    The aim of this work was to express the recombinant hexon protein of the hemorrhagic enteritis virus, to establish the diagnostic value of this protein for serological detection of antibodies in turkey serum samples and to assess seroprevalence of the infection in the Czech Republic. The N' terminal part of the hexon protein was expressed in a bacterial expression system and used as an antigen in an ELISA test for the detection of hemorrhagic enteritis virus specific antibodies in turkey sera. Validation of the test was performed by comparison with a commercially available ELISA test. Serological reactivity was assessed on a panel of 126 turkey sera by a newly developed ELISA test. Serum samples were taken from turkey farms with the history of hemorrhagic enteritis virus infection, from farms with animals free of infection, and from turkey farms following vaccination. Both ELISA kits gave identical results (100 %) with the tested sera. ELISA based on the recombinant hexon protein thus proved useful and cheaper for detection of antibodies in turkey flocks infected with the hemorrhagic enteritis virus.

  8. Prion protein expression in bovine podocytes and extraglomerular mesangial cells.

    PubMed

    Amselgruber, W M; Steffl, M; Didier, A; Märtlbauer, E; Pfaff, E; Büttner, M

    2006-06-01

    The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

  9. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  10. Protein Phosphatase-1 Regulates Expression of Neuregulin-1

    PubMed Central

    Ammosova, Tatiana; Washington, Kareem; Rotimi, Jamie; Kumari, Namita; Smith, Kahli A.; Niu, Xiaomei; Jerebtsova, Marina; Nekhai, Sergei

    2016-01-01

    Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner. PMID:27918433

  11. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  12. Tissue-Specific Protein Expression in Plant Mitochondria.

    PubMed Central

    Conley, C. A.; Hanson, M. R.

    1994-01-01

    Although the physiological role of plant mitochondria is thought to vary in different tissues at progressive stages of development, there has been little documentation that the complement of mitochondrial proteins is altered in different plant organs. Because the phenomenon of cytoplasmic male sterility suggests an unusual function for mitochondria in floral buds, we examined the tissue-specific expression of mitochondrial proteins in petunia buds at several stages of development, using both fertile and cytoplasmic male sterile plants. On tissue prints of cryostat-sectioned buds, antibodies recognizing subunit A of the mitochondrial ATPase (ATPA) localized very differently from antibodies recognizing subunit II of the cytochrome oxidase (COXII), which indicated that mitochondria in the same tissue could differentially express mitochondrially encoded proteins. The petunia cytoplasmic male sterility-associated fused (pcf) gene encodes a protein that colocalized with ATPA and the nuclear-encoded mitochondrial alternative oxidase (AOA) in sporogenous tissues, where little COXII protein was found. These overlapping and differential localization patterns may provide clues to the molecular mechanism of cytoplasmic male sterility. PMID:12244222

  13. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  14. Eosinophil granule proteins expressed in ocular cicatricial pemphigoid

    PubMed Central

    Heiligenhaus, A.; Schaller, J.; Mauss, S.; Engelbrecht, S.; Dutt, J.; Foster, C; Steuhl, K.

    1998-01-01

    BACKGROUND—Blister formation and tissue damage in bullous pemphigoid have been attributed to the release of eosinophil granule proteins—namely, to eosinophil derived cationic protein (ECP) and major basic protein (MBP). In the present investigation these eosinophil granule proteins were studied in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP).
METHODS—Conjunctival biopsy specimens obtained from patients with subacute (n=8) or chronic conjunctival disease (n=13) were analysed histologically and immunohistochemically using antibodies directed against EG1 (stored and secreted ECP), EG2 (secreted ECP), MBP, CD45 (common leucocyte antigen), CD3 (pan T cell marker), and HLA-DR (class II antigen).
RESULTS—Subepithelial mononuclear cells, mast cells, and neutrophils were detected in all specimens. The number of mononuclear cells, neutrophils, CD45+ cells, CD3+ cells, and the HLA-DR expression were significantly higher in the subacute than in the chronic disease group. Some eosinophils were found in specimens from five of eight patients with subacute OCP, but in none of the patients with chronic disease. The eosinophil granule proteins (ECP and MBP) were found in the epithelium and substantia propria in patients with subacute conjunctivitis.
CONCLUSIONS—Subepithelial cell infiltration in the conjunctiva greatly differs between subacute and chronic ocular cicatricial pemphigoid specimens. The findings suggest that eosinophil granule proteins may participate in tissue damage in acute phase of inflammation in OCP.

 Keywords: ocular cicatricial pemphigoid; conjunctivitis; eosinophil derived cationic protein; major basic protein PMID:9602632

  15. Somatostatin regulates tight junction proteins expression in colitis mice.

    PubMed

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P<0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P<0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P<0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P<0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression.

  16. Somatostatin regulates tight junction proteins expression in colitis mice

    PubMed Central

    Li, Xiao; Wang, Qian; Xu, Hua; Tao, Liping; Lu, Jing; Cai, Lin; Wang, Chunhui

    2014-01-01

    Tight junction plays a critical role in intestinal defence. The alteration and perturbation of tight junction proteins could induce intestine barrier damage, and lead to the malabsorption of electrolytes and water. Previous studies had showed that colonic infection and inflammation could lead to the alteration of tight junction function, and somatostatin could protect intestinal epithelia. Thus, this study could explore that whether somatostatin could regulate tight junction in colitis mice. Colitis mice with diarrhea were induced by Citrobacter rodentium (CR) and Dextran sulfate sodium (DSS). In CR infected model, cladudin-1 and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); after octreotide treatment, claudin-1 and claudin-3 expression significantly increased compared with untreated CR infected mice (P < 0.05). In DSS colitis model, occludin and claudin-3 expression significantly decreased compared with the control mice (P < 0.05); and octreotide treatment could only significantly upregulate claudin-3 expression compared with untreated DSS colitis mice (P < 0.05). To testify our results in vivo, we repeated the models in caco-2 cells by exposed with enteropathogenic Escherichia coli (E. Coli) and Tumor necrosis factor α (TNF-α). The results in vitro were consistent with in vivo study. The results suggested that somatostatin play a role in intestinal barrier protection by modulating tight junction proteins expression. PMID:24966923

  17. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  18. Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

    PubMed

    Welsch, Norma; Homuth, Georg; Schweder, Thomas

    2015-08-01

    In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

  19. BMP-7 PROTEIN EXPRESSION IS DOWNREGULATED IN HUMAN DIABETIC NEPHROPATHY.

    PubMed

    Ivanac-Janković, Renata; Ćorić, Marijana; Furić-Čunko, Vesna; Lovičić, Vesna; Bašić-Jukić, Nikolina; Kes, Petar

    2015-06-01

    Bone morphogenetic protein-7 (BMP-7) is expressed in all parts of the normal kidney parenchyma, being highest in the epithelium of proximal tubules. It protects kidney against acute and chronic injury, inflammation and fibrosis. Diabetic nephropathy is the leading cause of chronic kidney disease, and is characterized by decreased expression of BMP-7. The aim of our study was to analyze whether the expression of BMP-7 is significantly changed in advanced stages of human diabetic nephropathy. Immunohistochemical analysis of the expression of BMP-7 was performed on archival material of 30 patients that underwent renal biopsy and had confirmed diagnosis of diabetic nephropathy. Results showed that BMP-7 was differently expressed in the cytoplasm of epithelial cells of proximal tubules and podocytes among all stages of diabetic nephropathy. At early stages of diabetic nephropathy, BMP-7 was strongly positive in proximal tubules and podocytes, while low expression was recorded in the majority of samples at advanced stages. In conclusion, increased expression of BMP-7 at initial stages of diabetic nephropathy with subsequent decrease at advanced stage highlights the role of BMP-7 in the protection of kidney structure and function. Further investigations should be focused on disturbances of BMP-7 receptors and signaling pathways in patients with diabetic nephropathy.

  20. Epithelial membrane protein 1 expression in ovarian serous tumors.

    PubMed

    Demirag, Guzin Gonullu; Kefeli, Mehmet; Kemal, Yasemin; Yucel, Idris

    2016-03-01

    The present study aimed to analyze the clinical significance of epithelial membrane protein 1 (EMP1) expression in ovarian serous tumors. A total of 84 cases of ovarian serous tumor (50 patients with malignant ovarian serous tumors and 34 patients with borderline and benign serous tumors) were retrospectively analyzed. Differences in the expression levels of EMP1 between the malignant and non-malignant tumor groups were evaluated by immunohistochemical staining. In addition, the association between EMP1 expression and prognostic factors in malignant ovarian serous tumors was investigated. The expression levels of EMP1 were significantly reduced in all the 50 malignant ovarian serous tumors, compared with the 34 non-malignant ovarian serous tumors (P<0.000). Reduced expression of EMP1 was correlated with high grade (P=0.009) and stage (P<0.000) of malignant tumors. EMP1 expression was not observed to be correlated with any other investigated parameters, including surgery, type of operation and chemotherapy response (P>0.005). These results indicated that EMP1 may have a significant role as a negative regulator in ovarian serous tumors, and reduced EMP1 expression in serous tumors may be associated with increased disease severity.

  1. Generation of transgenic dogs that conditionally express green fluorescent protein.

    PubMed

    Kim, Min Jung; Oh, Hyun Ju; Park, Jung Eun; Kim, Geon A; Hong, So Gun; Jang, Goo; Kwon, Mo Sun; Koo, Bon Chul; Kim, Teoan; Kang, Sung Keun; Ra, Jeong Chan; Ko, Chemyong; Lee, Byeong Chun

    2011-06-01

    We report the creation of a transgenic dog that conditionally expresses eGFP (enhanced green fluorescent protein) under the regulation of doxycycline. Briefly, fetal fibroblasts infected with a Tet-on eGFP vector were used for somatic cell nuclear transfer. Subsequently reconstructed oocytes were transferred to recipients. Three clones having transgenes were born and one dog was alive. The dog showed all features of inducible expression of eGFP upon doxycycline administration, and successful breeding resulted in eGFP-positive puppies, confirming stable insertion of the transgene into the genome. This inducible dog model will be useful for a variety of medical research studies.

  2. Personalized treatment in advanced ALK-positive non-small cell lung cancer: from bench to clinical practice

    PubMed Central

    Passaro, Antonio; Lazzari, Chiara; Karachaliou, Niki; Spitaleri, Gianluca; Pochesci, Alessia; Catania, Chiara; Rosell, Rafael; de Marinis, Filippo

    2016-01-01

    The discovery of anaplastic lymphoma kinase (ALK) gene rearrangements and the development of tyrosine kinase inhibitors (TKI) that target them have achieved unprecedented success in the management of patients with ALK-positive non-small cell lung cancer (NSCLC). Despite the high efficacy of crizotinib, the first oral ALK TKI approved for the treatment of ALK-positive NSCLC, almost all patients inevitably develop acquired resistance, showing disease progression in the brain or in other parenchymal sites. Second- or third-generation ALK TKIs have shown to be active in crizotinib-pretreated or crizotinib-naïve ALK-positive patients, even in those with brain metastases. In this review, the current knowledge regarding ALK-positive NSCLC, focusing on the biology of the disease and the available therapeutic options are discussed. PMID:27799783

  3. Downregulation of hepatocyte nuclear factor-4{alpha} and its role in regulation of gene expression by TGF-{beta} in mammary epithelial cells

    SciTech Connect

    Ishikawa, Fumihiro; Nose, Kiyoshi; Shibanuma, Motoko

    2008-06-10

    We found that a specific isoform of hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), HNF-4{alpha}8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-{beta}. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4{alpha}8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4{alpha}8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4{alpha}8 in TGF-{beta} signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-{beta}1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-{beta}1 was attenuated by forced expression of HNF-4{alpha}8. HMGA2 has recently emerged as a transcriptional organizer of TGF-{beta} signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4{alpha} as a new target downstream of HMGA2 and assigned a new role to HNF-4{alpha} in the TGF-{beta} signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

  4. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  5. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    PubMed

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet.

  6. A novel Patient Derived Tumorgraft model with TRAF1-ALK Anaplastic Large Cell Lymphoma translocation

    PubMed Central

    Abate, Francesco; Todaro, Maria; van der Krogt, Jo-Anne; Boi, Michela; Landra, Indira; Machiorlatti, Rodolfo; Tabbo’, Fabrizio; Messana, Katia; Barreca, Antonella; Novero, Domenico; Gaudiano, Marcello; Aliberti, Sabrina; Di Giacomo, Filomena; Tousseyn, Thomas; Lasorsa, Elena; Crescenzo, Ramona; Bessone, Luca; Ficarra, Elisa; Acquaviva, Andrea; Rinaldi, Andrea; Ponzoni, Maurilio; Longo, Dario Livio; Aime, Silvio; Cheng, Mangeng; Ruggeri, Bruce; Piccaluga, Pier Paolo; Pileri, Stefano; Tiacci, Enrico; Falini, Brunangelo; Pera-Gresely, Benet; Cerchietti, Leandro; Iqbal, Javeed; Chan, Wing C; Shultz, Leonard D.; Kwee, Ivo; Piva, Roberto; Wlodarska, Iwona; Rabadan, Raul; Bertoni, Francesco; Inghirami, Giorgio

    2016-01-01

    Although Anaplastic Large Cell Lymphomas (ALCL) carrying Anaplastic Lymphoma Kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human Patient Derived Tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and of NFkB pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells lacking PRDM1/Blimp-1 and with c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to down-regulation of p50/p52 and lymphoma growth inhibition. Moreover a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Moreover, a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, but the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable to validate the role of druggable molecules, predict therapeutic responses and are helpful tools for the implementation of patient specific therapies. PMID:25533804

  7. Alectinib: a novel second generation anaplastic lymphoma kinase (ALK) inhibitor for overcoming clinically-acquired resistance

    PubMed Central

    Song, Zilan; Wang, Meining; Zhang, Ao

    2015-01-01

    The development of inhibitors for the tyrosine anaplastic lymphoma kinase (ALK) has advanced rapidly, driven by biology and medicinal chemistry. The first generation ALK inhibitor crizotinib was granted US FDA approval with only four years of preclinical and clinical testing. Although this drug offers significant clinical benefit to the ALK-positive patients, resistance has been developed through a variety of mechanisms. In addition to ceritinib, alectinib is another second-generation ALK inhibitor launched in 2014 in Japan. This drug has a unique chemical structure bearing a 5H-benzo[b]carbazol-11(6H)-one structural scaffold with an IC50 value of 1.9 nmol/L, and is highly potent against ALK bearing the gatekeeper mutation L1196M with an IC50 of 1.56 nmol/L. In the clinic, alectinib is highly efficacious in treatment of ALK-positive non-small cell lung cancer (NSCLC), and retains potency to combat crizotinib-resistant ALK mutations L1196M, F1174L, R1275Q and C1156Y. PMID:26579422

  8. French multicentric validation of ALK rearrangement diagnostic in 547 lung adenocarcinomas.

    PubMed

    Lantuejoul, Sylvie; Rouquette, Isabelle; Blons, Hélène; Le Stang, Nolwenn; Ilie, Marius; Begueret, Hugues; Grégoire, Valerie; Hofman, Paul; Gros, Audrey; Garcia, Stephane; Monhoven, Nathalie; Devouassoux-Shisheboran, Mojgan; Mansuet-Lupo, Audrey; Thivolet, Françoise; Antoine, Martine; Vignaud, Jean-Michel; Penault-Llorca, Frederique; Galateau-Sallé, Françoise; McLeer-Florin, Anne

    2015-07-01

    Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening. Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded. 140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (<15%). FISH was not interpretable for seven cases. ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p<0.0001). 13 cases were IHC negative but FISH ≥15%, including six cases with FISH between 15% and 20%; eight were IHC positive with FISH between 10% and 14%. Sensitivity and specificity for 5A4 and D5F3 were 87% and 92%, and 89% and 76%, respectively. False-negative IHC, observed in 2.4% of cases, dropped to 1.3% for FISH >20%. Variants were undetected in 36% of ALK tumours. Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.

  9. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  10. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  11. Disposable bioreactors for inoculum production and protein expression.

    PubMed

    Eibl, Regine; Löffelholz, Christian; Eibl, Dieter

    2014-01-01

    Disposable bioreactors have been increasingly implemented over the past ten years. This relates to both R & D and commercial manufacture, in particular, in animal cell-based processes. Among the numerous disposable bioreactors which are available today, wave-mixed bag bioreactors and stirred bioreactors are predominant. Whereas wave-mixed bag bioreactors represent the system of choice for inoculum production, stirred systems are often preferred for protein expression. For this reason, the authors present protocols instructing the reader how to use the wave-mixed BIOSTAT CultiBag RM 20 L for inoculum production and the stirred UniVessel SU 2 L for recombinant protein production at benchtop scale. All methods described are based on a Chinese hamster ovary (CHO) suspension cell line expressing the human placental secreted alkaline phosphatase (SEAP).

  12. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  13. Expression data on liver metabolic pathway genes and proteins

    PubMed Central

    Raja Gopal Reddy, Mooli; Pavan Kumar, Chodisetti; Mahesh, Malleswarapu; Sravan Kumar, Manchiryala; Jeyakumar, Shanmugam M.

    2016-01-01

    Here, we present the expression data on various metabolic pathways of liver with special emphasize on lipid and carbohydrate metabolism and long chain polyunsaturated fatty acid (PUFA) synthesis, both at gene and protein levels. The data were obtained to understand the effect of vitamin A deficiency on the expression status (both gene and protein levels) of some of the key factors involved in lipogenesis, fatty acid oxidation, triglyceride secretion, long chain PUFA, resolvin D1 synthesis, glucose transport and glycogen synthesis of liver, using modern biology tools, such as quantitative real-time PCR (RT-PCR) and immunoblotting techniques. This data article provides the supporting evidence to the article “Vitamin A deficiency suppresses high fructose-induced triglyceride synthesis and elevates resolvin D1 levels” [1] and therefore, these data may be referred back, for comprehensive understanding and interpretations and for future studies. PMID:26909377

  14. Hippocampal expression of the calcium sensor protein visinin-like protein-1 in schizophrenia.

    PubMed

    Bernstein, Hans-Gert; Braunewell, Karl-Heinz; Spilker, Christina; Danos, Peter; Baumann, Bruno; Funke, Sieglinde; Diekmann, Silvia; Gundelfinger, Eckart D; Bogerts, Bernhard

    2002-03-25

    Hippocampal cytoarchitectural abnormalities may be part of the cerebral substrate of schizophrenia. Amongst the chemical components being abnormal in brains of schizophrenics are altered calcium concentrations and reduced expression of the neurotrophin receptor, trkB. We studied by immunohistochemical methods the distribution of visinin-like protein-1 (VILIP-1), which is a calcium sensor protein and at the same time a trkB mRNA binding protein, in hippocampi of nine schizophrenic patients and nine matched control subjects. In normal hippocampi VILIP-1 immunoreactivity was found in multiple pyramidal cells and interneurons. A portion of VILIP-1 immunoreactive interneurons co-express calretinin (60%) and parvalbumin (<10%). In schizophrenics fewer pyramidal cells but more interneurons were immunostained. Our data point to an involvement of the protein in the altered hippocampal circuitry in schizophrenia.

  15. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

    PubMed

    Allen, Robert S; Tilbrook, Kimberley; Warden, Andrew C; Campbell, Peter C; Rolland, Vivien; Singh, Surinder P; Wood, Craig C

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

  16. Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix

    PubMed Central

    Allen, Robert S.; Tilbrook, Kimberley; Warden, Andrew C.; Campbell, Peter C.; Rolland, Vivien; Singh, Surinder P.; Wood, Craig C.

    2017-01-01

    The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia. PMID:28316608

  17. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression

    PubMed Central

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-01-01

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm’-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment. PMID:27231931

  18. Mutational Analysis of the Rift Valley Fever Virus Glycoprotein Precursor Proteins for Gn Protein Expression.

    PubMed

    Phoenix, Inaia; Lokugamage, Nandadeva; Nishiyama, Shoko; Ikegami, Tetsuro

    2016-05-24

    The Rift Valley fever virus (RVFV) M-segment encodes the 78 kD, NSm, Gn, and Gc proteins. The 1st AUG generates the 78 kD-Gc precursor, the 2nd AUG generates the NSm-Gn-Gc precursor, and the 3rd AUG makes the NSm'-Gn-Gc precursor. To understand biological changes due to abolishment of the precursors, we quantitatively measured Gn secretion using a reporter assay, in which a Gaussia luciferase (gLuc) protein is fused to the RVFV M-segment pre-Gn region. Using the reporter assay, the relative expression of Gn/gLuc fusion proteins was analyzed among various AUG mutants. The reporter assay showed efficient secretion of Gn/gLuc protein from the precursor made from the 2nd AUG, while the removal of the untranslated region upstream of the 2nd AUG (AUG2-M) increased the secretion of the Gn/gLuc protein. Subsequently, recombinant MP-12 strains encoding mutations in the pre-Gn region were rescued, and virological phenotypes were characterized. Recombinant MP-12 encoding the AUG2-M mutation replicated slightly less efficiently than the control, indicating that viral replication is further influenced by the biological processes occurring after Gn expression, rather than the Gn abundance. This study showed that, not only the abolishment of AUG, but also the truncation of viral UTR, affects the expression of Gn protein by the RVFV M-segment.

  19. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1^E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein's flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1^E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1^E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1^E4, these kinases regulate one of

  20. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression.

    PubMed

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M

    2010-06-01

    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  1. Phylogeny and expression of carbonic anhydrase-related proteins

    PubMed Central

    2010-01-01

    Background Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate α-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR. Results We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain

  2. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  3. Beyond ALK-RET, ROS1 and other oncogene fusions in lung cancer

    PubMed Central

    Nakaoku, Takashi; Tsuta, Koji; Tsuchihara, Katsuya; Matsumoto, Shingo; Yoh, Kiyotaka; Goto, Koichi

    2015-01-01

    Fusions of the RET and ROS1 protein tyrosine kinase oncogenes with several partner genes were recently identified as new targetable genetic aberrations in cases of non-small cell lung cancer (NSCLC) lacking activating EGFR, KRAS, ALK, BRAF, or HER2 oncogene aberrations. RET and ROS1 fusion-positive tumors are mainly observed in young, female, and/or never smoking patients. Studies based on in vitro and in vivo (i.e., mouse) models and studies of several fusion-positive patients indicate that inhibiting the kinase activity of the RET and ROS1 fusion proteins is a promising therapeutic strategy. Accordingly, there are several ongoing clinical trials aimed at examining the efficacy of tyrosine kinase inhibitors (TKIs) against RET and ROS1 proteins in patients with fusion-positive lung cancer. Other gene fusions (NTRK1, NRG1, and FGFR1/2/3) that are targetable by existing TKIs have also been identified in NSCLCs. Options for personalized lung cancer therapy will be increased with the help of multiplex diagnosis systems able to detect multiple druggable gene fusions. PMID:25870798

  4. Smac mimetic LCL161 supports neuroblastoma chemotherapy in a drug class-dependent manner and synergistically interacts with ALK inhibitor TAE684 in cells with ALK mutation F1174L

    PubMed Central

    Najem, Safiullah; Langemann, Doerte; Appl, Birgit; Trochimiuk, Magdalena; Hundsdoerfer, Patrick; Reinshagen, Konrad; Eschenburg, Georg

    2016-01-01

    Neuroblastoma is the most common extracranial solid tumor during infancy and childhood. Outcome of high-risk and late-stage disease remains poor despite intensive treatment regimens. Suppressing inhibitor of apoptosis proteins (IAPs) using Smac mimetics (SM) significantly sensitizes neuroblastoma (NB) cells for chemotherapy, however strongly dependent on the cytotoxic drug combined with SM. Therefore, a systematic analysis of the impact of SM in combination with different classes of chemotherapeutics was of crucial importance. Treatment of NB cell lines with SM LCL161 and vinca alkaloids revealed a strong synergistic inhibition of proliferation and significant induction of apoptosis in virtually all established and de novo NB cell lines (n=8). In contrast, combination of anthracyclines or topoisomerase inhibitors with LCL161 showed a synergism for single drugs and/or cell lines only. Furthermore, we could show that insensibility to LCL161-mediated sensitization for chemotherapeutics is associated with aberrant activation of anaplastic lymphoma kinase (ALK) by common mutation F1174L. Inhibition of ALK using TAE684 is able to overcome this resistance in a synergistic fashion, a finding that could be highly relevant for improvement of neuroblastoma therapy. PMID:27655666

  5. Smac mimetic LCL161 supports neuroblastoma chemotherapy in a drug class-dependent manner and synergistically interacts with ALK inhibitor TAE684 in cells with ALK mutation F1174L.

    PubMed

    Najem, Safiullah; Langemann, Doerte; Appl, Birgit; Trochimiuk, Magdalena; Hundsdoerfer, Patrick; Reinshagen, Konrad; Eschenburg, Georg

    2016-11-08

    Neuroblastoma is the most common extracranial solid tumor during infancy and childhood.Outcome of high-risk and late-stage disease remains poor despite intensive treatment regimens.Suppressing inhibitor of apoptosis proteins (IAPs) using Smac mimetics (SM) significantly sensitizes neuroblastoma (NB) cells for chemotherapy, however strongly dependent on the cytotoxic drug combined with SM.Therefore, a systematic analysis of the impact of SM in combination with different classes of chemotherapeutics was of crucial importance. Treatment of NB cell lines with SM LCL161 and vinca alkaloids revealed a strong synergistic inhibition of proliferation and significant induction of apoptosis in virtually all established and de novo NB cell lines (n=8).In contrast, combination of anthracyclines or topoisomerase inhibitors with LCL161 showed a synergism for single drugs and/or cell lines only.Furthermore, we could show that insensibility to LCL161-mediated sensitization for chemotherapeutics is associated with aberrant activation of anaplastic lymphoma kinase (ALK) by common mutation F1174L. Inhibition of ALK using TAE684 is able to overcome this resistance in a synergistic fashion, a finding that could be highly relevant for improvement of neuroblastoma therapy.

  6. An inducible expression system for high-level expression of recombinant proteins in slow growing mycobacteria.

    PubMed

    Leotta, Lisa; Spratt, Joanne M; Kong, Carlyn U; Triccas, James A

    2015-09-01

    A novel protein expression vector utilising the inducible hspX promoter of Mycobacterium tuberculosis was constructed and evaluated in this study. High-level induction of three mycobacterial antigens, comprising up to 9% of bacterial sonicate, was demonstrated in recombinant Mycobacterium bovis BCG when grown under low-oxygen tension, which serves to enhance hspX promoter activity. Recombinant proteins were efficiently purified from bacterial lysates in a soluble form by virtue of a C-terminal 6-histidine tag. Purification of the immunodominant M. tuberculosis Ag85B antigen using this system resulted in a recombinant protein that stimulated significant IFN-γ release from Ag85B-reactive T cells generated after vaccination of mice with an Ag85B-expressing vaccine. Further, the M. tuberculosis L-alanine dehydrogenase (Ald) protein purified from recombinant BCG displayed strong enzymatic activity in recombinant form. This study demonstrated that high levels of native-like recombinant mycobacterial proteins can be produced in mycobacterial hosts, and this may aid the analysis of mycobacterial protein function and the development of new treatments.

  7. Structure and Expression of a Novel Compact Myelin Protein - Small VCP-Interacting Protein (SVIP)

    PubMed Central

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-01-01

    SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes. PMID:24055875

  8. Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins

    PubMed Central

    2013-01-01

    Background Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. Results The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. Conclusion The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags

  9. Expression of P53 protein after exposure to ionizing radiation

    NASA Astrophysics Data System (ADS)

    Salazar, A. M.; Salvador, C.; Ruiz-Trejo, C.; Ostrosky, P.; Brandan, M. E.

    2001-10-01

    One of the most important tumor suppressor genes is p53 gene, which is involved in apoptotic cell death, cell differentiation and cell cycle arrest. The expression of p53 gene can be evaluated by determining the presence of P53 protein in cells using Western Blot assay with a chemiluminescent method. This technique has shown variabilities that are due to biological factors. Film developing process can influence the quality of the p53 bands obtained. We irradiated tumor cell lines and human peripheral lymphocytes with 137Cs and 60Co gamma rays to standardize irradiation conditions, to compare ionizing radiation with actinomycin D and to reduce the observed variability of P53 protein induction levels. We found that increasing radiation doses increase P53 protein induction while it decreases viability. We also conclude that ionizing radiation could serve as a positive control for Western Blot analysis of protein P53. In addition, our results show that the developing process may play an important role in the quality of P53 protein bands and data interpretation.

  10. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    PubMed

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  11. Protein expression in salivary glands of rats with streptozotocin diabetes

    PubMed Central

    Mednieks, Maija I; Szczepanski, Andrew; Clark, Brett; Hand, Arthur R

    2009-01-01

    Diabetes mellitus (DM) is a widespread disease with high morbidity and health care costs. An experimental animal model was employed, using morphological and biochemical methods, to investigate the effects of DM on the expression and compartmentation of salivary gland proteins. The distribution of proline-rich proteins (PRP), submandibular mucin (Muc10) and the regulatory (RI and RII) subunits of cyclic AMP-dependent protein kinase type I and type II was determined in the parotid and submandibular (SMG) glands of rats treated with streptozotocin. Quantitative immunocytochemistry of secretory granules in diabetic glands revealed decreases of 30% for PRP in both the parotid and SMG, and a 40% decrease in Muc10 in the SMG. Immunogold labelling showed that RII decreased in nuclei and the cytoplasm in diabetic acinar cells while labelling of secretory granules was similar in control and diabetic parotid. Electrophoresis and Western blotting of tissue extracts of two secretory proteins showed that the response to DM and insulin treatment was gland specific: PRP showed little change in the SMG, but decreased in the parotid in DM and was partially restored after insulin treatment. Photoaffinity labelling showed only RI present in the SMG and mainly RII in the parotid. The results of this and previous studies demonstrating highly specific changes in salivary protein expression indicate that the oral environment is significantly altered by DM, and that oral tissues and their function can be compromised. These findings may provide a basis for future studies to develop tests using saliva for diabetic status or progression in humans. PMID:19659899

  12. Quantum mechanics/molecular mechanics study on the oxygen binding and substrate hydroxylation step in AlkB repair enzymes.

    PubMed

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-07

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N(1) -methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)-oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ- and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.

  13. Common and specific signatures of gene expression and protein-protein interactions in autoimmune diseases.

    PubMed

    Tuller, T; Atar, S; Ruppin, E; Gurevich, M; Achiron, A

    2013-03-01

    The aim of this study is to understand intracellular regulatory mechanisms in peripheral blood mononuclear cells (PBMCs), which are either common to many autoimmune diseases or specific to some of them. We incorporated large-scale data such as protein-protein interactions, gene expression and demographical information of hundreds of patients and healthy subjects, related to six autoimmune diseases with available large-scale gene expression measurements: multiple sclerosis (MS), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), Crohn's disease (CD), ulcerative colitis (UC) and type 1 diabetes (T1D). These data were analyzed concurrently by statistical and systems biology approaches tailored for this purpose. We found that chemokines such as CXCL1-3, 5, 6 and the interleukin (IL) IL8 tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In addition, the anti-apoptotic gene BCL3, interferon-γ (IFNG), and the vitamin D receptor (VDR) gene physically interact with significantly many genes that tend to be differentially expressed in PBMCs of patients with the analyzed autoimmune diseases. In general, similar cellular processes tend to be differentially expressed in PBMC in the analyzed autoimmune diseases. Specifically, the cellular processes related to cell proliferation (for example, epidermal growth factor, platelet-derived growth factor, nuclear factor-κB, Wnt/β-catenin signaling, stress-activated protein kinase c-Jun NH2-terminal kinase), inflammatory response (for example, interleukins IL2 and IL6, the cytokine granulocyte-macrophage colony-stimulating factor and the B-cell receptor), general signaling cascades (for example, mitogen-activated protein kinase, extracellular signal-regulated kinase, p38 and TRK) and apoptosis are activated in most of the analyzed autoimmune diseases. However, our results suggest that in each of the analyzed diseases, apoptosis and chemotaxis are activated via

  14. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    PubMed

    Miranda, Alberto; Pericuesta, Eva; Ramírez, Miguel Ángel; Gutierrez-Adan, Alfonso

    2011-04-04

    Cellular prion protein (PRNP) is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB) differentiation in mouse Prnp-null (KO) and WT embryonic stem cell (ESC) lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC) markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5) in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel) and SPRN (Shadoo), whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  15. DNA bending and a flip-out mechanism for base excision by the helix–hairpin–helix DNA glycosylase, Escherichia coli AlkA

    PubMed Central

    Hollis, Thomas; Ichikawa, Yoshitaka; Ellenberger, Tom

    2000-01-01

    The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 Å crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1–azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1–azaribose in the enzyme active site suggests an SN1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin–helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner. PMID:10675345

  16. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity.

  17. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

    PubMed Central

    Cheng, Kevin P.; Kiernan, Elizabeth A.; Eliceiri, Kevin W.; Williams, Justin C.; Watters, Jyoti J.

    2016-01-01

    Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS. PMID:26883795

  18. The analysis of ALK gene rearrangement by fluorescence in situ hybridization in non-small cell lung cancer patients

    PubMed Central

    Krawczyk, Paweł Adam; Ramlau, Rodryg Adam; Szumiło, Justyna; Kozielski, Jerzy; Kalinka-Warzocha, Ewa; Bryl, Maciej; Knopik-Dąbrowicz, Alina; Spychalski, Łukasz; Szczęsna, Aleksandra; Rydzik, Ewelina; Milanowski, Janusz

    2013-01-01

    Introduction ALK gene rearrangement is observed in a small subset (3–7%) of non-small cell lung cancer (NSCLC) patients. The efficacy of crizotinib was shown in lung cancer patients harbouring ALK rearrangement. Nowadays, the analysis of ALK gene rearrangement is added to molecular examination of predictive factors. Aim of the study The frequency of ALK gene rearrangement as well as the type of its irregularity was analysed by fluorescence in situ hybridisation (FISH) in tissue samples from NSCLC patients. Material and methods The ALK gene rearrangement was analysed in 71 samples including 53 histological and 18 cytological samples. The analysis could be performed in 56 cases (78.87%), significantly more frequently in histological than in cytological materials. The encountered problem with ALK rearrangement diagnosis resulted from the scarcity of tumour cells in cytological samples, high background fluorescence noises and fragmentation of cell nuclei. Results The normal ALK copy number without gene rearrangement was observed in 26 (36.62%) patients ALK gene polysomy without gene rearrangement was observed in 25 (35.21%) samples while in 3 (4.23%) samples ALK gene amplification was found. ALK gene rearrangement was observed in 2 (2.82%) samples from males, while in the first case the rearrangement coexisted with ALK amplification. In the second case, signet-ring tumour cells were found during histopathological examination and this patient was successfully treated with crizotinib with partial remission lasting 16 months. Conclusions FISH is a useful technique for ALK gene rearrangement analysis which allows us to specify the type of gene irregularities. ALK gene examination could be performed in histological as well as cytological (cellblocks) samples, but obtaining a reliable result in cytological samples depends on the cellularity of examined materials. PMID:24592134

  19. Recombinant protein expression in Lactococcus lactis using the P170 expression system.

    PubMed

    Jørgensen, Casper M; Vrang, Astrid; Madsen, Søren M

    2014-02-01

    The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L(-1).

  20. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  1. Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F.

    PubMed

    Shaw, Alice T; Friboulet, Luc; Leshchiner, Ignaty; Gainor, Justin F; Bergqvist, Simon; Brooun, Alexei; Burke, Benjamin J; Deng, Ya-Li; Liu, Wei; Dardaei, Leila; Frias, Rosa L; Schultz, Kate R; Logan, Jennifer; James, Leonard P; Smeal, Tod; Timofeevski, Sergei; Katayama, Ryohei; Iafrate, A John; Le, Long; McTigue, Michele; Getz, Gad; Johnson, Ted W; Engelman, Jeffrey A

    2016-01-07

    In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

  2. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  3. Transforming Growth Factor β1 (TGF-β1) Activates Hepcidin mRNA Expression in Hepatocytes.

    PubMed

    Chen, Simeng; Feng, Teng; Vujić Spasić, Maja; Altamura, Sandro; Breitkopf-Heinlein, Katja; Altenöder, Jutta; Weiss, Thomas S; Dooley, Steven; Muckenthaler, Martina U

    2016-06-17

    The hepatic hormone hepcidin is the master regulator of systemic iron homeostasis. Its expression level is adjusted to alterations in iron levels, inflammatory cues, and iron requirements for erythropoiesis. Bone morphogenetic protein 6 (BMP6) contributes to the iron-dependent control of hepcidin. In addition, TGF-β1 may stimulate hepcidin mRNA expression in murine hepatocytes and human leukocytes. However, receptors and downstream signaling proteins involved in TGF-β1-induced hepcidin expression are still unclear. Here we show that TGF-β1 treatment of mouse and human hepatocytes, as well as ectopic expression of TGF-β1 in mice, increases hepcidin mRNA levels. The hepcidin response to TGF-β1 depends on functional TGF-β1 type I receptor (ALK5) and TGF-β1 type II receptor (TβRII) and is mediated by a noncanonical mechanism that involves Smad1/5/8 phosphorylation. Interestingly, increasing availability of canonical Smad2/3 decreases TGF-β1-induced hepcidin regulation, whereas the BMP6-hepcidin signal was enhanced, indicating a signaling component stoichiometry-dependent cross-talk between the two pathways. Although ALK2/3-dependent hepcidin activation by BMP6 can be modulated by each of the three hemochromatosis-associated proteins: HJV (hemojuvelin), HFE (hemochromatosis protein), and TfR2 (transferrin receptor 2), these proteins do not control the ALK5-mediated hepcidin response to TGF-β1. TGF-β1 mRNA levels are increased in mouse models of iron overload, indicating that TGF-β1 may contribute to hepcidin synthesis under these conditions. In conclusion, these data demonstrate that a complex regulatory network involving TGF-β1 and BMP6 may control the sensing of systemic and/or hepatic iron levels.

  4. Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

    PubMed Central

    Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

    2014-01-01

    Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

  5. Structure and expression of a novel compact myelin protein – Small VCP-interacting protein (SVIP)

    SciTech Connect

    Wu, Jiawen; Peng, Dungeng; Voehler, Markus; Sanders, Charles R.; Li, Jun

    2013-10-11

    Highlights: •SVIP (small p97/VCP-interacting protein) co-localizes with myelin basic protein (MBP) in compact myelin. •We determined that SVIP is an intrinsically disordered protein (IDP). •The helical content of SVIP increases dramatically during its interaction with negatively charged lipid membrane. •This study provides structural insight into interactions between SVIP and myelin membranes. -- Abstract: SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.

  6. Brevibacillus expression system: host-vector system for efficient production of secretory proteins.

    PubMed

    Mizukami, Makoto; Hanagata, Hiroshi; Miyauchi, Akira

    2010-04-01

    Brevibacillus expression system is an effective bacterial expression system for secretory proteins. The host bacterium, Brevibacillus choshinensis, a gram-positive bacterium, has strong capacity to secrete a large amount of proteins (approximately 30 g/L), which mostly consist of cell wall protein. A host-vector system that utilizes such high expression capacity has been constructed for the production of secretory proteins and tested for various heterologous proteins, including cytokines, enzymes, antigens, and adjuvants.

  7. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    PubMed

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  8. Transgenic expression of therapeutic proteins in Arabidopsis thaliana seed.

    PubMed

    Nykiforuk, Cory L; Boothe, Joseph G

    2012-01-01

    The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.

  9. Expression and putative role of mitochondrial transport proteins in cancer.

    PubMed

    Lytovchenko, Oleksandr; Kunji, Edmund R S

    2017-03-22

    Cancer cells undergo major changes in energy and biosynthetic metabolism. One of them is the Warburg effect, in which pyruvate is used for fermentation rather for oxidative phosphorylation. Another major one is their increased reliance on glutamine, which helps to replenish the pool of Krebs cycle metabolites used for other purposes, such as amino acid or lipid biosynthesis. Mitochondria are central to these alterations, as the biochemical pathways linking these processes run through these organelles. Two membranes, an outer and inner membrane, surround mitochondria, the latter being impermeable to most organic compounds. Therefore, a large number of transport proteins are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. Since the transport steps are relatively slow, it is expected that many of these transport steps are altered when cells become cancerous. In this review, changes in expression and regulation of these transport proteins are discussed as well as the role of the transported substrates.

  10. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus.

    PubMed

    Masseguin, C; Corcoran, M; Carcenac, C; Daunton, N G; Güell, A; Verkman, A S; Gabrion, J

    2000-03-01

    Aquaporin-1 (AQP1) is a water channel expressed abundantly at the apical pole of choroidal epithelial cells. The protein expression was quantified by immunocytochemistry and confocal microscopy in adult rats adapted to altered gravity. AQP1 expression was decreased by 64% at the apical pole of choroidal cells in rats dissected 5.5-8 h after a 14-day spaceflight. AQP1 was significantly overexpressed in rats readapted for 2 days to Earth's gravity after an 11-day flight (48% overshoot, when compared with the value measured in control rats). In a ground-based model that simulates some effects of weightlessness and alters choroidal structures and functions, apical AQP1 expression was reduced by 44% in choroid plexus from rats suspended head down for 14 days and by 69% in rats suspended for 28 days. Apical AQP1 was rapidly enhanced in choroid plexus of rats dissected 6 h after a 14-day suspension (57% overshoot, in comparison with control rats) and restored to the control level when rats were dissected 2 days after the end of a 14-day suspension. Decreases in the apical expression of choroidal AQP1 were also noted in rats adapted to hypergravity in the NASA 24-ft centrifuge: AQP1 expression was reduced by 47% and 85% in rats adapted for 14 days to 2 G and 3 G, respectively. AQP1 is downregulated in the apical membrane of choroidal cells in response to altered gravity and is rapidly restored after readaptation to normal gravity. This suggests that water transport, which is partly involved in the choroidal production of cerebrospinal fluid, might be decreased during spaceflight and after chronic hypergravity.

  11. Expression of S-100 protein in renal cell neoplasms.

    PubMed

    Lin, Fan; Yang, Wannian; Betten, Mark; Teh, Bin Tean; Yang, Ximing J

    2006-04-01

    Polyclonal antibody to S-100 protein has been routinely applied for initial screening of various types of tumors, including, melanocytic tumors and neurogenic tumors. S-100 protein has been shown to have a broad distribution in human tissues, including renal tubules. The potential utility of S-100 protein in renal cell neoplasms has not been extensively investigated. Using an EnVision-Horseradish Peroxidase (HRP; Dako, Carpinteria, Calif) kit, we evaluated the diagnostic value of S-100 protein on tissue microarray sections from 175 cases of renal epithelial neoplasm (145 primary renal neoplasms and 30 metastatic renal cell carcinomas) and 24 non-neoplastic renal tissues. Immunohistochemical stains for pancytokeratin, HMB-45, and Mart-1 were also performed. Western blot using the same antibody (anti-S-100 protein) was performed on 10 cases of renal cell neoplasm. The results demonstrated that nuclear and cytoplasmic staining pattern for S-100 protein was observed in 56 (69%) of 81 conventional (clear cell) renal cell carcinomas (RCCs), 10 (30%) of 33 papillary RCCs, 1 (6%) of 16 ChRCCs, and 13 (87%) of 15 oncocytomas. Among the 81 cases of CRCC, positivity for S-100 protein was seen in 41 (71%) of 58 and 15 (65%) of 23 cases with Furhman nuclear grade I/II and III/IV, respectively. Focal immunostaining was present in 22 (92%) of 24 normal renal tubules. Similar staining pattern was observed in 21 (70%) of 30 metastatic RCCs. Western blotting demonstrated the S-100 protein expression in both renal cell neoplasm and normal renal tissue. Overexpression of S-100 in oncocytomas compared with ChRCCs was confirmed by the data of Western blot and cDNA microarray analysis. Importantly, 14.8% (12/81) of clear cell RCC and 13.3% (4/30) of metastatic RCC revealed an immunostaining profile of pancytokeratin (-)/S-100 protein (+). These data indicate that caution should be taken in interpreting an unknown primary with S-100 positivity and cytokeratin negativity. In addition, it

  12. Plasmodium vivax merozoite surface protein 8 cloning, expression, and characterisation.

    PubMed

    Perez-Leal, Oscar; Sierra, Adriana Y; Barrero, Carlos A; Moncada, Camilo; Martinez, Pilar; Cortes, Jimena; Lopez, Yolanda; Torres, Elizabeth; Salazar, Luz M; Patarroyo, Manuel A

    2004-11-26

    Plasmodium vivax, one of the four parasite species causing malaria in humans, is the most widespread throughout the world, leading to nearly 80 million cases per year, mainly in Latin-America and Asia. An open reading frame encoding the Plasmodium falciparum merozoite surface protein 8 P. vivax homologue has been identified in the present study by screening the current data obtained from this parasite's partially sequenced genome. This new protein contains 487 amino-acids, two epidermal growth factor like domains, hydrophobic regions at the N- and C-termini compatible with a signal peptide, and a glycosylphosphatidylinositol anchor site, respectively. This gene's transcription and its encoded protein expression have been assessed, as well as its recognition by P. vivax-infected patients' sera. Based on this recognition, and a previous study showing that mice immunised with the Plasmodium yoelii homologous protein were protected, we consider the PvMSP8 a good candidate to be included in a multi-stage multi-antigen P. vivax vaccine.

  13. Expression and purification of recombinant human EGFL7 protein.

    PubMed

    Picuric, Srdjan; Friedrich, Matthias; Oess, Stefanie

    2009-11-01

    The secreted epidermal growth factor-like protein 7 (EGFL7) plays an important role in angiogenesis, especially in the recruitment of endothelial and smooth muscle cells to the site of the nascent vessel and their ordered assembly into functional vasculature. However, progress in the understanding of the underlying mechanisms is to date greatly hindered by the lack of recombinant EGFL7 protein in a stable, soluble, native state, thus preventing e.g. the characterization of the proposed functional receptor as well as investigation of additional biological effects of EGFL7. So far all attempts to produce sufficient amounts of recombinant EGFL7 protein by various groups have failed. In this study we describe a procedure for the expression and purification of human EGFL7 from Sf9 cells and for the first time provide means to isolate biologically functional EGFL7 protein in sufficient quantities for its further biological characterization. We believe that the availability of EGFL7 will greatly accelerate our understanding of the precise role of EGFL7 and the underlying molecular mechanisms of EGFL7 action in the fundamentally important process of angiogenesis.

  14. Expressed Protein Ligation: A Resourceful Tool to Study Protein Structure and Function

    PubMed Central

    Berrade, Luis; Camarero, Julio A.

    2013-01-01

    This review outlines the use of expressed protein ligation (EPL) to study protein structure, function and stability. EPL is a chemoselective ligation method that allows the selective ligation of unprotected polypeptides from synthetic and recombinant origin for the production of semi-synthetic protein samples of well-defined and homogeneous chemical composition. This method has been extensively used for the site-specific introduction of biophysical probes, unnatural amino acids, and increasingly complex post-translational modifications. Since it was introduced 10 years ago, EPL applications have grown increasingly more sophisticated in order to address even more complex biological questions. In this review we highlight how this powerful technology combined with standard biochemical analysis techniques has been used to improve our ability to understand protein structure and function. PMID:19685006

  15. Extracellular matrix protein expression is brain region dependent.

    PubMed

    Dauth, Stephanie; Grevesse, Thomas; Pantazopoulos, Harry; Campbell, Patrick H; Maoz, Ben M; Berretta, Sabina; Parker, Kevin Kit

    2016-05-01

    In the brain, extracellular matrix (ECM) components form networks that contribute to structural and functional diversity. Maladaptive remodeling of ECM networks has been reported in neurodegenerative and psychiatric disorders, suggesting that the brain microenvironment is a dynamic structure. A lack of quantitative information about ECM distribution in the brain hinders an understanding of region-specific ECM functions and the role of ECM in health and disease. We hypothesized that each ECM protein as well as specific ECM structures, such as perineuronal nets (PNNs) and interstitial matrix, are differentially distributed throughout the brain, contributing to the unique structure and function in the various regions of the brain. To test our hypothesis, we quantitatively analyzed the distribution, colocalization, and protein expression of aggrecan, brevican, and tenascin-R throughout the rat brain utilizing immunohistochemistry and mass spectrometry analysis and assessed the effect of aggrecan, brevican, and/or tenascin-R on neurite outgrowth in vitro. We focused on aggrecan, brevican, and tenascin-R as they are especially expressed in the mature brain, and have established roles in brain development, plasticity, and neurite outgrowth. The results revealed a differentiated distribution of all three proteins throughout the brain and indicated that their presence significantly reduces neurite outgrowth in a 3D in vitro environment. These results underline the importance of a unique and complex ECM distribution for brain physiology and suggest that encoding the distribution of distinct ECM proteins throughout the brain will aid in understanding their function in physiology and in turn assist in identifying their role in disease. J. Comp. Neurol. 524:1309-1336, 2016. © 2016 Wiley Periodicals, Inc.

  16. Use of minimal disseminated disease and immunity to NPM-ALK antigen to stratify ALK-positive ALCL patients with different prognosis.

    PubMed

    Mussolin, L; Damm-Welk, C; Pillon, M; Zimmermann, M; Franceschetto, G; Pulford, K; Reiter, A; Rosolen, A; Woessmann, W

    2013-02-01

    We studied the prognostic value of minimal disseminated disease (MDD) and anti-ALK immune response in children with NPM-ALK-positive anaplastic-large cell lymphoma (ALCL) and evaluated their potential for risk stratification. NPM-ALK transcripts were analyzed by RT-PCR in bone marrow/peripheral blood of 128 ALCL patients at diagnosis, whereas ALK antibody titers in plasma were assessed using an immunocytochemical approach. MDD was positive in 59% of patients and 96% showed an anti-ALK response. Using MDD and antibody titer results, patients could be divided into three biological risk groups (bRG) with different prognosis: high risk (bHR): MDD-positive and antibody titer ≤ 1/750, 26/128 (20%); low risk (bLR): MDD negative and antibody titer >1/750, 40/128 (31%); intermediate risk (bIR): all remaining patients, 62/128 (48%). Progression-free survival was 28% (s.e., 9%), 68% (s.e., 6%) and 93% (s.e., 4%) for bHR, bIR and bLR, respectively (P<0.0001). Survival was 71% (s.e., 9%), 83% (s.e., 5%) and 98% (s.e., 2%) for bHR, bIR and bLR (P=0.02). Only bHR and histology other than common type were predictive of higher risk of failure (hazard ratio 4.9 and 2.7, respectively) in multivariate analysis. Stratification of ALCL patients based on MDD and anti-ALK titer should be considered in future ALCL trials to optimize treatment.

  17. AR-v7 protein expression is regulated by protein kinase and phosphatase.

    PubMed

    Li, Yinan; Xie, Ning; Gleave, Martin E; Rennie, Paul S; Dong, Xuesen

    2015-10-20

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.

  18. Arteriovenous malformations in hereditary haemorrhagic telangiectasia: looking beyond ALK1-NOTCH interactions.

    PubMed

    Peacock, Hanna M; Caolo, Vincenza; Jones, Elizabeth A V

    2016-02-01

    Hereditary haemorrhagic telangiectasia (HHT) is characterized by the development of arteriovenous malformations--enlarged shunts allowing arterial flow to bypass capillaries and enter directly into veins. HHT is caused by mutations in ALK1 or Endoglin; however, the majority of arteriovenous malformations are idiopathic and arise spontaneously. Idiopathic arteriovenous malformations differ from those due to loss of ALK1 in terms of both location and disease progression. Furthermore, while arteriovenous malformations in HHT and Alk1 knockout models have decreased NOTCH signalling, some idiopathic arteriovenous malformations have increased NOTCH signalling. The pathogenesis of these lesions also differs, with loss of ALK1 causing expansion of the shunt through proliferation, and NOTCH gain of function inducing initial shunt enlargement by cellular hypertrophy. Hence, we propose that idiopathic arteriovenous malformations are distinct from those of HHT. In this review, we explore the role of ALK1-NOTCH interactions in the development of arteriovenous malformations and examine a possible role of two signalling pathways downstream of ALK1, TMEM100 and IDs, in the development of arteriovenous malformations in HHT. A nuanced understanding of the precise molecular mechanisms underlying idiopathic and HHT-associated arteriovenous malformations will allow for development of targeted treatments for these lesions.

  19. Distribution of alkB genes within n-alkane-degrading bacteria.

    PubMed

    Vomberg, A; Klinner, U

    2000-08-01

    Fifty-four bacterial strains belonging to 37 species were tested for their ability to assimilate short chain and/or medium chain liquid n-alkanes. A gene probe derived from the alkB gene of Pseudomonas oleovorans ATCC 29347 was utilized in hybridization experiments. Results of Southern hybridization of PCR-amplificates were compared with those of colony hybridization and dot blot hybridization. Strongest signals were received only from Gram-negative bacteria growing solely with short n-alkanes (C10). Hybridization results with soil isolates growing with n-alkanes of different chain lengths suggested as well that alkB genes seem to be widespread only in solely short-chain n-alkane-degrading pseudomonads. PCR products of Rhodococcus sp., Nocardioides sp., Gordona sp. and Sphingomonas sp. growing additionally or solely with medium-chain n-alkane as hexadecane had only few sequence identity with alkB though hybridizing with the gene probe. The derived amino acid sequence of the alkB-amplificate of Pseudomonas aureofaciens showed high homology (95%) with AlkB from Ps. oleovorans. alkB gene disruptants were not able to grow with decane.

  20. Trichohyalin-like 1 protein, a member of fused S100 proteins, is expressed in normal and pathologic human skin

    SciTech Connect

    Yamakoshi, Takako; Makino, Teruhiko; Ur Rehman, Mati; Yoshihisa, Yoko; Sugimori, Michiya; Shimizu, Tadamichi

    2013-03-01

    Highlights: ► Trichohyalin-like 1 protein is a member of the fused-type S100 protein gene family. ► Specific antibodies against the C-terminus of the TCHHL1 protein were generated. ► TCHHL1 proteins were expressed in the basal layer of the normal epidermis. ► TCHHL1 proteins were strongly expressed in tumor nests of BCC and SCC. ► The expression of TCHHL1 proteins increased in epidermis of psoriasis vulgaris. - Abstract: Trichohyalin-like 1 (TCHHL1) protein is a novel member of the fused-type S100 protein gene family. The deduced amino acid sequence of TCHHL1 contains an EF-hand domain in the N-terminus, one trans-membrane domain and a nuclear localization signal. We generated specific antibodies against the C-terminus of the TCHHL1 protein and examined the expression of TCHHL1 proteins in normal and pathological human skin. An immunohistochemical study showed that TCHHL1 proteins were expressed in the basal layer of the normal epidermis. In addition, signals of TCHHL1 proteins were observed around the nuclei of cultured growing keratinocytes. Accordingly, TCHHL1 mRNA has been detected in normal skin and cultured growing keratinocytes. Furthermore, TCHHL1 proteins were strongly expressed in the peripheral areas of tumor nests in basal cell carcinomas and squamous cell carcinomas. A dramatic increase in the number of Ki67 positive cells was observed in TCHHL1-expressing areas. The expression of TCHHL1 proteins also increased in non-cancerous hyperproliferative epidermal tissues such as those of psoriasis vulgaris and lichen planus. These findings highlight the possibility that TCHHL1 proteins are expressed in growing keratinocytes of the epidermis and might be associated with the proliferation of keratinocytes.

  1. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    NASA Technical Reports Server (NTRS)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  2. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment.

  3. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    PubMed

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

  4. An efficient protocol to enhance recombinant protein expression using ethanol in Escherichia coli.

    PubMed

    Chhetri, Gaurav; Kalita, Parismita; Tripathi, Timir

    2015-01-01

    Bacterial cells can be engineered to express non-native genes, resulting in the production of, recombinant proteins, which have various biotechnological and pharmaceutical applications. In eukaryotes, such as yeast or mammalian cells, which have large genomes, a higher recombinant protein expression can be troublesome. Comparatively, in the Escherichia coli (E. coli) expression system, although the expression is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG), studies have shown low expression levels of proteins. Irrespective of the purpose of protein production, the production process requires the accomplishment of three individual factors: expression, solubilization and purification. Although several efforts, including changing the host, vector, culture parameters of the recombinant host strain, co-expression of other genes and changing of the gene sequences, have been directed towards enhancing recombinant protein expression, the protein expression is still considered as a significant limiting step. Our protocol explains a simple method to enhance the recombinant protein expression that we have optimized using several unrelated proteins. It works with both T5 and T7 promoters. This protocol can be used to enhance the expressions of most of the proteins. The advantages of this technique are presented below:•It produces several fold increase in the expression of poorly expressed, less expressed or non-expressed recombinant proteins.•It does not employ any additional component such as chaperones, heat shock proteins or co-expression of other genes.•In addition to being inexpensive, easy to manage, universal, and quick to perform, the proposed method does not require any commercial kits and, can be used for various recombinant proteins expressed in the E. coli expression system.

  5. Generation of cloned transgenic cats expressing red fluorescence protein.

    PubMed

    Yin, Xi Jun; Lee, Hyo Sang; Yu, Xian Feng; Choi, Eugene; Koo, Bon Chul; Kwon, Mo Sun; Lee, Young S; Cho, Su Jin; Jin, Guang Zhen; Kim, Lyoung Hyo; Shin, Hyoung Doo; Kim, Teoan; Kim, Nam Hyung; Kong, Il Keun

    2008-03-01

    A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

  6. AGL15, a MADS domain protein expressed in developing embryos.

    PubMed Central

    Heck, G R; Perry, S E; Nichols, K W; Fernandez, D E

    1995-01-01

    To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo. PMID:7549483

  7. Teicoplanin-resistant Staphylococcus aureus expresses a novel membrane protein and increases expression of penicillin-binding protein 2 complex.

    PubMed Central

    Shlaes, D M; Shlaes, J H; Vincent, S; Etter, L; Fey, P D; Goering, R V

    1993-01-01

    In the recent clinical trials of teicoplanin therapy of endocarditis caused by Staphylococcus aureus, at least one instance of the emergence of teicoplanin-resistant strains during therapy has been reported (G.W. Kaatz, S. M. Seo, N. J. Dorman, and S. A. Lerner, J. Infect. Dis 162:103-108, 1990). We have confirmed, using conventional electrophoresis of EcoRI-digested chromosomal DNA and pulsed-field gel electrophoresis of SmaI-digested chromosomal DNA, that the resistant strain (12873) (MIC, 16 micrograms/ml) is genetically very similar to the susceptible parent (12871) (MIC, 4 micrograms/ml). Kaatz et al. were able to select spontaneous teicoplanin-resistant mutants (10(-9)), suggesting that a single gene might be involved. We have shown that the mutation is highly stable during growth in the absence of teicoplanin. Using Tn551, we have selected insertion mutants of 12873 that become teicoplanin susceptible. We have examined a number of aspects of cell wall physiology in strains 12871 and 12873 and the teicoplanin-susceptible Tn551 mutants of 12873. 12873 was more susceptible to lysostaphin lysis than 12871 and the susceptible Tn551 derivatives of 12873. Autolysis in phosphate buffer (pH 7.5) and cell wall turnover rates were similar in 12871 and 12873. An analysis of membrane proteins revealed the expression of a ca. 35-kDa protein and increased expression of both polypeptides of penicillin-binding protein (PBP) 2 (PBP2) in 12873 relative to 12871 and the Tn551 mutants of 12873. This increased expression was not related to PBP2', since both strains were susceptible to oxacillin in 2% NaCl (MIC, < or = 0.25 microgram/ml) and cellular DNA from neither strain hybridized with a specific mec gene probe. Two independent Tn551 inserts have been mapped to a ca. 117-kb SmaI fragment of the chromosome. These data suggest the possibility that the mutation resulting in resistance to teicoplanin involves the regulation of expression of both polypeptides of PBP2 and a 35-k

  8. Ras protein expression as a marker for breast cancer

    PubMed Central

    CALAF, GLORIA M.; ABARCA-QUINONES, JORGE

    2016-01-01

    Breast cancer, the most common neoplasm in women of all ages, is the leading cause of cancer-related mortality in women worldwide. Markers to help to predict the risk of progression and ultimately provide non-surgical treatment options would be of great benefit. At present, there are no available molecular markers to predict the risk of carcinoma in situ progression to invasive cancer; therefore, all women diagnosed with this type of malignancy must undergo surgery. Breast cancer is a heterogeneous complex disease, and different patients respond differently to different treatments. In breast cancer, analysis using immunohistochemical markers remains an essential component of routine pathological examinations, and plays an import role in the management of the disease by providing diagnostic and prognostic strategies. The aim of the present study was to identify a marker that can be used as a prognostic tool for breast cancer. For this purpose, we firstly used an established breast cancer model. MCF-10F, a spontaneously immortalized breast epithelial cell line was transformed by exposure to estrogen and radiation. MCF-10F cells were exposed to low doses of high linear energy transfer (LET) α particles (150 keV/μm) of radiation, and subsequently cultured in the presence of 17β-estradiol. Three cell lines were used: i) MCF-10F cells as a control; ii) Alpha5 cells, a malignant and tumorigenic cell line; and iii) Tumor2 cells derived from Alpha5 cells injected into nude mice. Secondly, we also used normal, benign and malignant breast specimens obtained from biopsies. The results revealed that the MCF-10F cells were negative for c-Ha-Ras protein expression; however, the Alpha5 and Tumor2 cell lines were positive for c-Ha-Ras protein expression. The malignant breast samples were also strongly positive for c-Ha-Ras expression. The findings of our study indicate that c-Ha-Ras protein expression may be used as a marker to predict the progression of breast cancer; this

  9. Production of Computationally Designed Small Soluble- and Membrane-Proteins: Cloning, Expression, and Purification.

    PubMed

    Tripathy, Barsa; Acharya, Rudresh

    2017-01-01

    This book chapter focuses on expression and purification of computationally designed small soluble proteins and membrane proteins that are ordinarily difficult to express in good amounts for experiments. Over-expression of such proteins can be achieved by using the solubility tag such as maltose binding protein (MBP), Thioredoxin (Trx), and Gultathione-S-transferase (GST) fused to the protein of interest. Here, we describe and provide the protocols for cloning, expression and purification of such proteins using the solubility tag.

  10. Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.

    PubMed

    Aymoz, Delphine; Wosika, Victoria; Durandau, Eric; Pelet, Serge

    2016-04-21

    Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.

  11. Functions of BET proteins in erythroid gene expression.

    PubMed

    Stonestrom, Aaron J; Hsu, Sarah C; Jahn, Kristen S; Huang, Peng; Keller, Cheryl A; Giardine, Belinda M; Kadauke, Stephan; Campbell, Amy E; Evans, Perry; Hardison, Ross C; Blobel, Gerd A

    2015-04-30

    Inhibitors of bromodomain and extraterminal motif proteins (BETs) are being evaluated for the treatment of cancer and other diseases, yet much remains to be learned about how BET proteins function during normal physiology. We used genomic and genetic approaches to examine BET function in a hematopoietic maturation system driven by GATA1, an acetylated transcription factor previously shown to interact with BETs. We found that BRD2, BRD3, and BRD4 were variably recruited to GATA1-regulated genes, with BRD3 binding the greatest number of GATA1-occupied sites. Pharmacologic BET inhibition impaired GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, BETs promoted chromatin occupancy of GATA1 and subsequently supported transcriptional activation. Using a combination of CRISPR-Cas9-mediated genomic engineering and shRNA approaches, we observed that depletion of either BRD2 or BRD4 alone blunted erythroid gene activation. Surprisingly, depletion of BRD3 only affected erythroid transcription in the context of BRD2 deficiency. Consistent with functional overlap among BET proteins, forced BRD3 expression substantially rescued defects caused by BRD2 deficiency. These results suggest that pharmacologic BET inhibition should be interpreted in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

  12. GPR37 Surface Expression Enhancement via N-Terminal Truncation or Protein-Protein Interactions1

    PubMed Central

    Dunham, Jill H.; Meyer, Rebecca C.; Garcia, Erin L.; Hall, Randy A.

    2009-01-01

    GPR37, also known as the parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that exhibits poor plasma membrane expression when expressed in most cell types. We sought to find ways to enhance GPR37 trafficking to the cell surface in order to facilitate studies of GPR37 functional activity in heterologous cells. In truncation studies, we found that removing the GPR37 N-terminus (NT) dramatically enhanced the receptor’s plasma membrane insertion. Further studies on sequential NT truncations revealed that removal of the first 210 amino acids increased surface expression nearly as much as removal of the entire NT. In studies examining the effects of co-expression of GPR37 with a variety of other GPCRs, we observed significant increases in GPR37 surface expression when the receptor was co-expressed with the adenosine receptor A2AR or the dopamine receptor D2R. Co-immunoprecipitation experiments revealed that full-length GPR37 and, to a greater extent, the truncated GPR37 were capable of robustly associating with D2R, resulting in modestly-altered D2R affinity for both agonists and antagonists. In studies examining potential interactions of GPR37 with PDZ scaffolds, we observed a specific interaction between GPR37 and syntenin-1, which resulted in a dramatic increase in GPR37 surface expression in HEK-293 cells. These findings reveal three independent approaches – N-terminal truncation, co-expression with other receptors and co-expression with syntenin-1 – by which GPR37 surface trafficking in heterologous cells can be greatly enhanced to facilitate functional studies on this orphan receptor. PMID:19799451

  13. SSAO/VAP-1 protein expression during mouse embryonic development.

    PubMed

    Valente, Tony; Solé, Montse; Unzeta, Mercedes

    2008-09-01

    SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation.

  14. Design, synthesis and pharmacological evaluation of 2-(thiazol-2-amino)-4-arylaminopyrimidines as potent anaplastic lymphoma kinase (ALK) inhibitors.

    PubMed

    Liu, Zhiqing; Yue, Xihua; Song, Zilan; Peng, Xia; Guo, Junfeng; Ji, Yinchun; Cheng, Zhen; Ding, Jian; Ai, Jing; Geng, Meiyu; Zhang, Ao

    2014-10-30

    A series of new 2,4-diarylaminopyrimidine analogues (DAAPalogues) was developed by incorporation of a substituted 2-aminothiazole component as the C-2 substituent of the center pyrimidine core. Compound 5i showed highest potency of 12.4 nM against ALK and 24.1 nM against ALK gatekeeper mutation L1196M. Although only having moderate cellular potency in the SUP-M2 cells harboring NPM-ALK, compound 5i showed good kinase selectivity and dose-dependently inhibited phosphorylation of ALK and its down-stream signaling pathways.

  15. Soluble expression and complex formation of proteins required for HCMV DNA replication using the SFV expression system.

    PubMed

    McCue, L A; Anders, D G

    1998-08-01

    Several of the viral proteins required for human cytomegalovirus (HCMV) DNA replication have been difficult to study due to their low abundance in infected cells and low solubility in bacterial or insect-cell expression systems. Therefore we used the Semliki Forest virus expression system to express these proteins in mammalian cells. All of the recombinant proteins were soluble, on the basis of ultracentrifugation properties and their ability to be immunoprecipitated from solution with specific antibodies. Pulse-chase analysis of the 86-kDa major immediate-early protein (IE86) revealed two expressed forms-a precursor and a product-indicating that this recombinant protein, like the native HCMV protein, is posttranslationally processed. The recombinant proteins (polymerase core and accessory as well as the IE86 and pUL84) formed stable complexes similar to those known to form in HCMV-infected cells. The recombinant DNA polymerase holoenzyme also exhibited enzyme activity that was phosphonoformic acid sensitive, as is the infected-cell DNA polymerase activity. This expression system offers many advantages for the expression and study of the HCMV replication proteins, including the expression of soluble, active proteins that are able to interact to form complexes. Additionally, the relative ease with which SFV recombinants can be made lends itself to the construction and evaluation of mutants.

  16. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy.

    PubMed

    Duwé, Sam; De Zitter, Elke; Gielen, Vincent; Moeyaert, Benjamien; Vandenberg, Wim; Grotjohann, Tim; Clays, Koen; Jakobs, Stefan; Van Meervelt, Luc; Dedecker, Peter

    2015-10-27

    "Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins.

  17. Developmental expression of odorant-binding proteins and chemosensory proteins in the embryos of Locusta migratoria.

    PubMed

    Yu, Yanxue; Zhang, Shangan; Zhang, Long; Zhao, Xingbo

    2009-06-01

    We have investigated the development of chemosensilla and the secretion of odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in the embryo of Locusta migratoria manilensis. We first report the changes of each sensillum in embryo just preceding hatch in detail and show that different sensilla have different developmental processes. Trichogen cells are first involved in forming the structure of pegs, and then, after retraction, they start secreting OBPs and CSPs in the sensillar lymph. The synthesis of LmigOBP1 starts during the embryogenesis about 0.5 h preceding hatching, specifically in sensilla trichodea and basiconica of the antenna. LmigOBP2, instead, was only found in the outer sensillum lymph (oSl) of sensilla chaetica of the antenna, while we could not detect LmigOBP3 in any type of sensilla of the antenna. The ontogenesis of CSPs in the embryos is similar to that of OBPs. Expression of CSPI homolog in Locusta migratoria is detected using the antiserum raised against SgreCSPI. CSPI is specifically expressed in the outer sensillum lymph of sensilla chaetica of the antenna, and anti-LmigCSPII dose not label any sensilla of the embryos. These data indicate that in locusts, OBPs and CSPs follow different temporal expression patterns, and also that OBPs are expressed in different types of sensilla.

  18. Bcl-2-related protein family gene expression during oligodendroglial differentiation.

    PubMed

    Itoh, Takayuki; Itoh, Aki; Pleasure, David

    2003-06-01

    Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.

  19. Recombinant protein production data after expression in the bacterium Escherichia coli

    PubMed Central

    Cantu-Bustos, J. Enrique; Cano del Villar, Kevin D.; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-01-01

    Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography. PMID:27014739

  20. Recombinant protein production data after expression in the bacterium Escherichia coli.

    PubMed

    Cantu-Bustos, J Enrique; Cano Del Villar, Kevin D; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

    2016-06-01

    Fusion proteins have become essential for the expression and purification of recombinant proteins in Escherichia coli. The metal-binding protein CusF has shown several features that make it an attractive fusion protein and affinity tag: "Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF" (Cantu-Bustos et al., 2016 [1]). Here we present accompanying data from protein expression experiments; we tested different protein tags, temperatures, expression times, cellular compartments, and concentrations of inducer in order to obtain soluble protein and low formation of inclusion bodies. Additionally, we present data from the purification of the green fluorescent protein (GFP) tagged with CusF, using Ag(I) metal affinity chromatography.

  1. New examples of membrane protein expression and purification using the yeast based Pdr1-3 expression strategy.

    PubMed

    Gupta, Rakeshkumar P; Kueppers, Petra; Schmitt, Lutz

    2014-12-10

    Overexpression and purification of membrane proteins has been a bottleneck for their functional and structural study for a long time. Both homologous and heterologous expression of membrane proteins with suitable tags for purification presents unique challenges for cloning and expression. Saccharomyces cerevisiae is a potential host system with significant closeness to higher eukaryotes and provides opportunity for attempts to express membrane proteins. In the past, bakers yeast containing mutations within the transcriptional regulator Pdr1 has been used to overexpress various membrane proteins including for example the ABC transporters Pdr5 and Yor1, respectively. In this study we exploited this system and tried to express and purify 3 membrane proteins in yeast along with Pdr5 and Yor1 viz. Rsb1, Mdl1 and Drs2 by virtue of an N-terminal 14-histidine affinity tag. Out of these five, we could express all membrane proteins although at different levels. Satisfactory yields were obtained for three examples i.e. Pdr5, Yor1 and Drs2. Rsb1 expression was comparatively low and Mdl1 was rather unsatisfactory. Thus, we demonstrate here the application of this yeast based expression system that is suitable for cloning, expression and purification of a wide variety of membrane proteins.

  2. Mycobacterium tuberculosis Rv1096 protein: gene cloning, protein expression, and peptidoglycan deacetylase activity

    PubMed Central

    2014-01-01

    Background Many bacteria modulate and evade the immune defenses of their hosts through peptidoglycan (PG) deacetylation. The PG deacetylases from Streptococcus pneumonia, Listeria monocytogenes and Lactococcus lactis have been characterized. However, thus far, the PG deacetylase of Mycobacterium tuberculosis has not been identified. Results In this study, we cloned the Rv1096 gene from the M. tuberculosis H37Rv strain and expressed Rv1096 protein in both Escherichia coli and M. smegmatis. The results showed that the purified Rv1096 protein possessed metallo-dependent PG deacetylase activity, which increased in the presence of Co2+. The kinetic parameters of the PG deacetylase towards M. smegmatis PG as a substrate were as follows: Km, 0.910 ± 0.007 mM; Vmax, 0.514 ± 0.038 μMmin-1; and Kcat = 0.099 ± 0.007 (S-1). Additionally, the viability of M. smegmatis in the presence of over-expressed Rv1096 protein was 109-fold higher than that of wild-type M. smegmatis after lysozyme treatment. Additionally, light microscopy and scanning electron microscopy showed that in the presence of over-expressed Rv1096 protein, M. smegmatis kept its regular shape, with an undamaged cell wall and smooth surface. These results indicate that Rv1096 caused deacetylation of cell wall PG, leading to lysozyme resistance in M. smegmatis. Conclusion We have determined that M. tuberculosis Rv1096 is a PG deacetylase. The PG deacetylase activity of Rv1096 contributed to lysozyme resistance in M. smegmatis. Our findings suggest that deacetylation of cell wall PG may be involved in evasion of host immune defenses by M. tuberculosis. PMID:24975018

  3. Lytic Promoters Express Protein during Herpes Simplex Virus Latency

    PubMed Central

    Russell, Tiffany A.; Tscharke, David C.

    2016-01-01

    Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency. PMID:27348812

  4. Anatomical profiling of G protein-coupled receptor expression

    PubMed Central

    Regard, Jean B.; Sato, Isaac T.; Coughlin, Shaun R.

    2008-01-01

    Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets. PMID:18984166

  5. Patagonfibrase modifies protein expression of tissue factor and protein disulfide isomerase in rat skin.

    PubMed

    Peichoto, María Elisa; Santoro, Marcelo Larami

    2016-09-01

    Patagonfibrase is a hemorrhagic metalloproteinase isolated from the venom of the South American rear-fanged snake Philodryas patagoniensis, and is an important contributor to local lesions inflicted by this species. The tissue factor (TF)-factor VIIa complex, besides triggering the coagulation cascade, has been demonstrated to be involved in inflammatory events. Our aim was to determine whether patagonfibrase affects the expression of TF and protein disulfide isomerase (PDI), an enzyme that controls TF biological activity, at the site of patagonfibrase injection, and thus if they may play a role in hemostatic and inflammatory events induced by snake venoms. Patagonfibrase (60 μg/kg) was administered s.c. to rats, and after 3 h blood was collected to evaluate hemostasis parameters, and skin fragments close to the site of injection were taken to assess TF and PDI expression. Patagonfibrase did not alter blood cell counts, plasma fibrinogen levels, or levels of TF activity in plasma. However, by semiquantitative Western blotting, patagonfibrase increased TF expression by 2-fold, and decreased PDI expression by 3-fold in skin samples. In agreement, by immunohistochemical analyses, prominent TF expression was observed in the subcutaneous tissue. Thus, patagonfibrase affects the local expression of TF and PDI without inducing any systemic hemostatic disturbance, although that they may be involved in the local inflammatory events induced by hemorrhagic metalloproteinases. Once antivenom therapy is not totally effective to treat the local injury induced by snake venoms, modulation of the activity and expression of TF and/or PDI might become a strategy for treating snake envenomation.

  6. Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

    PubMed

    Simmons, Jason; D'Souza, Olivia; Rheault, Mark; Donly, Cam

    2013-02-01

    Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

  7. Leptin responsiveness in mice that ectopically express agouti protein.

    PubMed

    Harris, Ruth B S; Mitchell, Tiffany D; Mynatt, Randall L

    Agouti protein is an endogenous antagonist of melanocortin receptors (MCR), including MCR3 and MCR4, which have been implicated as part of the hypothalamic mechanism that mediates leptin-induced hypophagia. In this experiment we examined the effects of peripheral and central leptin administration in male and female beta-actin promoter (BAPa) mice that express agouti protein ectopically and have a phenotype that includes obesity and diabetes which is exaggerated in males compared with females. Intraperitoneal infusion of 10 microg leptin/day for 13 days caused weight loss and a transient inhibition of food intake in wild-type mice, with a greater effect in males than females. Male BAPa mice were resistant to leptin infusion whereas female mice lost weight. All of the mice lost body weight following a single intracerebroventricular injection of leptin but the effect was greater in female BAPa mice than any other group. There also was a delayed suppression of food intake that was the same for wild-type and BAPa female mice, whereas food intake recovered faster in BAPa than wild-type males. The dissociation between food intake and body weight loss implies a significant effect of leptin on energy expenditure in BAPa mice. These results demonstrate that the effect of leptin on energy balance is not entirely dependent upon the melanocortin system.

  8. Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

    PubMed Central

    Townson, Sharon A.; Martinez-Hackert, Erik; Greppi, Chloe; Lowden, Patricia; Sako, Dianne; Liu, June; Ucran, Jeffrey A.; Liharska, Katia; Underwood, Kathryn W.; Seehra, Jasbir; Kumar, Ravindra; Grinberg, Asya V.

    2012-01-01

    Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies. PMID:22718755

  9. Neuroendocrine secretory protein 7B2: structure, expression and functions.

    PubMed Central

    Mbikay, M; Seidah, N G; Chrétien, M

    2001-01-01

    7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2-7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin ('ACTH') hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2 has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation. PMID:11439082

  10. Prion Protein Expression and Functional Importance in Skeletal Muscle

    PubMed Central

    Smith, Jeffrey D.; Moylan, Jennifer S.; Hardin, Brian J.; Chambers, Melissa A.; Estus, Steven; Telling, Glenn C.

    2011-01-01

    Abstract Skeletal muscle expresses prion protein (PrP) that buffers oxidant activity in neurons. Aims We hypothesize that PrP deficiency would increase oxidant activity in skeletal muscle and alter redox-sensitive functions, including contraction and glucose uptake. We used real-time polymerase chain reaction and Western blot analysis to measure PrP mRNA and protein in human diaphragm, five murine muscles, and muscle-derived C2C12 cells. Effects of PrP deficiency were tested by comparing PrP-deficient mice versus wild-type mice and morpholino-knockdown versus vehicle-treated myotubes. Oxidant activity (dichlorofluorescin oxidation) and specific force were measured in murine diaphragm fiber bundles. Results PrP content differs among mouse muscles (gastrocnemius>extensor digitorum longus, EDL>tibialis anterior, TA; soleus>diaphragm) as does glycosylation (di-, mono-, nonglycosylated; gastrocnemius, EDL, TA=60%, 30%, 10%; soleus, 30%, 40%, 30%; diaphragm, 30%, 30%, 40%). PrP is predominantly di-glycosylated in human diaphragm. PrP deficiency decreases body weight (15%) and EDL mass (9%); increases cytosolic oxidant activity (fiber bundles, 36%; C2C12 myotubes, 7%); and depresses specific force (12%) in adult (8–12 mos) but not adolescent (2 mos) mice. Innovation This study is the first to directly assess a role of prion protein in skeletal muscle function. Conclusions PrP content varies among murine skeletal muscles and is essential for maintaining normal redox homeostasis, muscle size, and contractile function in adult animals. Antioxid. Redox Signal. 15, 2465—2475. PMID:21453198

  11. Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF.

    PubMed

    Cantu-Bustos, J Enrique; Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Galbraith, David W; McEvoy, Megan M; Zarate, Xristo

    2016-05-01

    Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.

  12. Rift Valley fever virus structural and non-structural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Rift Valley fever virus (RVFV) encodes structural proteins, nucleoprotein (N), N-terminus glycoprotein (Gn), C-terminus glycoprotein (Gc) and L protein, 78-kDa and non-structural proteins NSm and NSs. Using the baculovirus system we expressed the full-length coding sequence of N, NSs, NSm, Gc an...

  13. C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

    PubMed

    Lee, Beom Seob; Kim, Soo Hyuk; Oh, Jaewon; Jin, Taewon; Choi, Eun Young; Park, Sungha; Lee, Sang-Hak; Chung, Ji Hyung; Kang, Seok-Min

    2014-01-01

    C-reactive protein (CRP) is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

  14. BMP9/ALK1 inhibits neovascularization in mouse models of age-related macular degeneration

    PubMed Central

    Ntumba, Kalonji; Akla, Naoufal; Oh, S. Paul; Eichmann, Anne; Larrivée, Bruno

    2016-01-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in aging populations of industrialized countries. The drawbacks of inhibitors of vascular endothelial growth factor (VEGFs) currently used for the treatment of AMD, which include resistance and potential serious side-effects, require the identification of new therapeutic targets to modulate angiogenesis. BMP9 signaling through the endothelial Alk1 serine-threonine kinase receptor modulates the response of endothelial cells to VEGF and promotes vessel quiescence and maturation during development. Here, we show that BMP9/Alk1 signaling inhibits neovessel formation in mouse models of pathological ocular angiogenesis relevant to AMD. Activating Alk1 signaling in laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy (OIR) inhibited neovascularization and reduced the volume of vascular lesions. Alk1 signaling was also found to interfere with VEGF signaling in endothelial cells whereas BMP9 potentiated the inhibitory effects of VEGFR2 signaling blockade, both in OIR and laser-induced CNV. Together, our data show that targeting BMP9/Alk1 efficiently prevents the growth of neovessels in AMD models and introduce a new approach to improve conventional anti-VEGF therapies. PMID:27517154

  15. Concurrent EGFR Mutation and ALK Translocation in Non-Small Cell Lung Cancer

    PubMed Central

    Thomas, Sachdev; Bank, Bruce; Fishkin, Paul; Mooney, Colin; Salgia, Ravi

    2016-01-01

    Epidermal growth factor receptor (EGFR) mutations and anaplastic large-cell lymphoma kinase (ALK) rearrangements are now routine biomarkers that have been incorporated into the practice of managing non-small cell lung cancer (NSCLC). Historically, the two molecular alterations have been viewed as mutually exclusive, but recent identified cases suggest otherwise. In this report, we describe cases of lung cancer with concurrent EGFR mutation and ALK rearrangement and identify their clinical characteristics. Non-small cell lung cancer patients with multiple molecular alterations were retrospectively analyzed from an academic referral center from 2011–2013. An additional review was conducted of reported cases with dual alterations. Four cases of NSCLC with alterations in both EGFR and ALK were identified and evaluated with 16 published cases for a total of 20 cases. The age of patients ranged from 37 to 77 years. Nine patients were never smokers. The disease control rates in patients treated with EGFR inhibitors and ALK inhibitors were 46% (6/13) and 71% (5/7), respectively. This series highlights the importance of comprehensive molecular profiling of newly diagnosed lung cancer, as NSCLC may be driven by concurrent molecular alterations. EGFR- and ALK-targeted therapies appear to have modest activity in patients with tumors possessing both alterations. Dual-altered NSCLC patients may have distinct clinical characteristics warranting further study. Combination targeted therapy or novel multi-targeted tyrosine kinase inhibitors may prove important in these patients, though necessary studies remain ongoing. PMID:27026837

  16. Spectrum of EGFR gene mutations and ALK rearrangements in lung cancer patients in Turkey.

    PubMed

    Sag, Sebnem Ozemri; Gorukmez, Ozlem; Ture, Mehmet; Gorukmez, Orhan; Deligonul, Adem; Sahinturk, Serdar; Topak, Ali; Gulten, Tuna; Kurt, Ender; Yakut, Tahsin

    2016-01-01

    The EGFR gene and ALK rearrangements are two genetic drivers of non-small cell lung cancer (NSCLC). The frequency of EGFR mutations and ALK rearrangement varies according to not only ethnicity but also gender, smoking status and the histological type of NSCLC. In the present study, we demonstrated the distribution of EGFR mutations in 132 NSCLC patients by using a pyrosequencing technique and the distribution of ALK rearrangements in 51 NSCLC patients by using fluorescent in situ hybridization technique in Turkey. Additionally, we compared the clinicopathological data of NSCLC patients with the mutation status of EGFR in their cancerous tissues. Both EGFR mutations and ALK rearrangements were identified in 19 (14.39 %) and 1 (1.96 %) patients, respectively. We found EGFR mutations in codon 861, 719 and 858 with the ratios of 10.52 % (2/19), 10.52 % (2/19) and 31.58 % (6/19), respectively, and deletion of exon 19 in 47.37 % (9/19) of the patients. We found the frequency of EGFR mutations to be significantly higher in female patients and nonsmokers (p = 0.043, p = 0.027, respectively). Consequently, we found EGFR mutations to be more frequent in female patients and nonsmokers. Future studies on larger patient groups would provide more accurate data to exhibit the relationship between EGFR mutations and ALK rearrangements and the clinicopathological status.

  17. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

    SciTech Connect

    Sun, Hui-Yong; Ji, Feng-Qin

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

  18. Differential dissolved protein expression throughout the life cycle of Giardia lamblia.

    PubMed

    Lingdan, Li; Pengtao, Gong; Wenchao, Li; Jianhua, Li; Ju, Yang; Chengwu, Liu; He, Li; Guocai, Zhang; Wenzhi, Ren; Yujiang, Chen; Xichen, Zhang

    2012-12-01

    Giardia lamblia (G. lamblia) has a simple life cycle that alternates between a cyst and a trophozoite, and this parasite is an important human and animal pathogen. To increase our understanding of the molecular basis of the G. lamblia encystment, we have analyzed the soluble proteins expressed by trophozoites and cysts extracted from feces by quantitative proteomic analysis. A total of 63 proteins were identified by isobaric tags for relative and absolute quantitation (iTRAQ) labeling, and were categorized as cytoskeletal proteins, a cell-cycle-specific kinase, metabolic enzymes and stress resistance proteins. Importantly, we demonstrated that the expression of seven proteins differed significantly between trophozoites and cysts. In cysts, the expression of three proteins (one variable surface protein (VSP), ornithine carbamoyltransferase (OTC), β-tubulin) increased, whereas the expression of four proteins (14-3-3 protein, α-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), protein disulfide isomerase 2 (PDI-2)) decreased significantly when compared with the levels of these proteins in trophozoites. The mRNA expression patterns of four of these proteins (OTC, α-tubulin, GAPDH, VSP) were similar to the expression levels of the proteins. These seven proteins appear to play an important role in the completion of the life cycle of G. lamblia.

  19. Expression and purification of toxic anti-breast cancer p28-NRC chimeric protein

    PubMed Central

    Soleimani, Meysam; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    Background: Chimeric proteins consisting of a targeting moiety and a cytotoxic moiety are now under intense research focus for targeted therapy of cancer. Here, we report cloning, expression, and purification of such a targeted chimeric protein made up of p28 peptide as both targeting and anticancer moiety fused to NRC peptide as a cytotoxic moiety. However, since the antimicrobial activity of the NRC peptide would intervene expression of the chimeric protein in Escherichia coli, we evaluated the effects of two fusion tags, that is, thioredoxin (Trx) and 6x-His tags, and various expression conditions, on the expression of p28-NRC chimeric protein. Materials and Methods: In order to express the chimeric protein with only 6x-His tag, pET28 expression plasmid was used. Cloning in pET32 expression plasmid was performed to add both Trx and 6x-His tags to the chimeric protein. Expression of the chimeric protein with both plasmids was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis following optimization of expression conditions and host strains. Results: Expression of the chimeric protein in pET28a was performed. However, expression yield of the chimeric protein was low. Optimization of culture conditions and host strains led to reasonable expression yield of the toxic chimeric protein in pET32a vector. In cases of both plasmids, approximately 10 kDa deviation of the apparent molecular weight from the theoretical one was seen in SDS-PAGE of purified chimeric proteins. Conclusions: The study leads to proper expression and purification yield of p28-NRC chimeric protein with Trx tag following optimizing culture conditions and host strains. PMID:27169101

  20. Statistical analysis of features associated with protein expression/solubility in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system.

    PubMed

    Hirose, Shuichi; Kawamura, Yoshifumi; Yokota, Kiyonobu; Kuroita, Toshihiro; Natsume, Tohru; Komiya, Kazuo; Tsutsumi, Takeshi; Suwa, Yorimasa; Isogai, Takao; Goshima, Naoki; Noguchi, Tamotsu

    2011-07-01

    Recombinant protein technology is an important tool in many industrial and pharmacological applications. Although the success rate of obtaining soluble proteins is relatively low, knowledge of protein expression/solubility under 'standard' conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we conducted a genome-scale experiment to assess the overexpression and the solubility of human full-length cDNA in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system. We evaluated the influences of sequence and structural features on protein expression/solubility in each system and estimated a minimal set of features associated with them. A comparison of the feature sets related to protein expression/solubility in the in vivo Escherichia coli expression system revealed that the structural information was strongly associated with protein expression, rather than protein solubility. Moreover, a significant difference was found in the number of features associated with protein solubility in the two expression systems.

  1. Mutations of the Wiskott-Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes.

    PubMed

    Ochs, Hans D

    2009-01-01

    Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and in rare instances congenital X-linked neutropenia (XLN). WASP is a regulator of actin polymerization in hematopoietic cells with well-defined functional domains that are involved in cell signaling and cell locomotion, immune synapse formation, and apoptosis. Mutations of WASP are located throughout the gene and either inhibit or disregulate normal WASP function. Analysis of a large patient population demonstrates a strong phenotype-genotype correlation. Classic WAS occurs when WASP is absent, XLT when mutated WASP is expressed and XLN when missense mutations occur in the Cdc42-binding site. However, because there are exceptions to this rule it is difficult to predict the long-term prognosis of a given affected boy solely based on the analysis of WASP expression.

  2. An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

    PubMed

    Linhoff, Michael W; Laurén, Juha; Cassidy, Robert M; Dobie, Frederick A; Takahashi, Hideto; Nygaard, Haakon B; Airaksinen, Matti S; Strittmatter, Stephen M; Craig, Ann Marie

    2009-03-12

    Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

  3. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions.

    PubMed

    Gez, Swetlana; Crossett, Ben; Christopherson, Richard I

    2007-09-01

    Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.

  4. Proteomic identification of abnormally expressed proteins in early-stage placenta derived from cloned cat embryos.

    PubMed

    Bang, Jae-Il; Lee, Hyo-Sang; Deb, Gautam Kumar; Ha, A-Na; Kwon, Young-Sang; Cho, Seong-Keun; Kim, Byeong-Woo; Cho, Kyu-Woan; Kong, Il-Keun

    2013-01-15

    It is unknown whether gene expression in cloned placenta during pre- and postimplantation is associated with early pregnancy failure in the cat. In this study, protein expression patterns were examined in early-stage (21-day-old) domestic cat placentas of fetuses derived from AI (CP; N = 4) and cloned embryo transfer (CEP; N = 2). Differentially expressed proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (TOF) mass spectrometry (MS). A total of 21 proteins were aberrantly expressed (P < 0.05) by >1.5-fold in CEP compared with CP. Compared with CP, 12 proteins were upregulated in CEP (peptidyl-prolyl cis-trans isomerase A, annexin A2, protein DJ-1, adenylate kinase isoenzyme 1, protein disulfide-isomerase A3, actin cytoplasmic 1, serum albumin, protein disulfide-isomerase A6, and triosephosphate isomerase), and nine proteins were downregulated (triosephosphate isomerase; heterogeneous nuclear ribonucleoprotein H; tropomyosin alpha-4; triosephosphate isomerase 1; 60 kDa heat shock protein, mitochondrial; serum albumin; calumenin; keratin type 1; and prohibitin). The identities of the differentially expressed proteins were validated by peptide mass fingerprinting using matrix-assisted laser desorption/ionization-TOF/TOF MS/MS. The abnormally expressed proteins identified in this study might be associated with impaired development and dysfunction of CEP during early pregnancy. Abnormal protein expression might also induce fetal loss and contribute to failure to maintain pregnancy to term.

  5. Identification of different ALK mutations in a pair of neuroblastoma cell lines established at diagnosis and relapse

    PubMed Central

    Chen, Lindi; Humphreys, Angharad; Turnbull, Lisa; Bellini, Angela; Schleiermacher, Gudrun; Salwen, Helen; Cohn, Susan L.; Bown, Nick; Tweddle, Deborah A.

    2016-01-01

    Anaplastic Lymphoma Kinase (ALK) is a transmembrane receptor kinase that belongs to the insulin receptor superfamily and has previously been shown to play a role in cell proliferation, migration and invasion in neuroblastoma. Activating ALK mutations are reported in both hereditary and sporadic neuroblastoma tumours, and several ALK inhibitors are currently under clinical evaluation as novel treatments for neuroblastoma. Overall, mutations at codons F1174, R1275 and F1245 together account for ∼85% of reported ALK mutations in neuroblastoma. NBLW and NBLW-R are paired cell lines originally derived from an infant with metastatic MYCN amplified Stage IVS (Evans Criteria) neuroblastoma, at diagnosis and relapse, respectively. Using both Sanger and targeted deep sequencing, this study describes the identification of distinct ALK mutations in these paired cell lines, including the rare R1275L mutation, which has not previously been reported in a neuroblastoma cell line. Analysis of the sensitivity of NBLW and NBLW-R cells to a panel of ALK inhibitors (TAE-684, Crizotinib, Alectinib and Lorlatinib) revealed differences between the paired cell lines, and overall NBLW-R cells with the F1174L mutation were more resistant to ALK inhibitor induced apoptosis compared with NBLW cells. This pair of cell lines represents a valuable pre-clinical model of clonal evolution of ALK mutations associated with neuroblastoma progression. PMID:27888620

  6. Targeting autophagy enhances the anti-tumoral action of crizotinib in ALK-positive anaplastic large cell lymphoma

    PubMed Central

    Desquesnes, Aurore; Le Gonidec, Sophie; AlSaati, Talal; Beau, Isabelle; Lamant, Laurence; Meggetto, Fabienne; Espinos, Estelle; Codogno, Patrice; Brousset, Pierre; Giuriato, Sylvie

    2015-01-01

    Anaplastic Lymphoma Kinase-positive Anaplastic Large Cell Lymphomas (ALK+ ALCL) occur predominantly in children and young adults. Their treatment, based on aggressive chemotherapy, is not optimal since ALCL patients can still expect a 30% 2-year relapse rate. Tumor relapses are very aggressive and their underlying mechanisms are unknown. Crizotinib is the most advanced ALK tyrosine kinase inhibitor and is already used in clinics to treat ALK-associated cancers. However, crizotinib escape mechanisms have emerged, thus preventing its use in frontline ALCL therapy. The process of autophagy has been proposed as the next target for elimination of the resistance to tyrosine kinase inhibitors. In this study, we investigated whether autophagy is activated in ALCL cells submitted to ALK inactivation (using crizotinib or ALK-targeting siRNA). Classical autophagy read-outs such as autophagosome visualization/quantification by electron microscopy and LC3-B marker turn-over assays were used to demonstrate autophagy induction and flux activation upon ALK inactivation. This was demonstrated to have a cytoprotective role on cell viability and clonogenic assays following combined ALK and autophagy inhibition. Altogether, our results suggest that co-treatment with crizotinib and chloroquine (two drugs already used in clinics) could be beneficial for ALK-positive ALCL patients. PMID:26338968

  7. Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.

    PubMed

    Kim, Hyun; Yamanouchi, Keitaro; Matsuwaki, Takashi; Nishihara, Masugi

    2012-01-01

    The Ski protein is implicated in the proliferation/differentiation of a variety of cells. We previously reported that the Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. However, granulosa cells cannot only undergo apoptosis but can alternatively differentiate into luteal cells. It is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to determine the localization of the Ski protein in the rat ovary during luteinization to examine if Ski might play a role in this process. In order to examine the Ski protein expression during the progression of luteinization, follicular growth was induced in immature female rats by administration of equine chorionic gonadotropin, and luteinization was induced by human chorionic gonadotropin treatment to mimic the luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in the preovulatory follicle, Ski protein expression was induced in response to the LH surge and was maintained after formation of the corpus luteum (CL). Although the Ski protein is absent from the granulosa cells of the preovulatory follicle, its mRNA (c-ski) was expressed, and the level of c-ski mRNA was unchanged even after the LH surge. The combined results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated posttranscriptionally.

  8. An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

    PubMed Central

    Round, Ekaterina; Shevchenko, Vitaly; Gushchin, Ivan; Polovinkin, Vitaly; Borshchevskiy, Valentin; Gordeliy, Valentin

    2015-01-01

    Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. PMID:26046789

  9. Cell-free protein expression in a microchannel array with passive pumping.

    PubMed

    Khnouf, Ruba; Beebe, David J; Fan, Z Hugh

    2009-01-07

    We report in vitro (cell-free) protein expression in a microfluidic device using passive pumping. The polystyrene device contains 192 microchannels, each of which is connected to two wells positioned in a 384-well microplate format. A larger droplet of an expression solution was placed at one well of each channel while a smaller droplet of a nutrient solution was at the other well. Protein expression took place in the larger droplet and we found the expression yield in the expression solution is enhanced due to the replenishment of the nutrient solution supplied by passive pumping via the channel. The pumping pressure was generated from the difference in the surface tension between two different sized droplets at the two wells. We demonstrated expression of luciferase in the device and the expression yield was measured using luminescence assay. Different experimental conditions were investigated to achieve maximum protein yield with the least amount of reagents. Protein expression yields were found to be dependent on the amount of the nutrient solution pumped, independent of the amount of the expression solution within the experimental conditions studied. A higher feeding frequency or delivery rate of the nutrient solution resulted in higher protein expression yield. The work demonstrated the feasibility of using the microchannel array for protein expression with the following advantages: (1) simultaneous production of the same protein with different conditions to optimize the expression process; (2) simultaneous production of different proteins for high-throughput protein expression with high yield; (3) low reagent cost due to the fact that it consumes 125-800 times less than the amount used in a protein expression instrument commercially available.

  10. Role of a TPA-responsive element in hepcidin transcription induced by the bone morphogenetic protein pathway.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2015-10-16

    Systemic iron balance is governed by the liver-derived peptide hormone hepcidin. The transcription of hepcidin is primarily regulated by the bone morphogenetic protein (BMP) and inflammatory cytokine pathways through the BMP-response element (BMP-RE) and STAT-binding site, respectively. In addition to these elements, we previously identified a TPA-responsive element (TRE) in the hepcidin promoter and showed that it mediated the transcriptional activation of hepcidin through activator protein (AP)-1 induced by serum. In the present study, we examined the role of TRE in the BMP-induced transcription of hepcidin in HepG2 liver cells. The serum treatment increased the basal transcription of hepcidin; however, responsiveness to the expression of ALK3(QD), a constitutively active BMP type I receptor, was unaffected. Consistent with these results, mutations in TRE in the hepcidin promoter decreased basal transcription, whereas responsiveness to the expression of ALK3(QD) remained unchanged. HepG2 cells significantly expressed AP-1 components in the basal state, whereas BMP did not up-regulate the expression of these components. The expression of c-fos enhanced the basal transcription of hepcidin as well as ALK3(QD)-mediated hepcidin transcription, whereas that of dominant-negative c-fos decreased hepcidin transcription. The results of the present study suggested that the cis-elements of the hepcidin promoter, BMP-RE and TRE, individually transmitted BMP-mediated and AP-1-mediated signals, respectively, whereas transcription was synergistically increased by the stimulation of BMP-RE and TRE.

  11. Expression of liver fatty acid binding protein in hepatocellular carcinoma☆

    PubMed Central

    Cho, Soo-Jin; Ferrell, Linda D.; Gill, Ryan M.

    2017-01-01

    Summary Loss of expression of liver fatty acid binding protein (LFABP) by immunohistochemistry has been shown to be characteristic of a subset of hepatocellular adenomas (HCAs) in which HNF1A is inactivated. Transformation to hepatocellular carcinoma is thought to be a very rare phenomenon in the HNF1A-inactivated variant of HCA. However, we recently observed 2 cases at our institution, 1 definite hepatocellular carcinoma and 1 possible hepatocellular carcinoma, with loss of LFABP staining, raising the possibility that LFABP down-regulation may be associated with hepatocellular carcinogenesis. Our aim was to evaluate hepatocellular carcinomas arising in various backgrounds and with varying degrees of differentiation for loss of LFABP staining. Twenty total cases of hepatocellular carcinoma were examined. Thirteen cases arose in a background of cirrhosis due to hepatitis C (n = 8) or steatohepatitis (n = 5); 7 cases arose in a noncirrhotic background, with 2 cases arising within HNF1A-inactivated variant HCA and 2 cases arising within inflammatory variant HCA. Complete loss of expression of LFABP was seen in 6 of 20 cases, including 2 cases of hepatocellular carcinoma arising within HNF1A-inactivated variant HCA. Thus, loss of staining for LFABP appears to be common in hepatocellular carcinoma and may be seen in well-differentiated hepatocellular carcinoma. Therefore, LFABP loss should not be interpreted as evidence for hepatocellular adenoma over carcinoma, when other features support a diagnosis of hepatocellular carcinoma. The findings raise consideration for a role of HNF1A inactivation in hepatocellular carcinogenesis, particularly in less differentiated tumors. PMID:26997447

  12. Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

    PubMed

    Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

    2015-12-30

    Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains.

  13. In silico studies on the interaction between bioactive ligands and ALK5, a biological target related to the cancer treatment.

    PubMed

    Almeida, Michell O; Trossini, Gustavo H G; Maltarollo, Vinícius G; Silva, Danielle da C; Honorio, Kathia M

    2016-09-01

    Studies have showed that there are many biological targets related to the cancer treatment, for example, TGF type I receptor (TGF-βRI or ALK5). The ALK5 inhibition is a strategy to treat some types of cancer, such as breast, lung, and pancreas. Here, we performed CoMFA and CoMSIA studies for 70 ligands with ALK5 inhibition. The internal validation for both models (q(2)LOO = 0.887 and 0.822, respectively) showed their robustness, while the external validations showed their predictive power (CoMFA: r(2)test = 0.998; CoMSIA: r(2)test = 0.975). After all validations, CoMFA and CoMSIA maps indicated physicochemical evidences on the main factors involved in the interaction between bioactive ligands and ALK5. Therefore, these results suggest molecular modifications to design new ALK5 inhibitors.

  14. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  15. [The heterologous expression and purification of membrane protein from Mycobacterium tuberculosis].

    PubMed

    Liao, Dan; Xie, Jian-Ping; Wang, Hong-Hai

    2007-10-01

    Membrane proteins fulfill a wide range of central functions in the cell, but their structure determination remains one of the great challenges in structural biology. The heterologous overexpression is a demanding task. Here, we provide an overview of recent advance to heterologous expression and purification of membrane protein from Mycobacterium tuberculosis, whose membrane proteins represent the majority of the new potential drug targets in this bacillus, which is ranked as the number1 cause of infectious disease mortality in the world. A detailed structural and functional understanding of the membranes protein of Mycobacterium tuberculosis will be critical both for an understanding of the biology of infection and for the rational development of novel therapeutics. The procedures for functional expression followed by purification of membranes protein are reviewed here together with nonfunctional expression in inclusion bodies and subsequent refolding to produce functional proteins. The new expression systems, new approaches to soluble expression of recombinant proteins, new methods for membrane protein folding in vitro and new purification technology will provide a basis for choosing the best expression and purification protocol for a given membrane protein. The goal of this review is to aid researchers in the choice of a suitable expression system for their favourite proteins and make overproduction of functional membrane proteins becomes easier.

  16. Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1▿

    PubMed Central

    Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

    2010-01-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs. PMID:20038711

  17. Inhibition of bone morphogenetic protein signaling attenuates anemia associated with inflammation.

    PubMed

    Steinbicker, Andrea U; Sachidanandan, Chetana; Vonner, Ashley J; Yusuf, Rushdia Z; Deng, Donna Y; Lai, Carol S; Rauwerdink, Kristen M; Winn, Julia C; Saez, Borja; Cook, Colleen M; Szekely, Brian A; Roy, Cindy N; Seehra, Jasbir S; Cuny, Gregory D; Scadden, David T; Peterson, Randall T; Bloch, Kenneth D; Yu, Paul B

    2011-05-05

    Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

  18. Heat shock protein expression enhances heat tolerance of reptile embryos.

    PubMed

    Gao, Jing; Zhang, Wen; Dang, Wei; Mou, Yi; Gao, Yuan; Sun, Bao-Jun; Du, Wei-Guo

    2014-09-22

    The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes.

  19. Porcine bladder acellular matrix (ACM): protein expression, mechanical properties.

    PubMed

    Farhat, Walid A; Chen, Jun; Haig, Jennifer; Antoon, Roula; Litman, Jessica; Sherman, Christopher; Derwin, Kathleen; Yeger, Herman

    2008-06-01

    Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

  20. Helicobacter pylori infection and expression of DNA mismatch repair proteins

    PubMed Central

    Mirzaee, Vahid; Molaei, Mahsa; Shalmani, Hamid Mohaghegh; Zali, Mohammad Reza

    2008-01-01

    AIM: To determine the expression of DNA (MMR) proteins, including hMLH1 and hMSH2, in gastric epithelial cells in the patients with or without Helicobacter pylori (H pylori)-infected gastritis. METHODS: Fifty H pylori-positive patients and 50 H pylori-negative patients were enrolled in the study. During endoscopy of patients with non-ulcer dyspepsia, two antral and two corpus biopsies were taken for histological examination (Giemsa stain) and for immunohistochemical staining of hMLH1 and hMSH2. RESULTS: The percentage of epithelial cell nuclei that demonstrated positivity for hMLH1 staining was 84.14 ± 7.32% in H pylori-negative patients, while it was 73.34 ± 10.10% in H pylori-positive patients (P < 0.0001). No significant difference was seen between the two groups regarding the percentage of epithelial cell nuclei that demonstrated positivity for hMSH2 staining (81.16 ± 8.32% in H pylori-negative versus 78.24 ± 8.71% in H pylori-positive patients; P = 0.09). CONCLUSION: This study indicates that H pylori might promote development of gastric carcinoma at least in part through its ability to affect the DNA MMR system. PMID:19034977

  1. Protein kinase Cmu plays an essential role in hypertonicity-induced heat shock protein 70 expression.

    PubMed

    Lim, Yun Sook; Lee, Jae Seon; Huang, Tai Qin; Seo, Jeong Sun

    2008-12-31

    Heat shock protein 70 (HSP70), which evidences important functions as a molecular chaperone and anti-apoptotic molecule, is substantially induced in cells exposed to a variety of stresses, including hypertonic stress, heavy metals, heat shock, and oxidative stress, and prevents cellular damage under these conditions. However, the molecular mechanism underlying the induction of HSP70 in response to hypertonicity has been characterized to a far lesser extent. In this study, we have investigated the cellular signaling pathway of HSP70 induction under hypertonic conditions. Initially, we applied a variety of kinase inhibitors to NIH3T3 cells that had been exposed to hypertonicity. The induction of HSP70 was suppressed specifically by treatment with protein kinase C (PKC) inhibitors (Gö6976 and GF109203X). As hypertonicity dramatically increased the phosphorylation of PKCmu, we then evaluated the role of PKCmu in hypertonicity-induced HSP70 expression and cell viability. The depletion of PKCmu with siRNA or the inhibition of PKCmu activity with inhibitors resulted in a reduction in HSP70 induction and cell viability. Tonicity-responsive enhancer binding protein (TonEBP), a transcription factor for hypertonicity-induced HSP70 expression, was translocated rapidly into the nucleus and was modified gradually in the nucleus under hypertonic conditions. When we administered treatment with PKC inhibitors, the mobility shift of TonEBP was affected in the nucleus. However, PKCmu evidenced no subcellular co-localization with TonEBP during hypertonic exposure. From our results, we have concluded that PKCmu performs a critical function in hypertonicity-induced HSP70 induction, and finally cellular protection, via the indirect regulation of TonEBP modification.

  2. A complete approach for recombinant protein expression training: From gene cloning to assessment of protein functionality*.

    PubMed

    Novo, M Teresa Marques; Soares-Costa, Andrea; de Souza, Antonia Q L; Figueira, Ana Carolina M; Molina, Gustavo C; Palacios, Carlos A; Kull, Claudia R; Monteiro, Izabel F; Baldan-Pineda, Paulo H; Henrique-Silva, Flavio

    2005-01-01

    A practical course was given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering" at the Federal University of São Carlos (UFSCar), São Paulo, Brazil. The goal of the course was to teach current molecular biology tools applied to a real research situation that could be reported by the students themselves. The purpose was to produce a plant recombinant protein and demonstrate a heretofore unreported biological activity. Cystatins, natural inhibitors of cysteine proteases, were proposed for these studies. Initially, the students searched for plant cystatin cDNA sequences in the NCBI databases and selected the Oryzacystatin I gene (ocI) from rice, Oriza sativa, as the target gene for this study. Total RNA was extracted from rice-germinating seeds and primers containing restriction sites for NdeI and EcoRI were designed based on the ocI cDNA sequence and then used to amplify the open reading frame (ORF). RT-PCR amplification provided a band of the expected size for ocI ORF (309 bp). The PCR product was cut with NdeI and EcoRI restriction enzymes and cloned directly in the pET28a expression vector digested with the same enzymes. A pET28-ocI recombinant clone was selected, checked by sequencing, and used to transform Escherichia coli BL21 (DE3) expression strain. After induction of the bacteria with isopropylthiogalactoside and cellular disruption, the His-tagged OCI protein, present mainly in the soluble fraction, was purified by affinity chromatography in a nickel column. The purified protein was successfully used to inhibit fungal growth (Trichoderma reesei). The results were discussed extensively and the students contributed to the writing of this article, of which they are co-authors.

  3. The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

    PubMed

    Koyama-Nasu, R; Haruta, R; Nasu-Nishimura, Y; Taniue, K; Katou, Y; Shirahige, K; Todo, T; Ino, Y; Mukasa, A; Saito, N; Matsui, M; Takahashi, R; Hoshino-Okubo, A; Sugano, H; Manabe, E; Funato, K; Akiyama, T

    2014-04-24

    Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

  4. ALK Positive Anaplastic Large Cell Lymphoma Presenting as Extensive Bone Involvement

    PubMed Central

    Gajendra, Smeeta; Lipi, Lipika; Goel, Shalini; Misra, Ruchira

    2015-01-01

    Anaplastic lymphoma kinase (ALK) positive Anaplastic large cell lymphoma (ALCL) represents approximately 2% of all Non-Hodgkin’s lymphomas that commonly involves nodal as well as a wide variety of extra nodal sites, as skin, soft tissue, bones and lungs, although primary or secondary involvement of bone is rare. Herein, we report a case of 14-year-old female child presented as extensive bony involvement with a clinical diagnosis of bone tumour/ small round cell tumour, which was proved to be ALK positive ALCL on histopathological examination. PMID:25738071

  5. The major form of hepatitis C virus alternate reading frame protein is suppressed by core protein expression

    PubMed Central

    Wolf, Marie; Dimitrova, Maria; Baumert, Thomas F.; Schuster, Catherine

    2008-01-01

    Hepatitis C virus (HCV) is a human RNA virus encoding 10 proteins in a single open reading frame. In the +1 frame, an ‘alternate reading frame’ (ARF) overlaps with the core protein-encoding sequence and encodes the ARF protein (ARFP). Here, we investigated the molecular regulatory mechanisms of ARFP expression in HCV target cells. Chimeric HCV-luciferase reporter constructs derived from the infectious HCV prototype isolate H77 were transfected into hepatocyte-derived cell lines. Translation initiation was most efficient at the internal AUG codon at position 86/88, resulting in the synthesis of a truncated ARFP named 86/88ARFP. Interestingly, 86/88ARFP synthesis was markedly enhanced in constructs containing an inactivated core protein reading frame. This enhancement was reversed by co-expression of core protein in trans, demonstrating suppression of ARFP synthesis by HCV core protein. In conclusion, our results indicate that translation of ARFP occurs mainly by alternative internal initiation at position 86/88 and is regulated by HCV core protein expression. The suppression of ARFP translation by HCV core protein suggests that ARFP expression is inversely linked to the level of viral replication. These findings define key mechanisms regulating ARFP expression and set the stage for further studies addressing the function of ARFP within the viral life cycle. PMID:18400784

  6. Differential expression of hemolymph proteins between susceptible and insecticide-resistant Blattella germanica (Blattodea: Blattellidae).

    PubMed

    Zhang, F; Wang, X J; Huang, Y H; Zhao, Z G; Zhang, S S; Gong, X S; Xie, L; Kang, D M; Jing, X

    2014-08-01

    A proteomic approach combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain and a beta-cypermethrin-susceptible strain. Twenty-eight hemolymph proteins were differentially expressed in the resistant cockroach strain; 19 proteins were upregulated and 9 proteins were downregulated compared with the susceptible strain. Protein identification indicated that expression of putative cuticular protein, nitric oxide synthase, triosephosphate isomerase, alpha-amylase, ABC transporter, and Per a 3 allergen was elevated, and expression of arginine kinase and glycosidase was reduced. The differential expression of these proteins reflects the overall change in cellular structure and metabolism related to the resistance of pyrethroid insecticides.

  7. Glucose enhances collectrin protein expression in insulin-producing MIN6 {beta} cells

    SciTech Connect

    Saisho, Kenji; Fukuhara, Atsunori; Yasuda, Tomoko; Sato, Yoshifumi; Fukui, Kenji; Iwahashi, Hiromi; Imagawa, Akihisa; Hatta, Mitsutoki; Shimomura, Iichiro; Yamagata, Kazuya

    2009-11-06

    Collectrin is a novel target gene of hepatocyte nuclear factor-1{alpha} in pancreatic {beta}-cells and controls insulin exocytosis. Although glucose is known to stimulate the expression of genes of the insulin secretory pathway, there is no information on how glucose regulates collectrin expression. We investigated the effects of glucose on the expression of collectrin in MIN6 {beta}-cell line. Glucose, in a dose-dependent manner, increased collectrin protein levels without changing collectrin mRNA levels and protein stability, indicating that glucose stimulation of collectrin protein expression is primarily mediated at a translational level. Although mannose and pyruvate also increased collectrin protein expression level, neither 2-deoxyglucose, mitochondrial fuels leucine and glutamate, sulphonylurea nor Ca{sup 2+} channel blockers, mimicked the effects of glucose. These data indicate the involvement of mitochondrial TCA cycle intermediates, distal to pyruvate, in the regulation of collectrin protein expression in {beta}-cells.

  8. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

    PubMed Central

    Sørensen, Hans Peter; Mortensen, Kim Kusk

    2005-01-01

    Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags. PMID:15629064

  9. p73 expression is regulated by ribosomal protein RPL26 through mRNA translation and protein stability

    PubMed Central

    Yan, Wensheng; Chen, Xinbin

    2016-01-01

    p73, a p53 family tumor suppressor, is regulated by multiple mechanisms, including transcription and mRNA and protein stability. However, whether p73 expression is regulated via mRNA translation has not been explored. To test this, we examined whether ribosomal protein 26 (RPL26) plays a role in p73 expression. Here, we showed that p73 expression is controlled by RPL26 via protein stability and mRNA translation. To examine whether MDM2 mediates RPL26 to regulate p73 protein stability, we generated multiple MDM2-knockout cell lines by CRISPR-cas9. We found that in the absence of MDM2, the half-life of p73 protein is markedly increased. Interestingly, we also found that RPL26 is still capable of regulating p73 expression, albeit to a lesser extent, in MDM2-KO cells compared to that in isogenic control cells, suggesting that RPL26 regulates p73 expression via multiple mechanisms. Indeed, we found that RPL26 is necessary for efficient assembly of polysomes on p73 mRNA and de novo synthesis of p73 protein. Consistently, we found that RPL26 directly binds to p73 3′ untranslated region (3′UTR) and that RPL26 is necessary for efficient expression of an eGFP reporter that carries p73 3′UTR. We also found that RPL26 interacts with cap-binding protein eIF4E and enhances the association of eIF4E with p73 mRNA, leading to increased p73 mRNA translation. Finally, we showed that knockdown of RPL26 promotes, whereas ectopic expression of RPL26 inhibits, cell growth in a TAp73-dependent manner. Together, our data indicate that RPL26 regulates p73 expression via two distinct mechanisms: protein stability and mRNA translation. PMID:27825141

  10. The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

    PubMed Central

    Haillot, Emmanuel; Molina, Maria Dolores; Lapraz, François; Lepage, Thierry

    2015-01-01

    Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial res