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Sample records for ccnd1 gene over-expression

  1. Mutual antagonism of the paired-type homeobox genes, vsx2 and dmbx1, regulates retinal progenitor cell cycle exit upstream of ccnd1 expression.

    PubMed

    Wong, Loksum; Power, Namita; Miles, Amanda; Tropepe, Vincent

    2015-06-15

    Understanding the mechanisms that regulate the transition between the proliferative and a post-mitotic state of retinal progenitor cells (RPCs) is key to advancing our knowledge of retinal growth and maturation. In the present study we determined that during zebrafish embryonic retinal neurogenesis, two paired-type homeobox genes - vsx2 and dmbx1 - function in a mutually antagonistic manner. We demonstrate that vsx2 gene expression requires active Fgf signaling and that this in turn suppresses dmbx1 expression and maintains cells in an undifferentiated, proliferative RPC state. This vsx2-dependent RPC state can be prolonged cell-autonomously by knockdown of dmbx1, or it can be suppressed prematurely by the over-expression of dmbx1, which we show can inhibit vsx2 expression and lead to precocious neuronal differentiation. dmbx1 loss of function also results in altered expression of canonical cell cycle genes, and in particular up-regulation of ccnd1, which correlates with our previous finding of a prolonged RPC cell cycle. By knocking down ccnd1 and dmbx1 simultaneously, we show that RPCs can overcome this phenotype to exit the cell cycle on time and differentiate normally into retinal neurons. Collectively, our data provide novel insight into the mechanism that enables RPCs to exit the cell cycle through a previously unrecognized antagonistic interaction of two paired-type homeobox genes that are central regulators of an Fgf-vsx2-dmbx1-ccnd1 signaling axis.

  2. Polymorphisms of cell cycle regulator genes CCND1 G870A and TP53 C215G: Association with colorectal cancer susceptibility risk in a Malaysian population

    PubMed Central

    ZAHARY, MOHD NIZAM; AHMAD AIZAT, ABDUL AZIZ; KAUR, GURJEET; YEONG YEH, LEE; MAZUWIN, MAYA; ANKATHIL, RAVINDRAN

    2015-01-01

    Colorectal cancer (CRC) occurs as a more common sporadic form and a less common familial form. Our earlier analysis of germline mutations of mismatch repair genes confirmed only 32% of familial CRC cases as Lynch syndrome cases. It was hypothesized that the remaining familial aggregation may be ‘polygenic’ due to single nucleotide polymorphisms (SNPs) of low penetrance genes involved in cancer predisposition pathways, such as cell cycle regulation and apoptosis pathways. The current case-control study involving 104 CRC patients (52 sporadic and 52 familial) and 104 normal healthy controls investigated the contribution of the SNPs cyclin D1 (CCND1) G870A and tumor protein p53 (TP53) C215G in modulating familial and sporadic CRC susceptibility risk. DNA was extracted from peripheral blood and the polymorphisms were genotyped by employing a polymerase chain reaction-restriction fragment length polymorphism method. The association between these polymorphisms and CRC susceptibility risk was calculated using a binary logistic regression analysis and deriving odds ratios (ORs). The A/A variant genotype of CCND1 and G/G variant genotype of TP53 exhibited a significantly greater association with the risk of sporadic CRC [CCND1: OR, 3.471; 95% confidence interval (CI), 1.443–8.350; P=0.005. TP53: OR, 2.829; CI, 1.119–7.152; P=0.026] as well as familial CRC susceptibility (CCND1: OR, 3.086; CI, 1.270–7.497; P=0.019. TP53: OR, 3.048; CI, 1.147–8.097; P=0.030). The results suggest a potential role of the SNPs CCND1 G870A and TP53 C215G in the modulation of sporadic and familial CRC susceptibility risk. PMID:26722315

  3. MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1.

    PubMed

    Han, Kang; Chen, Xiang; Bian, Na; Ma, Baoan; Yang, Tongtao; Cai, Chengkui; Fan, QingYu; Zhou, Yong; Zhao, Ting Bao

    2015-04-20

    Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma.

  4. MicroRNA profiling identifies MiR-195 suppresses osteosarcoma cell metastasis by targeting CCND1.

    PubMed

    Han, Kang; Chen, Xiang; Bian, Na; Ma, Baoan; Yang, Tongtao; Cai, Chengkui; Fan, QingYu; Zhou, Yong; Zhao, Ting Bao

    2015-04-20

    Metastasis is a leading cause of mortality for osteosarcoma patients. The molecular pathological mechanism remains to be elucidated. In the previously study, we established two osteosarcoma cell lines with different metastatic potentials. Differential expressed genes and proteins regarding metastatic ability have been identified. MicroRNAs are important regulators in tumorigenesis and tumor progression. In this study, microRNA microarray was used to assess the differential expressed miRNAs level between these two cell lines. One of the top ranked miRNAs-miR-195 was identified highly expressing in lowly metastatic cells. It was showed that over-expression of miR-195 substantially inhibits migration and invasion of osteosarcoma cells in vitro and pulmonary metastasis formation in vivo. Meanwhile, CCND1 was identified as the target gene of miR-195 and further studied. More importantly, using real-time PCR, we evaluated the expression of miR-195 and CCND1 in osteosarcoma samples from 107 frozen biopsy tissues and 99 formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues. Results indicated lowly expressed miR-195 or highly CCND1 correlated with positive overall survival and their expression inversely related to each other. In summary, our study suggests miR-195 functions as a tumor metastasis suppressor gene by down-regulating CCND1 and can be used as a potential target in the treatment of osteosarcoma. PMID:25823925

  5. Mammary analogue secretory carcinoma of salivary glands with high-grade transformation: report of 3 cases with the ETV6-NTRK3 gene fusion and analysis of TP53, β-catenin, EGFR, and CCND1 genes.

    PubMed

    Skálová, Alena; Vanecek, Tomas; Majewska, Hanna; Laco, Jan; Grossmann, Petr; Simpson, Roderick H W; Hauer, Lukas; Andrle, Pavel; Hosticka, Lubor; Branžovský, Jindrich; Michal, Michal

    2014-01-01

    Mammary analogue secretory carcinoma of salivary gland origin (MASC) is a recently described tumor resembling secretory carcinoma of the breast characterized by strong S-100 protein, mammaglobin, and vimentin immunoexpression and which harbors a t(12;15) (p13;q25) translocation resulting in ETV6-NTRK3 fusion product. Histologically, conventional MASC displays bland histomorphology and a lobulated growth pattern and is often composed of microcystic, tubular, and solid structures with abundant eosinophilic homogenous or bubbly secretions. Colloid-like secretory material stains positively for periodic acid-Schiff with and without diastase as well as for Alcian Blue. We present for the first time, 3 patients with MASC of the parotid gland in which high-grade (HG) transformation developed in each case characterized by an accelerated clinical course and poor outcome. The HG component revealed strong membrane staining for EGFR and β-catenin, cytoplasmic/nuclear staining for S-100 protein, and nuclear staining for cyclin-D1, whereas HER-2/neu was absent. Analysis for the presence of the ETV6-NTRK3 fusion transcript revealed positivity in both HG and low-grade component of MASC in 2 of the 3 studied cases. The tumor in case 2 was negative in both its elements for the t(12;15) translocation, but ETV6 gene rearrangement was detected in both components in all 3 cases. Analysis of TP53 and CTNNB1 gene mutations in the HG component of MASCs as well as detection of copy number aberration of EGFR and CCND1 gene did not harbor any abnormalities. All 3 patients with HG-transformed MASC died of disseminated disease within 2 to 6 years after diagnosis. Recognizing HG-transformed MASC and testing for ETV6 rearrangement may be of potential value in patient treatment, because the presence of the ETV6-NTRK3 translocation may represent a therapeutic target in MASC. PMID:24145651

  6. Promoter-associated noncoding RNA from the CCND1 promoter.

    PubMed

    Song, Xiaoyuan; Wang, Xiangting; Arai, Shigeki; Kurokawa, Riki

    2012-01-01

    More than 90% of the human genome have been found to be transcribed and most of the transcripts are noncoding (nc) RNAs (Willingham et al., Science 309:1570-1573, 2005; ENCODE-consortium, Science 306:636-640, 2004; Carninci et al., Science 309:1559-1563, 2005; Bertone et al., Science 306:2242-2246, 2004). Studies on ncRNAs have been radically progressed mainly regarding microRNAs, piRNAs, siRNAs, and related small ncRNAs of which length are relatively short nucleotides (Fire et al., Nature 391:806-811, 1998; Filipowicz et al., Nat Rev Genet 9:102-114, 2008; Lau et al., Science 313:363-367, 2006; Brennecke et al., Science 322:1387-1392, 2008; Siomi and Siomi, Nature 457:396-404, 2009). These small RNAs play roles in regulation of translation and gene silencing while long ncRNAs with length more than 200 nucleotides have been emerging and turn out to be involved in regulation of transcription (Kapranov et al., Science 316:1484-1488, 2007; Ponting et al., Cell 136:629-641, 2009; Kurokawa et al., RNA Biol 6:233-236, 2009). Recently, we have identified novel, long ncRNAs bearing capability of repression of transcription (Wang et al., Nature 454:126-130, 2008).RNA-binding protein, translocated in liposarcoma (TLS), binds CREB-binding protein CBP/adenovirus p300 and inhibits their histone acetyltransferase (HAT) activities (Wang et al., Nature 454:126-130, 2008). The HAT inhibitory activity of TLS requires specific binding of RNA. The systematic evolution of ligands by exponential enrichment experiments with randomized sequences revealed that TLS specifically recognizes RNA oligonucleotides containing GGUG as a consensus sequence although the GGUG sequence is not an absolute requirement for the TLS binding (Lerga et al., J Biol Chem 276:6807-6816, 2001). TLS is specifically recruited to the CBP/p300-associated binding sites of the cyclin D1 gene (CCND1) and the cyclin E1 gene (CCNE1) promoters (Wang et al., Nature 454:126-130, 2008; Impey et al., Cell 119:1041-1054, 2004

  7. Detection of CCND1 amplification using laser capture microdissection coupled with real-time polymerase chain reaction in human esophageal squamous cell carcinoma.

    PubMed

    Huang, Chen; Yang, Lin; Li, Zhongfang; Yang, Jun; Zhao, Junjie; Dehui, Xu; Liu, Liying; Wang, Quanli; Song, Tusheng

    2007-05-01

    Several methods have been used to detect CCND1 amplification or overexpression in esophageal squamous cell carcinoma (ESCC), but problems remain, associated with heterogeneity of tumor tissue and quantification of gene copies. Laser capture microdissection coupled with real-time polymerase chain reaction (PCR) is a reliable method for the molecular analysis of gene profiles in specific tissues. All 35 specimens of ESCC studied were paraffin-embedded, cut into tissue slides, and stained by hematoxylin-eosin. The pure ESCC cell and normal squamous epithelia populations were separated by LCM and then genomic DNA was extracted from the dissected cells. CCND1 amplification was detected with real-time FQ-PCR and with PCR. Amplification was calculated by the formula X = 2(-DeltaDeltaCt) and R = (CCND1/ACTB) CANCER/(CCND1/ACTB) NORMAL. Twenty (57%) of primary ESCC cancer cell groups had a detectable CCND1 amplification (range, 2.06-fold to 25.9-fold) with real-time FQ-PCR, but only 2 of 15 primary ESCC cancer cell groups had detectable CCND1 amplification by PCR. CCND1 amplification was not correlated with age, sex, size of tumor, histological grade, and lymph node metastasis. In conclusion, LCM coupled with real-time fluorescence quantitative-PCR technique is more precise than PCR for the identifying amplified oncogenes; The role of CCND1 amplification in ESCC development and progression needs more extensive study.

  8. Expression of Cyclin d1 protein and CCND1 та PNKP genes in peripheral blood mononuclear cells in clean up worker of Chornobyl accident with different state of immune system.

    PubMed

    Bazyka, D A; Kubashko, A V; Ilyenko, I M; Belyaev, O A; Pleskach, O J

    2015-12-01

    Meta. Doslidyty zminy rivniv Cyclin D1+ klityn ta asotsiyovanykh geniv CCND1 ta PNKP u mononuklearakh peryfe rychnoI krovi v uchasnykiv likvidatsiI naslidkiv avariI (ULNA) na ChAES z riznym imunnym statusom v zalezhnosti vid dozy oprominennia.Materialy i metody. Proanalizovano vidnosnyy riven' Cyclin D1+ klityn u mononuklearakh peryferychnoI krovi 39 ULNA na ChAES, cholovikiv, oprominenykh u dozi u diapazoni (0,01–2,00) Gr. Imunologichnyy status obstezhenykh vyz nachavsia za rivnem CD3/19, CD4/8, CD3/HLA DR, SD3/16/56 metodom protochnoI tsytofluorymetriI ta za vmistom Ig klasiv A,M,G metodom imunofermentnogo analizu u krovi. Ekspresiia geniv CCND1 ta PNKP, iaki pov’iazani z Syclin D1, provodylos' za metodom polimeraznoI lantsiugovoI reaktsiI u real'nomu chasi. Porivniannia rezul'tativ zdiysniuva los' iz vidpovidnymy danymy, otrymanymy vid 18 zdorovykh cholovikiv, iaki ne maly kontaktu z ionizuiuchym vyp rominiuvanniam vyshche pryrodn'ogo fonu.Rezul'taty. Pokazano, shcho vidsotok Suclin D1+ klityn zbil'shuiet'sia za normu v osib, oprominenykh u dozi > 0,1 Gr, ta koreliuie z dozoiu oprominennia (rs = 0,417, p = 0,048). Vidkhylennia rivnia Cyclin D1+ klityn za mezhi kontrol'nykh zna chen' pov’iazuiet'sia zi zminamy v klitynniy ta gumoral'niy lankakh imunitetu. Zmenshennia vidsotku Cyclin D1+ klityn za mezhi kontrol'nykh znachen' v ULNA na ChAES iz dozoiu < 0,35 Gr suprovodzhuiet'sia znyzhenniam rivniv CD3+ ta pidvy shchenniam CD3 16+56+ limfotsytiv; u osib, oprominenykh u dozi > 0,35 Gr, zbil'shennia vidsotku Cyclin D1+ klityn asotsiiuiet'sia zi znyzhenniam CD3+ ta tendentsiieiu shchodo znyzhennia CD3+16+56+ limfotsytiv u poiednanni zi zbil'shen niam rivnia IgG. Zbil'shennia rivniv CD4+, CD19+, Ireg. ta IgG suprovodzhuiet'sia poiavoiu koreliatsiynykh zv’iazkiv mizh Cyclin D1+ ta CD3 16+56+ klitynamy (rs = 0,872, p = 0,049), Cyclin D1+ ta CD8+ i IgG (rs = 0,683, p = 0,042; rs = 0,809, p = 0,014), Cyclin D1+ ta CD4+ (rs = 0,602, p = 0,029), Cyclin D1+ ta CD19+ i

  9. Heterogeneous abnormalities of CCND1 and RB1 in primary cutaneous T-Cell lymphomas suggesting impaired cell cycle control in disease pathogenesis.

    PubMed

    Mao, Xin; Orchard, Guy; Vonderheid, Eric C; Nowell, Peter C; Bagot, Martine; Bensussan, Armand; Russell-Jones, Robin; Young, Bryan D; Whittaker, Sean J

    2006-06-01

    Upregulation of cyclin D1/B-cell leukemia/lymphoma 1 (CCND1/BCL1) is present in most mantle cell lymphomas with the t(11;14)(q13;q32) translocation. However, little is known about the abnormalities of CCND1 and its regulator RB1 in primary cutaneous T-cell lymphomas (CTCL). We analyzed CCND and RB status in CTCL using fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), and Affymetrix expression microarray. FISH revealed loss of CCND1/BCL1 in five of nine Sézary syndrome (SS) cases but gain in two cases, and RB1 loss in four of seven SS cases. IHC showed absent CCND1/BCL1 expression in 18 of 30 SS, 10 of 23 mycosis fungoides (MF), and three of 10 primary cutaneous CD30+ anaplastic large-cell lymphoma (C-ALCL). Increased CCND1/BCL1 expression was seen in nine MF, seven C-ALCL, and six SS cases. Absent RB1 expression was detected in 8 of 12 MF and 7 of 9 SS cases, and raised RB1 expression in 7 of 8 C-ALCL. Affymetrix revealed increased gene expression of CCND2 in four of eight CTCL cases, CCND3 in three cases, and CDKN2C in two cases with a normal expression of CCND1 and RB1. These findings suggest heterogeneous abnormalities of CCND and RB in CTCL, in which dysregulated CCND and RB1 may lead to impaired cell cycle control.

  10. Prognostic value of differential CCND1 expression in patients with resected gastric adenocarcinoma.

    PubMed

    Ma, Liqiang; Wang, Xiaoting; Lan, Fenghua; Yu, Yinghao; Ouyang, Xuenong; Liu, Wei; Xie, Feilai; Huang, Qiaojia

    2015-01-01

    Cyclin D1 (CCND1) plays essential roles in cancer progression. In this study, CCND1 expression patterns in 211 cases of resected gastric adenocarcinoma (RGA) tissue were determined by immunohistochemistry, and the association between CCND1 expression levels and RGA prognosis was analyzed. RGA tissues displayed differential CCND1 expression (high expression, 52.1 %; n = 110, and low expression, 47.9 %; n = 101). CCND1 expression levels were related with median overall survival time (MST). MST in patients with high CCND1 expression was 43 months, whereas with low CCND1 expression it was 62 months (P = 0.013). When data were stratified by postoperative treatments and CCND1 expression levels, the MST for patients treated with fluoropyrimidine plus platinum (n = 140) was significantly longer than for those treated with fluoropyrimidine only (n = 71) in both high and low CCND1 expression groups (65.0 vs. 29.0 months, P = 0.041; and 74.5 vs. 33.0 months, P = 0.003, respectively). Cox multivariate analyses further confirmed that high CCND1 expression was related with poor prognosis in both treatment groups [hazard ratio (HR) 1.91, 95 % confidence interval (CI) 1.12-3.23; P = 0.017, and HR 2.14, 95 % CI 1.08-4.25; P = 0.029] and that fluoropyrimidine plus platinum was more effective than fluoropyrimidine only in high CCND1 (HR 0.47, 95 % CI 0.28-0.78; P = 0.004) and low CCND1 (HR 0.44; 95 % CI 0.23-0.82; P = 0.01) expression patients. Therefore, CCND1 may be used as a prognostic biomarker for patients with RGA.

  11. Over-expression of a protein kinase gene enhances the defense of tobacco against Rhizoctonia solani.

    PubMed

    Chacón, Osmany; González, Marleny; López, Yunior; Portieles, Roxana; Pujol, Merardo; González, Ernesto; Schoonbeek, Henk-Jan; Métraux, Jean-Pierre; Borrás-Hidalgo, Orlando

    2010-03-01

    To identify Nicotiana tabacum genes involved in resistance and susceptibility to Rhizoctonia solani, suppression subtractive hybridization was used to generate a cDNA library from transcripts that are differentially expressed during a compatible and incompatible interaction. This allowed the isolation of a protein kinase cDNA that was down-regulated during a compatible and up-regulated during an incompatible interaction. Quantitative RT-PCR analysis of this gene confirmed the differential expression patterns between the compatible and incompatible interactions. Over-expression of this gene in tobacco enhanced the resistance to damping-off produced by an aggressive R. solani strain. Furthermore, silencing of this protein kinase gene reduced the resistance to a non-aggressive R. solani strain. A set of reported tobacco-resistant genes were also evaluated in tobacco plants over-expressing and silencing the protein kinase cDNA. Several genes previously associated with resistance in tobacco, like manganese superoxide dismutase, Hsr203J, chitinases and phenylalanine ammonia-lyase, were up-regulated in tobacco plants over-expressing the protein kinase cDNA. Potentially, the protein kinase gene could be used to engineer resistance to R. solani in tobacco cultivars susceptible to this important pathogen.

  12. Reduced seed germination in Arabidopsis over-expressing SWI/SNF2 ATPase genes.

    PubMed

    Leeggangers, Hendrika A C F; Folta, Adam; Muras, Aleksandra; Nap, Jan-Peter; Mlynarova, Ludmila

    2015-02-01

    In the life of flowering plants, seed germination is a critical step to ensure survival into the next generation. Generally the seed prior to germination has been in a dormant state with a low rate of metabolism. In the transition from a dormant seed to a germinating seed, various epigenetic mechanisms play a regulatory role. Here, we demonstrate that the over-expression of chromatin remodeling ATPase genes (AtCHR12 or AtCHR23) reduced the frequency of seed germination in Arabidopsis thaliana up to 30% relative to the wild-type seeds. On the other hand, single loss-of-function mutations of the two genes did not affect seed germination. The reduction of germination in over-expressing mutants was more pronounced in stress conditions (salt or high temperature), showing the impact of the environment. Reduced germinations upon over-expression coincided with increased transcript levels of seed maturation genes and with reduced degradation of their mRNAs stored in dry seeds. Our results indicate that repression of AtCHR12/23 gene expression in germinating wild-type Arabidopsis seeds is required for full germination. This establishes a functional link between chromatin modifiers and regulatory networks towards seed maturation and germination.

  13. Strategies used for genetically modifying bacterial genome: ite-directed mutagenesis, gene inactivation, and gene over-expression*

    PubMed Central

    Xu, Jian-zhong; Zhang, Wei-guo

    2016-01-01

    With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

  14. Over-expression of poplar transcription factor ERF76 gene confers salt tolerance in transgenic tobacco.

    PubMed

    Yao, Wenjing; Wang, Lei; Zhou, Boru; Wang, Shengji; Li, Renhua; Jiang, Tingbo

    2016-07-01

    Ethylene response factors (ERFs) belong to a large plant-specific transcription factor family, which play a significant role in plant development and stress responses. Poplar ERF76 gene, a member of ERF TF family, can be up-regulated in response to salt stress, osmotic stress, and ABA treatment. The ERF76 protein was confirmed to be targeted preferentially in the nucleus of onion cell by particle bombardment. In order to understand the functions of ERF76 gene in salt stress response, we conducted temporal and spatial expression analysis of ERF76 gene in poplar. Then the ERF76 cDNA fragment containing an ORF was cloned from di-haploid Populus simonii×P. nigra and transferred into tobacco (Nicotiana tobacum) genome by Agrobacterium-mediated leaf disc method. Under salt stress, transgenic tobacco over-expressing ERF76 gene showed a significant increase in seed germination rate, plant height, root length, and fresh weight, as well as in relative water content (RWC), superoxide dismutase (SOD) activity, peroxidase (POD) activity, and proline content, compared to control tobacco lines. In contrast, transgenic tobacco lines displayed a decrease in malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and reactive oxygen species (ROS) accumulation in response to salt stress, compared to control tobacco lines. Over all, the results indicated that ERF76 gene plays a critical role in salt tolerance in transgenic tobacco. PMID:27123829

  15. Frequent occurrence of CCND1 deregulation in patients with early stages of plasma cell dyscrasia.

    PubMed

    Miura, Kazuhisa; Iida, Shinsuke; Hanamura, Ichiro; Kato, Miyuki; Banno, Shogo; Ishida, Takashi; Kusumoto, Shigeru; Takeuchi, Genji; Miwa, Hiroshi; Nitta, Masakazu; Inagaki, Hiroshi; Eimoto, Tadaaki; Nomura, Kenichi; Taniwaki, Masafumi; Ueda, Ryuzo

    2003-04-01

    Chromosomal translocations involving the immunoglobulin heavy chain gene (IgH) and nonrandom protooncogene loci are the hallmark of genetic alterations found not only in multiple myeloma (MM), but also in premalignant stages of MM, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). We studied the frequency of IgH (14q32) rearrangements and their partner chromosomes in 16 Japanese patients with MGUS (13 cases), and SMM (3 cases) by means of interphase double-color fluorescence in situ hybridization (DCFISH) applied to purified plasma cells and using CD138-bead selection. IgH rearrangement was recognized in nine of the patients (56.3%). Protooncogene loci juxtaposed to IgH were identified in seven cases including CCND1 (11q13) in six cases and FGFR3 (4p16) in one. Four out of the six t(11;14)-positive cases showed nuclear staining of the cyclin D1 protein, whereas none of the seven t(11;14)-negative cases did. Moreover, neither MUM1(6p25)-IgH nor MAFB(20q11)-IgH fusion signals were observed. This suggests to us that cyclin D1 deregulation due to the presence of t(11;14) is involved in the early development of plasma cell neoplasms, and that this event alone is not enough for the development of symptomatic myeloma. PMID:12824903

  16. Dataset of differentially expressed genes from SOX9 over-expressing NT2/D1 cells.

    PubMed

    Ludbrook, Louisa; Alankarage, Dimuthu; Bagheri-Fam, Stefan; Harley, Vincent

    2016-12-01

    The data presents the genes that are differentially up-regulated or down-regulated in response to SOX9 in a human Sertoli-like cell line, NT2/D1. The dataset includes genes that may be implicated in gonad development and are further explored in our associated article, "SOX9 Regulates Expression of the Male Fertility Gene Ets Variant Factor 5 (ETV5) during Mammalian Sex Development" (D. lankarage, R. Lavery, T. Svingen, S. Kelly, L.M. Ludbrook, S. Bagheri-Fam, et al., 2016) [1]. The necessity of SOX9 for male sex development is evident in instances where SOX9 is lost, as in 46, XY DSD where patients are sex reversed or in mouse knock-out models, where mice lacking Sox9 are sex reversed. Despite the crucial nature of this transcriptional activator, downstream target genes of SOX9 remain largely undiscovered. Here, we have utilized NT2/D1 cells to transiently over-express SOX9 and performed microarray analysis of the RNA. Microarray data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-3378. PMID:27656672

  17. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    PubMed

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants.

  18. JARID2 inhibits leukemia cell proliferation by regulating CCND1 expression.

    PubMed

    Su, Chang-Liang; Deng, Tao-Ran; Shang, Zhen; Xiao, Yi

    2015-07-01

    It has recently been shown that JARID2 contributes to the malignant character of solid tumors, such as epithelial-mesenchymal transition in lung and colon cancer cell lines, but its role in leukemia progression is unexplored. In this study, we explored the effect and underlying molecular mechanism of JARID2 on leukemia cell proliferation. Real-time PCR and Western assay were carried out to detect JARID2 and CCND1 expression. Cell number and cell cycle change were detected using hemocytometer and flow cytometry, and a ChIP assay was utilized to investigate JARID2 and H3K27me3 enrichment on the CCND1 promoter. JARID2 is down-regulated in B-chronic lymphocytic leukemia (B-CLL) and acute monocytic leukemia (AMOL), and knockdown of JARID2 promotes leukemia cell proliferation via acceleration of the G1/S transition. Conversely, ectopic expression of JARID2 inhibits these malignant phenotypes. Mechanistic studies show that JARID2 negatively regulates CCND1 expression by increasing H3K27 trimethylation on the CCND1 promoter. Our findings indicate that JARID2 is a negative regulator of leukemia cell proliferation, and functions as potential tumor suppressor in leukemia.

  19. Decreased PM10 Exposure Attenuates Age-Related Lung Function Decline: Genetic Variants in p53, p21, and CCND1 Modify This Effect

    PubMed Central

    Imboden, Medea; Schwartz, Joel; Schindler, Christian; Curjuric, Ivan; Berger, Wolfgang; Liu, Sally L.J.; Russi, Erich W.; Ackermann-Liebrich, Ursula; Rochat, Thierry; Probst-Hensch, Nicole M.

    2009-01-01

    Background Decreasing exposure to airborne particulates was previously associated with reduced age-related decline in lung function. However, whether the benefit from improved air quality depends on genetic background is not known. Recent evidence points to the involvement of the genes p53 and p21 and of the cell cycle control gene cyclin D1 (CCND1) in the response of bronchial cells to air pollution. Objective We determined in 4,326 participants of the Swiss Cohort Study on Air Pollution and Lung and Heart Diseases in Adults (SAPALDIA) whether four single-nucleotide polymorphisms in three genes [CCND1 (rs9344 [P242P], rs667515), p53 (rs1042522 [R72P]), and p21 (rs1801270 [S31R])] modified the previously observed attenuation of the decline in the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF25–75) associated with improved air quality. Methods Subjects of the prospective population-based SAPALDIA cohort were assessed in 1991 and 2002 by spirometry, questionnaires, and biological sample collection for genotyping. We assigned spatially resolved concentrations of particulate matter with aerodynamic diameter ≤ 10 μm (PM10) to each participant’s residential history 12 months before the baseline and follow-up assessments. Results The effect of diminishing PM10 exposure on FEF25–75 decline appeared to be modified by p53 R72P, CCND1 P242P, and CCND1 rs667515. For example, a 10-μg/m3 decline in aver-age PM10 exposure over an 11-year period attenuated the average annual decline in FEF25–75 by 21.33 mL/year (95% confidence interval, 10.57–32.08) among participants homozygous for the CCND1 (P242P) GG genotype, by 13.72 mL/year (5.38–22.06) among GA genotypes, and by 6.00 mL/year (−4.54 to 16.54) among AA genotypes. Conclusions Our results suggest that cell cycle control genes may modify the degree to which improved air quality may benefit respiratory function in adults. PMID:19750108

  20. PROFILES OF GENE EXPRESSION ASSOCIATED WITH TETRACYCLINE OVER EXPRESSION OF HSP70 IN MCF-7 BREAST CANCER CELLS

    EPA Science Inventory

    Profiles of gene expression associated with tetracycline over expression of HSP70 in MCF-7 breast cancer cells.

    Heat shock proteins (HSPs) protect cells from damage through their function as molecular chaperones. Some cancers reveal high levels of HSP70 expression in asso...

  1. Over-expression of TaEXPB23, a wheat expansin gene, improves oxidative stress tolerance in transgenic tobacco plants.

    PubMed

    Han, Yangyang; Chen, Yanhui; Yin, Suhong; Zhang, Meng; Wang, Wei

    2015-01-15

    Expansins are cell wall proteins inducing cell wall loosening and participate in all plant growth and development processes which are associated with cell wall modifications. Here, TaEXPB23, a wheat expansin gene, was investigated and the tolerance to oxidative stress was strongly enhanced in over-expression tobacco plants. Our results revealed that over-expressing TaEXPB23 influenced the activity of antioxidant enzymes: in particular, the activity of the cell wall-bound peroxidase. The enhanced tolerance to oxidative stress and increased cell wall-bound peroxidase activity were partly inhibited by an anti-expansin antibody. The Arabidopsis expansin mutant atexpb2 showed reduced cell wall-bound peroxidase activity and decreased oxidative stress tolerance. In addition, atexpb2 exhibited lower chlorophyll contents and the germination rate compared to wild type (WT). Taken together, these results provided a new insight on the role of expansin proteins in plant stress tolerance by cell wall bound peroxidase.

  2. Enhanced skin carcinogenesis and lack of thymus hyperplasia in transgenic mice expressing human cyclin D1b (CCND1b)

    PubMed Central

    Rojas, Paola; Benavides, Fernando; Blando, Jorge; Perez, Carlos; Cardenas, Kim; Richie, Ellen; Knudsen, Erik S.; Johnson, David G.; Senderowicz, Adrian M.; Rodriguez-Puebla, Marcelo L.; Conti, Claudio J.

    2009-01-01

    Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties. PMID:18942117

  3. Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility.

    PubMed

    Jiang, Zhou; Sharfstein, Susan T

    2008-05-01

    Sodium butyrate treatment can increase the specific productivity of recombinant proteins in mammalian cells; however, it dramatically decreases cell growth and frequently leads to apoptosis. We have studied the responses of several Chinese hamster ovary (CHO) cells lines with different specific productivities (qP) to sodium butyrate treatment. Cell clones with lower productivities exhibited greater enhancement from butyrate treatment than cells with higher productivities. As we observed previously in cell clone characterization (Jiang et al., 2006. Biotechnol Prog 22: 313-318), heavy chain (HC) mRNA levels correlate very well with specific productivity and are amplified by butyrate treatment, indicating that sodium butyrate regulates the HC transcription. Sodium butyrate is an inhibitor of histone deacetylation, and possibly, increases gene transcription by enhancing gene accessibility to transcription factors. In this study, we applied DNase I footprinting to probe the HC and LC gene accessibility. We determined that more HC and LC gene copies are accessible by DNase I in sodium butyrate-treated CHO cells than in untreated controls, demonstrating that sodium butyrate regulates gene transcription by improving gene accessibility. However, the increase in accessibility did not correlate with the increase in transcript abundance, suggesting that butyrate enhances transcription by other mechanisms as well.

  4. Prognostic Importance of Cell Cycle Regulators Cyclin D1 (CCND1) and Cyclin-Dependent Kinase Inhibitor 1B (CDKN1B/p27) in Sporadic Gastric Cancers

    PubMed Central

    Minarikova, Petra; Halkova, Tereza; Belsanova, Barbora; Tuckova, Inna; Belina, Frantisek; Dusek, Ladislav; Zavoral, Miroslav

    2016-01-01

    Background. Gastric cancer is known for a notable variety in the course of the disease. Clinical factors, such as tumor stage, grade, and localization, are key in patient survival. It is expected that molecular factors such as somatic mutations and gene amplifications are also underlying tumor biological behavior and may serve as factors for prognosis estimation. Aim. The purpose of this study was to examine gene amplifications from a panel of genes to uncover potential prognostic marker candidates. Methods. A panel of gene amplifications including 71 genes was tested by multiplex ligation-dependent probe amplification (MLPA) technique in 76 gastric cancer samples from a Caucasian population. The correlation of gene amplification status with patient survival was determined by the Kaplan-Meier method. Results. The amplification of two cell cycle regulators, CCND1 and CDKN1B, was identified to have a negative prognostic role. The medial survival of patients with gastric cancer displaying amplification compared to patients without amplification was 192 versus 725 days for CCND1 (P = 0.0012) and 165 versus 611 days for CDKN1B (P = 0.0098). Conclusion. Gene amplifications of CCND1 and CDKN1B are potential candidates to serve as prognostic markers for the stratification of patients based on the estimate of survival in the management of gastric cancer patients. PMID:27781065

  5. Chilling temperature stimulates growth, gene over-expression and podophyllotoxin biosynthesis in Podophyllum hexandrum Royle.

    PubMed

    Yang, De Long; Sun, Ping; Li, Meng Fei

    2016-10-01

    Podophyllotoxin (PPT) and its derivatives, isolated from the rhizome of Podophyllum hexandrum Royle (P. hexandrum), are typically used in clinical settings for anti-cancer and anti-virus treatments. Empirical studies have verified that P. hexandrum had stronger tolerance to chilling, due to involving PPT accumulation in rhizome induced by cold stress. However, the cold-adaptive mechanism and its association with PPT accumulation at a molecular level in P. hexandrum are still limited. In this study, the morpho-physiological traits related to plant growth, PPT accumulation and key gene expressions controlling PPT biosynthesis were assessed by exposing P. hexandrum seedlings to different temperatures (4 °C and 10 °C as chilling stress and 22 °C as the control). The results showed that chilling significantly increased chlorophyll content, net photosynthetic rate, stomatal conductance, and plant biomass, whereas it greatly decreased transpiration rates and intercellular CO2 concentration. Compared to the control, the chilling treatments under 4 °C and 10 °C conditions induced a 5.00- and 3.33-fold increase in PPT contents, respectively. The mRNA expressions of six key genes were also up-regulated by chilling stresses. The findings are useful in understanding the molecular basis of P. hexandrum response to chilling.

  6. Chilling temperature stimulates growth, gene over-expression and podophyllotoxin biosynthesis in Podophyllum hexandrum Royle.

    PubMed

    Yang, De Long; Sun, Ping; Li, Meng Fei

    2016-10-01

    Podophyllotoxin (PPT) and its derivatives, isolated from the rhizome of Podophyllum hexandrum Royle (P. hexandrum), are typically used in clinical settings for anti-cancer and anti-virus treatments. Empirical studies have verified that P. hexandrum had stronger tolerance to chilling, due to involving PPT accumulation in rhizome induced by cold stress. However, the cold-adaptive mechanism and its association with PPT accumulation at a molecular level in P. hexandrum are still limited. In this study, the morpho-physiological traits related to plant growth, PPT accumulation and key gene expressions controlling PPT biosynthesis were assessed by exposing P. hexandrum seedlings to different temperatures (4 °C and 10 °C as chilling stress and 22 °C as the control). The results showed that chilling significantly increased chlorophyll content, net photosynthetic rate, stomatal conductance, and plant biomass, whereas it greatly decreased transpiration rates and intercellular CO2 concentration. Compared to the control, the chilling treatments under 4 °C and 10 °C conditions induced a 5.00- and 3.33-fold increase in PPT contents, respectively. The mRNA expressions of six key genes were also up-regulated by chilling stresses. The findings are useful in understanding the molecular basis of P. hexandrum response to chilling. PMID:27314513

  7. Transgenic Over Expression of Nicotinic Receptor Alpha 5, Alpha 3, and Beta 4 Subunit Genes Reduces Ethanol Intake in Mice

    PubMed Central

    Gallego, Xavier; Ruiz, Jessica; Valverde, Olga; Molas, Susanna; Robles, Noemí; Sabrià, Josefa; Crabbe, John C.; Dierssen, Mara

    2012-01-01

    Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol’s as well as nicotine’s effects. PMID:22459873

  8. Transgenic over expression of nicotinic receptor alpha 5, alpha 3, and beta 4 subunit genes reduces ethanol intake in mice.

    PubMed

    Gallego, Xavier; Ruiz-Medina, Jessica; Valverde, Olga; Molas, Susanna; Robles, Noemí; Sabrià, Josefa; Crabbe, John C; Dierssen, Mara

    2012-05-01

    Abuse of alcohol and smoking are extensively co-morbid. Some studies suggest partial commonality of action of alcohol and nicotine mediated through nicotinic acetylcholine receptors (nAChRs). We tested mice with transgenic over expression of the alpha 5, alpha 3, beta 4 receptor subunit genes, which lie in a cluster on human chromosome 15, that were previously shown to have increased nicotine self-administration, for several responses to ethanol. Transgenic and wild-type mice did not differ in sensitivity to several acute behavioral responses to ethanol. However, transgenic mice drank less ethanol than wild-type in a two-bottle (ethanol vs. water) preference test. These results suggest a complex role for this receptor subunit gene cluster in the modulation of ethanol's as well as nicotine's effects.

  9. An Adult Male Presenting with Concurrent Plasma Cell Myeloma Involving a CCND1-IGH Translocation and Chronic Myelogenous Leukemia with a Variant (9;22) Translocation.

    PubMed

    Lee, Peter M; Siangchin, Ken; Song, Sophie; Shabsovich, David; Naeini, Yalda; Tirado, Carlos A

    2016-01-01

    The t(11;14)(q13;q32) involving IGH and CCND1 a nd t(9;22) (q34;q11.2) involving BCR and ABL1 are common abnormalities in plasma cell myeloma (PCM) and chronic myelogenous leukemia (CML), respectively. However, the concurrence of the two malignancies is extremely rare. Herein, we present a case of an 87-year-old male who presented with anemia and monocytosis. FISH studies on a bone marrow sample enriched for plasma cells detected a t(11;14) positive for IGH and CCND1 fusion in 92% of nuclei. However, cytogenetic analysis of the bone marrow revealed a t(9;22)(q34;q11.2) in 40% of the metaphases. Interphase and metaphase FISH studies on the sample confirmed the presence of the BCR-ABL1 fusion in 88% of nuclei but did not show any signals corresponding to the derivative 9, suggesting a variant t(9;22) with a deletion or additional material of unknown origin at the 9q34 band of the derivative 9 and a derivative 22 bearing the BCR-ABL1 fusion gene. The concurrence of plasma cell myeloma and chronic myelogenous leukemia is extremely rare with less than 20 cases reported. The molecular pathway in which the multiple malignancies arise is still poorly understood, and this case provides insight into the concurrence of PCM and CML. PMID:27584682

  10. Over-expression of the cucumber expansin gene (Cs-EXPA1) in transgenic maize seed for cellulose deconstruction.

    PubMed

    Yoon, Sangwoong; Devaiah, Shivakumar P; Choi, Seo-eun; Bray, Jeff; Love, Robert; Lane, Jeffrey; Drees, Carol; Howard, John H; Hood, Elizabeth E

    2016-04-01

    Plant cell wall degradation into fermentable sugars by cellulases is one of the greatest barriers to biofuel production. Expansin protein loosens the plant cell wall by opening up the complex of cellulose microfibrils and polysaccharide matrix components thereby increasing its accessibility to cellulases. We over-expressed cucumber expansin in maize kernels to produce enough protein to assess its potential to serve as an industrial enzyme for applications particularly in biomass conversion. We used the globulin-1 embryo-preferred promoter to express the cucumber expansin gene in maize seed. Expansin protein was targeted to one of three sub-cellular locations: the cell wall, the vacuole, or the endoplasmic reticulum (ER). To assess the level of expansin accumulation in seeds of transgenic kernels, a high throughput expansin assay was developed. The highest expressing plants were chosen and enriched crude expansin extract from those plants was tested for synergistic effects with cellulase on several lignocellulosic substrates. Activity of recombinant cucumber expansin from transgenic kernels was confirmed on these pretreated substrates. The best transgenic lines (ER-targeted) can now be used for breeding to increase expansin expression for use in the biomass conversion industry. Results of these experiments show the success of expansin over-expression and accumulation in transgenic maize seed without negative impact on growth and development and confirm its synergistic effect with cellulase on deconstruction of complex cell wall substrates.

  11. Drought tolerance through over-expression of the expansin gene TaEXPB23 in transgenic tobacco.

    PubMed

    Li, Feng; Xing, Shichao; Guo, Qifang; Zhao, Meirong; Zhang, Jin; Gao, Qiang; Wang, Guiping; Wang, Wei

    2011-06-15

    Expansins are proteins that are the key regulators of wall extension during plant growth. To investigate the role of TaEXPB23, a wheat expansin gene, we analyzed TaEXPB23 mRNA expression levels in response to water stress in wheat and examined the drought resistance of transgenic tobaccos over-expressing TaEXPB23. We found that the expression of TaEXPB23 corresponded to wheat coleoptile growth and the response to water stress. The results also indicated that the transgenic tobacco lines lost water more slowly than the wild-type (WT) plants under drought stress; their cells could sustain a more integrated structure under water stress than that of WT. Other physiological and biochemical parameters under water stress, such as electrolyte leakage, malondialdehyde (MDA) level, photosynthetic rate, F(v)/F(m) and ΦPSII, also suggested that the transgenic tobaccos were more drought resistant than WT plants.

  12. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance.

    PubMed

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  13. Gain-of-function analysis of poplar CLE genes in Arabidopsis by exogenous application and over-expression assays.

    PubMed

    Liu, Yisen; Yang, Shaohui; Song, Yingjin; Men, Shuzhen; Wang, Jiehua

    2016-04-01

    Among 50 CLE gene family members in the Populus trichocarpa genome, three and six PtCLE genes encode a CLE motif sequence highly homologous to Arabidopsis CLV3 and TDIF peptides, respectively, which potentially make them functional equivalents. To test and compare their biological activity, we first chemically synthesized each dodecapeptide and analysed itsi n vitro bioactivity on Arabidopsis seedlings. Similarly, but to a different extent, three types of poplar CLV3-related peptides caused root meristem consumption, phyllotaxis disorder, anthocyanin accumulation and failure to enter the bolting stage. In comparison, application of two poplar TDIF-related peptides led to root length promotion in a dose-dependent manner with an even stronger effect observed for poplar TDIF-like peptide than TDIF. Next, we constructed CaMV35S:PtCLE transgenic plants for each of the nine PtCLE genes. Phenotypic abnormalities exemplified by arrested shoot apical meristem and abnormal flower structure were found to be more dominant and severe in 35S:PtCLV3 and 35S:PtCLV3-like2 lines than in the 35S:PtCLV3-like line. Disordered vasculature was detected in both stem and hypocotyl cross-sections in Arabidopsis plants over-expressing poplar TDIF-related genes with the most defective vascular patterning observed for TDIF2 and two TDIF-like genes. Phenotypic difference consistently observed in peptide application assay and transgenic analysis indicated the functional diversity of nine poplar PtCLE genes under investigation. This work represents the first report on the functional analysis of CLE genes in a tree species and constitutes a basis for further study of the CLE peptide signalling pathway in tree development. PMID:26912800

  14. Over-expression of a novel JAZ family gene from Glycine soja, increases salt and alkali stress tolerance

    SciTech Connect

    Zhu, Dan; Cai, Hua; Luo, Xiao; Bai, Xi; Deyholos, Michael K.; Chen, Qin; Chen, Chao; Ji, Wei; Zhu, Yanming

    2012-09-21

    Highlights: Black-Right-Pointing-Pointer We isolated and characterized a novel JAZ family gene, GsJAZ2, from Glycine soja. Black-Right-Pointing-Pointer Overexpression of GsJAZ2 enhanced plant tolerance to salt and alkali stress. Black-Right-Pointing-Pointer The transcriptions of stress marker genes were higher in GsJAZ2 overexpression lines. Black-Right-Pointing-Pointer GsJAZ2 was localized to nucleus. -- Abstract: Salt and alkali stress are two of the main environmental factors limiting crop production. Recent discoveries show that the JAZ family encodes plant-specific genes involved in jasmonate signaling. However, there is only limited information about this gene family in abiotic stress response, and in wild soybean (Glycine soja), which is a species noted for its tolerance to alkali and salinity. Here, we isolated and characterized a novel JAZ family gene, GsJAZ2, from G. soja. Transcript abundance of GsJAZ2 increased following exposure to salt, alkali, cold and drought. Over-expression of GsJAZ2 in Arabidopsis resulted in enhanced plant tolerance to salt and alkali stress. The expression levels of some alkali stress response and stress-inducible marker genes were significantly higher in the GsJAZ2 overexpression lines as compared to wild-type plants. Subcellular localization studies using a GFP fusion protein showed that GsJAZ2 was localized to the nucleus. These results suggest that the newly isolated wild soybean GsJAZ2 is a positive regulator of plant salt and alkali stress tolerance.

  15. Simultaneous translocations of FGFR3/MMSET and CCND1 into two different IGH alleles in multiple myeloma: lack of concurrent activation of both proto-oncogenes.

    PubMed

    Sáez, Borja; Martín-Subero, José I; Lahortiga, Idoya; Largo, Cristina; Larrayoz, María J; Odero, María D; Prosper, Felipe; Cigudosa, Juan C; Siebert, Reiner; Calasanz, María J

    2007-05-01

    The simultaneous occurrence of two different translocations affecting both alleles of the IGH gene has rarely been reported in multiple myeloma. In such a case, two different oncogenes might become transcriptionally deregulated. To investigate this hypothesis, we have characterized the plasma cell leukemia cell line SK-MM2 and a primary myeloma both carrying simultaneous IGH-FGFR3/MMSET and IGH-CCND1 fusions as shown by multicolor fluorescence in situ hybridization. Remarkably, quantitative real-time polymerase chain reaction demonstrated that only one of the oncogene loci was transcriptionally upregulated in both instances. Moreover, the upregulated oncogenes differed between both samples. Thus, biallelic IGH translocations might exert different pathogenetic effects in plasma cell disorders.

  16. Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference

    PubMed Central

    Mitra, Mautusi; Kirst, Henning; Dewez, David; Melis, Anastasios

    2012-01-01

    Truncated light-harvesting antenna 1 (TLA1) is a nuclear gene proposed to regulate the chlorophyll (Chl) antenna size in Chlamydomonas reinhardtii. The Chl antenna size of the photosystems and the chloroplast ultrastructure were manipulated upon TLA1 gene over-expression and RNAi downregulation. The TLA1 over-expressing lines possessed a larger chlorophyll antenna size for both photosystems and contained greater levels of Chl b per cell relative to the wild type. Conversely, TLA1 RNAi transformants had a smaller Chl antenna size for both photosystems and lower levels of Chl b per cell. Western blot analyses of the TLA1 over-expressing and RNAi transformants showed that modulation of TLA1 gene expression was paralleled by modulation in the expression of light-harvesting protein, reaction centre D1 and D2, and VIPP1 genes. Transmission electron microscopy showed that modulation of TLA1 gene expression impacts the organization of thylakoid membranes in the chloroplast. Over-expressing lines showed well-defined grana, whereas RNAi transformants possessed loosely held together and more stroma-exposed thylakoids. Cell fractionation suggested localization of the TLA1 protein in the inner chloroplast envelope and potentially in association with nascent thylakoid membranes, indicating a role in Chl antenna assembly and thylakoid membrane biogenesis. The results provide a mechanistic understanding of the Chl antenna size regulation by the TLA1 gene. PMID:23148270

  17. Comparative transcriptional and proteomic profiling of bread wheat cultivar and its derived transgenic line over-expressing a low molecular weight glutenin subunit gene in the endosperm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have carried out a parallel transcriptional and proteomic comparison of seeds from a transformed bread wheat line that over-expresses a transgenic low molecular weight glutenin subunit gene relative to the corresponding non-transformed genotype. Proteomic analyses showed that, during seed develop...

  18. Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum).

    PubMed

    Balaji, Vasudevan; Smart, Christine D

    2012-02-01

    Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T(0) transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T(0) lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T(0) lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T(1) plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100-10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.

  19. Ferritin as a reporter gene for MRI: chronic liver over expression of H-ferritin during dietary iron supplementation and aging.

    PubMed

    Ziv, Keren; Meir, Gila; Harmelin, Alon; Shimoni, Eyal; Klein, Eugenia; Neeman, Michal

    2010-06-01

    The iron storage protein, ferritin, provides an important endogenous MRI contrast that can be used to determine the level of tissue iron. In recent years the impact of modulating ferritin expression on MRI contrast and relaxation rates was evaluated by several groups, using genetically modified cells, viral gene transfer and transgenic animals. This paper reports the follow-up of transgenic mice that chronically over-expressed the heavy chain of ferritin (h-ferritin) in liver hepatocytes (liver-hfer mice) over a period of 2 years, with the aim of investigating the long-term effects of elevated level of h-ferritin on MR signal and on the well-being of the mice. Analysis revealed that aging liver-hfer mice, exposed to chronic elevated expression of h-ferritin, have increased R(2) values compared to WT. As expected for ferritin, R(2) difference was strongly enhanced at high magnetic field. Histological analysis of these mice did not reveal liver changes with prolonged over expression of ferritin, and no differences could be detected in other organs. Furthermore, dietary iron supplementation significantly affected MRI contrast, without affecting animal wellbeing, for both wildtype and ferritin over expressing transgenic mice. These results suggest the safety of ferritin over-expression, and support the use of h-ferritin as a reporter gene for MRI.

  20. MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1

    SciTech Connect

    Gu, Haihua; Yang, Tao; Fu, Shaozi; Chen, Xiaofan; Guo, Lei; Ni, Yiming

    2014-01-31

    Highlights: • We examined the level of miR-490-3p in A549 lung cancer cells compared with normal bronchial epithelial cell line. • We are the first to show the function of miR-490-3p in A549 lung cancer cells. • We demonstrate CCND1 may be one of the targets of miR-490-3p. - Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3′UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation.

  1. Enhanced morphinan alkaloid production in hairy root cultures of Papaver bracteatum by over-expression of salutaridinol 7-o-acetyltransferase gene via Agrobacterium rhizogenes mediated transformation.

    PubMed

    Sharafi, Ali; Hashemi Sohi, Haleh; Mousavi, Amir; Azadi, Pejman; Dehsara, Bahareh; Hosseini Khalifani, Bahman

    2013-11-01

    Papaver bracteatum is an important medicinal plant valued for its high content of thebaine and an alternative to P. somniferum for benzylisoquinoline alkaloid production. Salutaridinol 7-o-acetyltransferase (SalAT) is a key gene in morphinan alkaloids biosynthesis pathway. Over expression of SalAT gene was used for metabolic engineering in P. bracteatum hairy root cultures. Transcript level of the salutaridinol 7-o-acetyltransferase gene in transgenic hairy root lines increased up to 154 and 128 % in comparison with hairy roots without SalAT over expression and wild type roots, respectively. High performance liquid chromatography analysis showed that the transgenic hairy roots relatively improved levels of thebaine (1.28 % dry weight), codeine (0.02 % dry weight) and morphine (0.03 % dry weight) compared to those hairy roots without SalAT over expression. This suggests that P. bracteatum hairy roots expressing the SalAT gene could be potentially used for the production of valuable morphinan alkaloids.

  2. Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development

    PubMed Central

    Li, Shujun; Yang, Cuili; Xi, Yongyong; Wang, Liang; Zhang, Feng; Fu, Yunfeng; Li, Dejia

    2016-01-01

    Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1. PMID:26840018

  3. Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development.

    PubMed

    Sun, Chengcao; Huang, Chuanfeng; Li, Shujun; Yang, Cuili; Xi, Yongyong; Wang, Liang; Zhang, Feng; Fu, Yunfeng; Li, Dejia

    2016-02-16

    Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4 and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

  4. Effects of the deletion and over-expression of Fusarium graminearum gene FgHal2 on host response to mycovirus Fusarium graminearum virus 1.

    PubMed

    Yu, Jisuk; Lee, Kyung-Mi; Son, Moonil; Kim, Kook-Hyung

    2015-09-01

    The mycovirus Fusarium graminearum virus 1 (FgV1) is associated with reduced virulence (hypovirulence) of Fusarium graminearum. Transcriptomic and proteomic expression profiling have shown that many F. graminearum genes are differentially expressed as a consequence of FgV1 infection. Several of these genes may be related to the maintenance of the virus life cycle. The host gene, FgHal2, which has a highly conserved 3'-phosphoadenosine 5'-phosphatase (PAP phosphatase-like) domain or inositol monophosphatase (IMPase) superfamily domain, shows reduced expression in response to FgV1 infection. We generated targeted gene deletion and over-expression mutants to clarify the possible function(s) of FgHal2 and its relationship to FgV1. The gene deletion mutant showed retarded growth, reduced aerial mycelia formation and reduced pigmentation, whereas over-expression mutants were morphologically similar to the wild-type (WT). Furthermore, compared with the WT, the gene deletion mutant produced fewer conidia and these showed abnormal morphology. The FgHal2 expression level was decreased by FgV1 infection at 120 h post-inoculation (hpi), whereas the levels were nine-fold greater for both the virus-free and virus-infected over-expression mutant than for the WT. FgV1 RNA accumulation was decreased in the deletion mutant at 48, 72 and 120 hpi. FgV1 RNA accumulation in the over-expression mutant was reduced relative to that of the WT at 48 and 120 hpi, but was similar to that of the WT at 72 hpi. The vertical transmission rate of FgV1 in the gene deletion mutant was low, suggesting that FgHal2 may be required for the maintenance of FgV1 in the host cell. Together, these results indicate that the putative 3'(2'),5'-bisphosphate nucleotidase gene, FgHal2, has diverse biological functions in the host fungus and may affect the viral RNA accumulation and transmission of FgV1.

  5. Over-Expression of a Tobacco Nitrate Reductase Gene in Wheat (Triticum aestivum L.) Increases Seed Protein Content and Weight without Augmenting Nitrogen Supplying

    PubMed Central

    Zhao, Xiao-Qiang; Nie, Xuan-Li; Xiao, Xing-Guo

    2013-01-01

    Heavy nitrogen (N) application to gain higher yield of wheat (Triticum aestivum L.) resulted in increased production cost and environment pollution. How to diminish the N supply without losing yield and/or quality remains a challenge. To meet the challenge, we integrated and expressed a tobacco nitrate reductase gene (NR) in transgenic wheat. The 35S-NR gene was transferred into two winter cultivars, “Nongda146” and “Jimai6358”, by Agrobacterium-mediation. Over-expression of the transgene remarkably enhanced T1 foliar NR activity and significantly augmented T2 seed protein content and 1000-grain weight in 63.8% and 68.1% of T1 offspring (total 67 individuals analyzed), respectively. Our results suggest that constitutive expression of foreign nitrate reductase gene(s) in wheat might improve nitrogen use efficiency and thus make it possible to increase seed protein content and weight without augmenting N supplying. PMID:24040315

  6. Over-expression of a Zea mays L. protein phosphatase 2C gene (ZmPP2C) in Arabidopsis thaliana decreases tolerance to salt and drought.

    PubMed

    Liu, Lixia; Hu, Xiaoli; Song, Jian; Zong, Xiaojuan; Li, Dapeng; Li, Dequan

    2009-03-15

    ZmPP2C (AY621066) is a protein phosphatase type-2c previously isolated from roots of Zea mays (LD9002). In this study, constitutive expression of ZmPP2C in Arabidopsis thaliana under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter decreased plant tolerance to salt and drought during seed germination and vegetative growth. When growing on media with NaCl or mannitol, the ZmPP2C-overexpressed plants displayed more severe damages, with weaker growth phenotypes corresponding to a series of physiological changes: lower net photosynthesis rate (Pn) and free proline content, higher malondialdehyde (MDA) level, higher relative membrane permeability (RMP), and water loss. Under these stress conditions, they also showed decreased transcription of the stress-related genes RD29A, RD29B, P5CS1, and P5CS2, and ABA-related genes ABI1 and ABI2. Further, the transgenic plants became less sensitive to abscisic acid (ABA). ZmPP2C over-expression significantly attenuated ABA inhibition on seed germination and root growth of the transgenic plants. These results demonstrate that ZmPP2C is involved in plant stress signal transduction, and ZmPP2C gene over-expression in Arabidopsis thaliana may be exploited to study its potential roles in stress-induced signaling pathway.

  7. Over-expression of the AtGA2ox8 gene decreases the biomass accumulation and lignification in rapeseed (Brassica napus L.)*

    PubMed Central

    Zhao, Xiao-ying; Zhu, Deng-feng; Zhou, Bo; Peng, Wu-sheng; Lin, Jian-zhong; Huang, Xing-qun; He, Re-qing; Zhuo, Yu-hong; Peng, Dan; Tang, Dong-ying; Li, Ming-fang; Liu, Xuan-ming

    2010-01-01

    Gibberellin 2-oxidase (GA 2-oxidase) plays very important roles in plant growth and development. In this study, the AtGA2ox8 gene, derived from Arabidopsis (Arabidopsis thaliana), was transformed and over-expressed in rapeseed (Brassica napus L.) to assess the role of AtGA2ox8 in biomass accumulation and lignification in plants. The transgenic plants, identified by resistant selection, polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) analyses, and green fluorescence examination, showed growth retardation, flowering delay, and dwarf stature. The fresh weight and dry weight in transgenic lines were about 21% and 29% lower than those in wild type (WT), respectively, and the fresh to dry weight ratios were higher than that of WT. Quantitative measurements demonstrated that the lignin content in transgenic lines decreased by 10%–20%, and histochemical staining results also showed reduced lignification in transgenic lines. Quantitative real-time PCR analysis indicated that the transcript levels of lignin biosynthetic genes in transgenic lines were markedly decreased and were consistent with the reduced lignification. These results suggest that the reduced biomass accumulation and lignification in the AtGA2ox8 over-expression rapeseed might be due to altered lignin biosynthetic gene expression. PMID:20593511

  8. Over-expressing a yeast ornithine decarboxylase gene in transgenic roots of Nicotiana rustica can lead to enhanced nicotine accumulation.

    PubMed

    Hamill, J D; Robins, R J; Parr, A J; Evans, D M; Furze, J M; Rhodes, M J

    1990-07-01

    Transformed root cultures of Nicotiana rustica have been generated in which the gene from the yeast Saccharomyces cerevisiae coding for ornithine decarboxylase has been integrated. The gene, driven by the powerful CaMV35S promoter with an upstream duplicated enhancer sequence, shows constitutive expression throughout the growth cycle of some lines, as demonstrated by the analysis of mRNA and enzyme activity. The presence of the yeast gene and enhanced ornithine decarboxylase activity is associated with an enhanced capacity of cultures to accumulate both putrescine and the putrescine-derived alkaloid, nicotine. Even, however, with the very powerful promoter used in this work the magnitude of the changes seen is typically only in the order of 2-fold, suggesting that regulatory factors exist which limit the potential increase in metabolic flux caused by these manipulations. Nevertheless, it is demonstrated that flux through a pathway to a plant secondary product can be elevated by means of genetic manipulation. PMID:2103440

  9. The Evaluation of Nerve Growth Factor Over Expression on Neural Lineage Specific Genes in Human Mesenchymal Stem Cells

    PubMed Central

    Mortazavi, Yousef; Sheikhsaran, Fatemeh; Khamisipour, Gholamreza Khamisipour; Soleimani, Masoud; Teimuri, Ali; Shokri, Somayeh

    2016-01-01

    Objective Treatment and repair of neurodegenerative diseases such as brain tumors, spinal cord injuries, and functional disorders, including Alzheimer’s disease, are challenging problems. A common treatment approach for such disorders involves the use of mesenchymal stem cells (MSCs) as an alternative cell source to replace injured cells. However, use of these cells in hosts may potentially cause adverse outcomes such as tumorigenesis and uncontrolled differentiation. In attempt to generate mesenchymal derived neural cells, we have infected MSCs with recombinant lentiviruses that expressed nerve growth factor (NGF) and assessed their neural lineage genes. Materials and Methods In this experimental study, we cloned the NGF gene sequence into a helper dependent lentiviral vector that contained the green fluorescent protein (GFP) gene. The recombinant vector was amplified in DH5 bacterial cells. Recombinant viruses were generated in the human embryonic kidney 293 (HEK-293) packaging cell line with the helper vectors and analyzed under fluorescent microscopy. Bone marrow mesenchymal cells were infected by recombinant viruses for three days followed by assessment of neural differentiation. We evaluated expression of NGF through measurement of the NGF protein in culture medium by ELISA; neural specific genes were quantified by real-time polymerase chain reaction (PCR). Results We observed neural morphological changes after three days. Quantitative PCR showed that expressions of NESTIN, glial derived neurotrophic factor (GDNF), glial fibrillary acidic protein (GFAP) and Microtubule-associated protein 2 (MAP2) genes increased following induction of NGF overexpression, whereas expressions of endogenous NGF and brain derived neural growth factor (BDNF) genes reduced. Conclusion Ectopic expression of NGF can induce neurogenesis in MSCs. Direct injection of MSCs may cause tumorigenesis and an undesirable outcome. Therefore an alternative choice to overcome this obstacle may

  10. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    PubMed

    Mishra, Smrati; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides.

  11. Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence

    PubMed Central

    Christiansen, Michael W.; Matthewman, Colette; Podzimska-Sroka, Dagmara; O’Shea, Charlotte; Lindemose, Søren; Møllegaard, Niels Erik; Holme, Inger B.; Hebelstrup, Kim; Skriver, Karen; Gregersen, Per L.

    2016-01-01

    The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein–DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley. PMID:27436280

  12. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera

    PubMed Central

    Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S.

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides. PMID:26919744

  13. RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

    PubMed

    Mishra, Smrati; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh; Asha; Jadaun, Jyoti Singh; Sangwan, Neelam S

    2016-01-01

    Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides. PMID:26919744

  14. Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence.

    PubMed

    Christiansen, Michael W; Matthewman, Colette; Podzimska-Sroka, Dagmara; O'Shea, Charlotte; Lindemose, Søren; Møllegaard, Niels Erik; Holme, Inger B; Hebelstrup, Kim; Skriver, Karen; Gregersen, Per L

    2016-09-01

    The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley. PMID:27436280

  15. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα

    PubMed Central

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-01-01

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer. PMID:27576898

  16. Promising Nanocarriers for PEDF Gene Targeting Delivery to Cervical Cancer Cells Mediated by the Over-expressing FRα.

    PubMed

    Yang, Yuhan; He, Lili; Liu, Yongmei; Xia, Shan; Fang, Aiping; Xie, Yafei; Gan, Li; He, Zhiyao; Tan, Xiaoyue; Jiang, Chunling; Tong, Aiping; Song, Xiangrong

    2016-01-01

    Cervical cancer presents extremely low PEDF expression which is associated with tumor progression and poor prognosis. In this study, folate receptor α (FRα)-targeted nano-liposomes (FLP) were designed to enhance the anti-tumor effect by targeting delivery of exogenous PEDF gene to cervical cancer cells. The targeting molecule F-PEG-Chol was firstly synthesized by a novel simpler method. FLP encapsulating PEDF gene (FLP/PEDF) with a typical lipid-membrane structure were prepared by a film dispersion method. The transfection experiment found FLP could effectively transfect human cervical cancer cells (HeLa cells). FLP/PEDF significantly inhibited the growth of HeLa cells and human umbilical vein endothelial cells (HUVEC cells) and suppressed adhension, invasion and migration of HeLa cells in vitro. In the abdominal metastatic tumor model of cervical cancer, FLP/PEDF administered by intraperitoneal injection exhibited a superior anti-tumor effect probably due to the up-regulated PEDF. FLP/PEDF could not only sharply reduce the microvessel density but also dramatically inhibit proliferation and markedly induce apoptosis of tumor cells in vivo. Moreover, the preliminary safety investigation revealed that FLP/PEDF had no obvious toxicity. These results clearly showed that FLP were desired carriers for PEDF gene and FLP/PEDF might represent a potential novel strategy for gene therapy of cervical cancer. PMID:27576898

  17. Increased nitrogen-use efficiency in transgenic rice plants over-expressing a nitrogen-responsive early nodulin gene identified from rice expression profiling.

    PubMed

    Bi, Yong-Mei; Kant, Surya; Clarke, Joseph; Clark, Joseph; Gidda, Satinder; Ming, Feng; Xu, Jianyao; Rochon, Amanda; Shelp, Barry J; Hao, Lixin; Zhao, Rong; Mullen, Robert T; Zhu, Tong; Rothstein, Steven J

    2009-12-01

    Development of genetic varieties with improved nitrogen-use efficiency (NUE) is essential for sustainable agriculture. In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified N-responsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops.

  18. Over-expression of a glutathione S-transferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco.

    PubMed

    Ji, Wei; Zhu, Yanming; Li, Yong; Yang, Liang; Zhao, Xiaowen; Cai, Hua; Bai, Xi

    2010-08-01

    Glycine soja is a species of soybean that survives in adverse environments including high salt and drought conditions. We constructed a cDNA library from G. soja seedlings treated with NaCl and isolated a glutathione S-transferase gene (GsGST: GQ265911) from the library. The cDNA encoding GsGST contains an open reading frame of 660 bp and the predicted protein belongs to the tau class of GST family proteins. Tobacco plants over-expressing the GsGST gene showed sixfold higher GST activity than wild-type plants. Transgenic tobacco plants exhibited enhanced dehydration tolerance. T(2) transgenic tobacco plants showed higher tolerance at the seedling stage than wild-type plants to salt and mannitol as demonstrated by longer root length and less growth retardation.

  19. Over-expression of a subgroup 4 R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco.

    PubMed

    Omer, Sumita; Kumar, Santosh; Khan, Bashir M

    2013-01-01

    KEY MESSAGE : LlMYB1 , a subgroup 4 R2R3-type MYB transcription factor gene from Leucaena leucocephala appears to be a repressor of lignin biosynthesis pathway by regulating the transcription of general phenylpropanoid pathway genes. R2R3MYB transcription factors are known to play a wide role in regulating the phenylpropanoid pathway in plants. In this study, we report isolation, cloning and characterization of an R2R3MYB transcription factor gene (LlMYB1) from an economically important tree species, Leucaena leucocephala. LlMYB1 consists of 705 bp coding sequence corresponding to 235 amino acids. Sequence alignment revealed that the N-terminal (MYB) domain of the gene shares up to 95 % similarity with subgroup 4 (Sg4) members of R2R3Myb gene family functionally known to be lignin repressors. Highly divergent C-terminal region of the gene carried an ERF-associated amphiphilic repression (EAR) motif, another characteristic of the Sg4. The gene was phylogenetically grouped closest with AmMYB308, a known repressor of monolignol biosynthetic pathway genes. Spatio-temporal expression studies at different ages of seedlings using quantitative real-time PCR (QRT-PCR) showed highest transcript level of the gene in 10 day old stem tissues. Over-expression of the gene in transgenic tobacco showed statistically significant decline in the transcript levels of the general phenylpropanoid pathway genes and reduction in lignin content. Our study suggests that LlMYB1 might be playing the role of a repressor of lignin biosynthesis in L. leucocephala. PMID:23052594

  20. Biochemical and molecular characterization of transgenic Lotus japonicus plants constitutively over-expressing a cytosolic glutamine synthetase gene

    PubMed Central

    Ortega, Jose Luis; Temple, Stephen J.; Bagga, Suman; Ghoshroy, Soumitra; Sengupta-Gopalan, Champa

    2013-01-01

    Higher plants assimilate nitrogen in the form of ammonia through the concerted activity of glutamine synthetase (GS) and glutamate synthase (GOGAT). The GS enzyme is either located in the cytoplasm (GS1) or in the chloroplast (GS2). To understand how modulation of GS activity affects plant performance, Lotus japonicus L. plants were transformed with an alfalfa GS1 gene driven by the CaMV 35S promoter. The transformants showed increased GS activity and an increase in GS1 polypeptide level in all the organs tested. GS was analyzed by non-denaturing gel electrophoresis and ion-exchange chromatography. The results showed the presence of multiple GS isoenzymes in the different organs and the presence of a novel isoform in the transgenic plants. The distribution of GS in the different organs was analyzed by immunohistochemical localization. GS was localized in the mesophyll cells of the leaves and in the vasculature of the stem and roots of the transformants. Our results consistently showed higher soluble protein concentration, higher chlorophyll content and a higher biomass accumulation in the transgenic plants. The total amino acid content in the leaves and stems of the transgenic plants was 22–24% more than in the tissues of the non-transformed plants. The relative abundance of individual amino acid was similar except for aspartate/asparagine and proline, which were higher in the transformants. PMID:15197594

  1. Over-expression of a grape stilbene synthase gene in tomato induces parthenocarpy and causes abnormal pollen development.

    PubMed

    Ingrosso, Ilaria; Bonsegna, Stefania; De Domenico, Stefania; Laddomada, Barbara; Blando, Federica; Santino, Angelo; Giovinazzo, Giovanna

    2011-10-01

    A novel strategy to induce parthenocarpy in tomato fruits by the induction of resveratrol biosynthesis in flower tissues was exploited. Two transgenic tomato lines were considered: a higher resveratrol-producing (35SS) line, constitutively expressing a grape stilbene synthase cDNA, and a lower resveratrol-producing (LoxS) line, expressing stilbene synthase under a fruit-specific promoter. The expression of the stilbene synthase gene affected flavonoid metabolism in a different manner in the transgenic lines, and in one of these, the 35SS line, resulted in complete male sterility. Resveratrol was synthesised either in 35SS or LoxS tomato flowers, at an even higher extent (about 8-10 times) in the former line. We further investigated whether stilbene synthase expression may have resulted in impaired naringenin accumulation during flower development. In the 35SS flowers, naringenin was significantly impaired by about 50%, probably due to metabolic competition. Conversely, the amount of glycosylated flavonols increased in transgenic flowers, thereby excluding the diminished production of flavonols as a reason for parthenocarpy in tomato. We further investigated whether resveratrol synthesis may have resulted changes to pollen structure. Microscopic observations revealed the presence of few and abnormal flake-like pollen grains in 35SS flowers with no germination capability. Finally, the analysis of coumaric and ferulic acids, the precursors of lignin and sporopollenin biosynthesis, revealed significant depletion of these compounds, therefore suggesting an impairment in structural compounds as a reason for pollen ablation. These overall outcomes, to the best of our knowledge, reveal for the first time the major role displayed by resveratrol synthesis on parthenocarpy in tomato fruits. PMID:21843947

  2. Comparative analysis of chemical compositions between non-transgenic soybean seeds and those from plants over-expressing AtJMT, the gene for jasmonic acid carboxyl methyltransferase.

    PubMed

    Nam, Kyong-Hee; Kim, Do Young; Pack, In-Soon; Park, Jung-Ho; Seo, Jun Sung; Choi, Yang Do; Cheong, Jong-Joo; Kim, Chung Ho; Kim, Chang-Gi

    2016-04-01

    Transgenic overexpression of the Arabidopsis gene for jasmonic acid carboxyl methyltransferase (AtJMT) is involved in regulating jasmonate-related plant responses. To examine its role in the compositional profile of soybean (Glycine max), we compared the seeds from field-grown plants that over-express AtJMT with those of the non-transgenic, wild-type (WT) counterpart. Our analysis of chemical compositions included proximates, amino acids, fatty acids, isoflavones, and antinutrients. Overexpression of AtJMT in the seeds resulted in decreased amounts of tryptophan, palmitic acid, linolenic acid, and stachyose, but increased levels of gadoleic acid and genistein. In particular, seeds from the transgenic soybeans contained 120.0-130.5% more genistein and 60.5-82.1% less stachyose than the WT. A separate evaluation of ingredient values showed that all were within the reference ranges reported for commercially available soybeans, thereby demonstrating the substantial equivalence of these transgenic and non-transgenic seeds.

  3. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice.

    PubMed

    Azizi, Parisa; Rafii, Mohd Y; Abdullah, Siti N A; Hanafi, Mohamed M; Maziah, M; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g(-1) in transgenic plants. The M. oryzae population was constant at 31, 48

  4. Over-Expression of the Pikh Gene with a CaMV 35S Promoter Leads to Improved Blast Disease (Magnaporthe oryzae) Tolerance in Rice

    PubMed Central

    Azizi, Parisa; Rafii, Mohd Y.; Abdullah, Siti N. A.; Hanafi, Mohamed M.; Maziah, M.; Sahebi, Mahbod; Ashkani, Sadegh; Taheri, Sima; Jahromi, Mohammad F.

    2016-01-01

    Magnaporthe oryzae is a rice blast fungus and plant pathogen that causes a serious rice disease and, therefore, poses a threat to the world's second most important food security crop. Plant transformation technology has become an adaptable system for cultivar improvement and to functionally analyze genes in plants. The objective of this study was to determine the effects (through over-expressing and using the CaMV 35S promoter) of Pikh on MR219 resistance because it is a rice variety that is susceptible to the blast fungus pathotype P7.2. Thus, a full DNA and coding DNA sequence (CDS) of the Pikh gene, 3172 bp, and 1206 bp in length, were obtained through amplifying the gDNA and cDNA template from a PH9-resistant rice variety using a specific primer. Agrobacterium-mediated transformation technology was also used to introduce the Pikh gene into the MR219 callus. Subsequently, transgenic plants were evaluated from the DNA to protein stages using polymerase chain reaction (PCR), semi-quantitative RT-PCR, real-time quantitative PCR and high performance liquid chromatography (HPLC). Transgenic plants were also compared with a control using a real-time quantification technique (to quantify the pathogen population), and transgenic and control plants were challenged with the local most virulent M. oryzae pathotype, P7.2. Based on the results, the Pikh gene encodes a hydrophilic protein with 18 sheets, 4 helixes, and 21 coils. This protein contains 401 amino acids, among which the amino acid sequence from 1 to 376 is a non-cytoplasmic region, that from 377 to 397 is a transmembrane region, and that from 398 to 401 is a cytoplasmic region with no identified disordered regions. The Pikh gene was up-regulated in the transgenic plants compared with the control plants. The quantity of the amino acid leucine in the transgenic rice plants increased significantly from 17.131 in the wild-type to 47.865 mg g−1 in transgenic plants. The M. oryzae population was constant at 31, 48

  5. High throughput quantitative reverse transcription PCR assays revealing over-expression of cancer testis antigen genes in multiple myeloma stem cell-like side population cells.

    PubMed

    Wen, Jianguo; Li, Hangwen; Tao, Wenjing; Savoldo, Barbara; Foglesong, Jessica A; King, Lauren C; Zu, Youli; Chang, Chung-Che

    2014-09-01

    Multiple myeloma (MM) stem cells, proposed to be responsible for the tumourigenesis, drug resistance and recurrence of this disease, are enriched in the cancer stem cell-like side population (SP). Cancer testis antigens (CTA) are attractive targets for immunotherapy because they are widely expressed in cancers but only in limited types of normal tissues. We designed a high throughput assay, which allowed simultaneous relative quantifying expression of 90 CTA genes associated with MM. In the three MM cell lines tested, six CTA genes were over-expressed in two and LUZP4 and ODF1 were universally up-regulated in all three cell lines. Subsequent study of primary bone marrow (BM) from eight MM patients and four healthy donors revealed that 19 CTA genes were up-regulated in SP of MM compared with mature plasma cells. In contrast, only two CTA genes showed a moderate increase in SP cells of healthy BM. Furthermore, knockdown using small interfering RNA (siRNA) revealed that LUZP4 expression is required for colony-forming ability and drug resistance in MM cells. Our findings indicate that multiple CTA have unique expression profiles in MM SP, suggesting that CTA may serve as targets for immunotherapy that it specific for MM stem cells and which may lead to the long-term cure of MM.

  6. Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol production by over-expressing dhaT gene in Klebsiella pneumoniae TUAC01.

    PubMed

    Hao, Jian; Wang, Wei; Tian, Jiesheng; Li, Jilun; Liu, Dehua

    2008-07-01

    Glycerol can be biologically converted to 1,3-propanediol, a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. In 1,3-propanediol synthesis pathway, 3-hydroxypropionaldehyde (3-HPA) was an inhibitory intermediary metabolite. The accumulation of 3-HPA in broth would cause an irreversible cessation of the fermentation process. With the object of reducing 3-HPA level in the fermentation broth, dhaT gene which encodes 1,3-propanediol oxidoreductase (PDOR) was cloned and over expressed in 1,3-propanediol producing bacterium Klebsiella pneumoniae TUAC01. dhaT gene was linked downstream of the ptac promoter in an expressing vector pDK6 to form plasmid pDK-dhaT. The newly formed pDK-dhaT was transformed to K. pneumoniae TUAC01. Under the inducement of IPTG, PDOR was over-expressed when the constructed strain was cultured on an LB medium or a fermentation medium. A 5 L scale-up fermentation experiment was done to test the 3-HPA accumulation in broth, with the initial substrate glycerol 30 g/L; the peak levels of 3-HPA in broth were 7.55 and 1.49 mmol/L for control host strain and the constructed strain, respectively. In 50 g/L initial glycerol experiment, the peak level of 3-HPA in broth was 12.57 and 2.02 mmol/l for the control host strain and the constructed strain, respectively. Thus the fermentation cessation caused by the toxicity of 3-HPA was alleviated in the constructed strain.

  7. MiR-503 inhibited cell proliferation of human breast cancer cells by suppressing CCND1 expression.

    PubMed

    Long, Jianting; Ou, Caiwen; Xia, Haoming; Zhu, Yifan; Liu, Dayue

    2015-11-01

    Breast cancer is one of the most common malignancies and a major cause of cancer-related mortality all over the world. A growing body of reports revealed that microRNAs play essential roles in the progression of cancers. Aberrant expression of miR-503 has been reported in several kinds of cancer. The aim of the current study was to elucidate the role of miR-503 in the pathogenesis of breast cancer. In the present study, our results suggested that miR-503 expression was markedly downregulated in breast cancer tissues and cells. Overexpression of miR-503 in breast cancer cell lines reduced cell proliferation through inducing G0/G1 cell cycle arrest by targeting CCND1. Together, our findings provide new knowledge regarding the role of miR-503 in the progression of breast cancer and indicate the role of miR-503 as a tumor suppressor microRNA (miRNA) in breast cancer.

  8. Over-expression of the Gerbera hybrida At-SOC1-like1 gene Gh-SOC1 leads to floral organ identity deterioration

    PubMed Central

    Ruokolainen, Satu; Ng, Yan Peng; Albert, Victor A.; Elomaa, Paula; Teeri, Teemu H.

    2011-01-01

    Background and Aims The family of MADS box genes is involved in a number of processes besides controlling floral development. In addition to supplying homeotic functions defined by the ABC model, they influence flowering time and transformation of vegetative meristem into inflorescence meristem, and have functions in roots and leaves. Three Gerbera hybrida At-SOC1-like genes (Gh-SOC1–Gh-SOC3) were identified among gerbera expressed sequence tags. Methods Evolutionary relationships between SOC1-like genes from gerbera and other plants were studied by phylogenetic analysis. The function of the gerbera gene Gh-SOC1 in gerbera floral development was studied using expression analysis, protein–protein interaction assays and reverse genetics. Transgenic gerbera lines over-expressing or downregulated for Gh-SOC1 were obtained using Agrobacterium transformation and investigated for their floral phenotype. Key Results Phylogenetic analysis revealed that the closest paralogues of At-SOC1 are Gh-SOC2 and Gh-SOC3. Gh-SOC1 is a more distantly related paralogue, grouping together with a number of other At-SOC1 paralogues from arabidopsis and other plant species. Gh-SOC1 is inflorescence abundant and no expression was seen in vegetative parts of the plant. Ectopic expression of Gh-SOC1 did not promote flowering, but disturbed the development of floral organs. The epidermal cells of ray flower petals appeared shorter and their shape was altered. The colour of ray flower petals differed from that of the wild-type petals by being darker red on the adaxial side and greenish on the abaxial surface. Several protein–protein interactions with other gerbera MADS domain proteins were identified. Conclusions The At-SOC1 paralogue in gerbera shows a floral abundant expression pattern. A late petal expression might indicate a role in the final stages of flower development. Over-expression of Gh-SOC1 led to partial loss of floral identity, but did not affect flowering time. Lines where Gh

  9. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato.

    PubMed

    Pandey, Roopali; Gupta, Aarti; Chowdhary, Anuj; Pal, Ram Krishna; Rajam, Manchikatla Venkat

    2015-02-01

    Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches. PMID:25537646

  10. Over-expression of mouse ornithine decarboxylase gene under the control of fruit-specific promoter enhances fruit quality in tomato.

    PubMed

    Pandey, Roopali; Gupta, Aarti; Chowdhary, Anuj; Pal, Ram Krishna; Rajam, Manchikatla Venkat

    2015-02-01

    Diamine putrescine (Put) and polyamines; spermidine (Spd) and spermine (Spm) are essential component of every cell because of their involvement in the regulation of cell division, growth and development. The aim of this study is to enhance the levels of Put during fruit development and see its implications in ripening and quality of tomato fruits. Transgenic tomato plants over-expressing mouse ornithine decarboxylase gene under the control of fruit-specific promoter (2A11) were developed. Transgenic fruits exhibited enhanced levels of Put, Spd and Spm, with a concomitant reduction in ethylene levels, rate of respiration and physiological loss of water. Consequently such fruits displayed significant delay of on-vine ripening and prolonged shelf life over untransformed fruits. The activation of Put biosynthetic pathway at the onset of ripening in transgenic fruits is also consistent with the improvement of qualitative traits such as total soluble solids, titratable acids and total sugars. Such changes were associated with alteration in expression pattern of ripening specific genes. Transgenic fruits were also fortified with important nutraceuticals like lycopene, ascorbate and antioxidants. Therefore, these transgenic tomatoes would be useful for the improvement of tomato cultivars through breeding approaches.

  11. Perinucleolar relocalization and nucleolin as crucial events in the transcriptional activation of key genes in mantle cell lymphoma.

    PubMed

    Allinne, Jeanne; Pichugin, Andrei; Iarovaia, Olga; Klibi, Manel; Barat, Ana; Zlotek-Zlotkiewicz, Ewa; Markozashvili, Diana; Petrova, Natalia; Camara-Clayette, Valérie; Ioudinkova, Elena; Wiels, Joëlle; Razin, Sergey V; Ribrag, Vincent; Lipinski, Marc; Vassetzky, Yegor S

    2014-03-27

    In mantle cell lymphoma (MCL), one allele of the cyclin D1 (Ccnd1) gene is translocated from its normal localization on chromosome 11 to chromosome 14. This is considered as the crucial event in the transformation process of a normal naive B-cell; however, the actual molecular mechanism leading to Ccnd1 activation remains to be deciphered. Using a combination of three-dimensional and immuno-fluorescence in situ hybridization experiments, the radial position of the 2 Ccnd1 alleles was investigated in MCL-derived cell lines and malignant cells from affected patients. The translocated Ccnd1 allele was observed significantly more distant from the nuclear membrane than its nontranslocated counterpart, with a very high proportion of IgH-Ccnd1 chromosomal segments localized next to a nucleolus. These perinucleolar areas were found to contain active RNA polymerase II (PolII) clusters. Nucleoli are rich in nucleolin, a potent transcription factor that we found to bind sites within the Ccnd1 gene specifically in MCL cells and to activate Ccnd1 transcription. We propose that the Ccnd1 transcriptional activation in MCL cells relates to the repositioning of the rearranged IgH-Ccnd1-carrying chromosomal segment in a nuclear territory with abundant nucleolin and active PolII molecules. Similar transforming events could occur in Burkitt and other B-cell lymphomas.

  12. The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

    PubMed Central

    Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

    2015-01-01

    Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

  13. Over-expression of genes and proteins of ubiquitin specific peptidases (USPs) and proteasome subunits (PSs) in breast cancer tissue observed by the methods of RFDD-PCR and proteomics.

    PubMed

    Deng, Shishan; Zhou, Hongying; Xiong, Ruohong; Lu, Youguang; Yan, Dazhong; Xing, Tianyong; Dong, Lihua; Tang, Enjie; Yang, Huijun

    2007-07-01

    The ubiquitin-proteasome system facilitates the degradation of damaged proteins and regulators of growth and stress response. Alterations in this proteolytic system are associated with a variety of human pathologies. By restriction fragment differential display polymerase chain reaction (RFDD-PCR) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF MS) based on two-dimensional polyacrylamide gel electrophoresis (2-DE), differentially expressed genes and proteins of ubiquitin specific proteases (USPs), proteasome subuinits (PSs) and ubiquitin protein ligase E3A (UBE3A) were analyzed between breast cancer and adjacent normal tissues. Some of them were further verified as over-expression by immunohistochemical stain. Five genes of proteasome subunits (PSs), including PSMB5, PSMD1, PSMD2, PSMD8 and PSMD11, four genes of USPs, including USP9X, USP9Y, USP10 and USP25, and ubiquitin protein ligase E3A (UBE3A) were over-expressed (>3-fold) in breast cancer tissue compared to adjacent normal tissue, and over-expression (>4-fold) of proteins of PSMA1 and SMT3A were observed in breast cancer tissue. PSMD8, PSMD11 and UBE3A were further verified as over-expression by immunohistochemical stain. The action of ubiquitin-proteasome system were obviously enhanced in breast cancer, and selectively intervention in action of ubiquitin-proteasome system may be a useful method of treating human breast cancer.

  14. MiRNA-20 and MiRNA-106a Regulate Spermatogonial Stem Cell Renewal at the Post-transcriptional Level via Targeting STAT3 and Ccnd1

    PubMed Central

    He, Zuping; Jiang, Jiji; Kokkinaki, Maria; Tang, Lin; Zeng, Wenxian; Gallicano, Ian; Dobrinski, Ina; Dym, Martin

    2013-01-01

    Studies onspermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction. PMID:23836497

  15. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6.

    PubMed

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-07-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer.

  16. miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

    PubMed Central

    Xu, Junfen; Wan, Xiaoyun; Chen, Xiaojing; Fang, Yifeng; Cheng, Xiaodong; Xie, Xing; Lu, Weiguo

    2016-01-01

    Persistent infection with oncogenic human papillomavirus viruses (HPVs) is a casual factor for cervical cancer and its precursors, and the abnormal constitutive expression of viral oncoprotein E6 is a key event during the malignant transformation. Here, we performed miRNA microarray to identify changes of miRNAs following ectopic HPV16 E6 overexpression in HEK293T cells and found miR-2861 was greatly decreased in both HEK293T and HaCaT cells expressing HPV16 E6 compared to vector control. Further, we demonstrated a biological link among HPV16 E6, miR-2861, EGFR, AKT2, and CCND1 in cervical cancer cells. We showed that miR-2861 was downregulated in cervical cancer tissues and negatively correlated with advanced tumor stage and lymph node metastasis. Overexpression of miR-2861 suppressed cervical cancer cell proliferation and invasion and enhanced apoptosis. Subsequent investigation revealed that EGFR, AKT2, and CCND1 were all the direct targets of miR-2861. Importantly, silencing EGFR, AKT2, and/or CCND1 recapitulated the cellular effects seen upon miR-2861 overexpression. Restoration of EGFR, AKT2, and/or CCND1 counteracted the effects of miR-2861 expression. Thus, we identified a new pathway employing miR-2861, EGFR, AKT2, and CCND1 that may mediate HPV16 E6 induced initiation and progression of cervical cancer. PMID:27364926

  17. Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis.

    PubMed

    Guan, Chunfeng; Ji, Jing; Zhang, Xuqiang; Li, Xiaozhou; Jin, Chao; Guan, Wenzhu; Wang, Gang

    2015-03-01

    Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress.

  18. Mobility and subcellular localization of endogenous, gene-edited Tau differs from that of over-expressed human wild-type and P301L mutant Tau

    PubMed Central

    Di Xia; Gutmann, Julia M.; Götz, Jürgen

    2016-01-01

    Alzheimer’s disease (AD) and a subset of frontotemporal dementia termed FTLD-Tau are characterized by a massive, yet incompletely characterized and understood redistribution of Tau. To establish a framework for understanding this pathology, we used the genome-editing tool TALEN and generated Tau-mEOS2 knock-in mice to determine the mobility and subcellular localization of endogenous Tau in hippocampal cultures. We analysed Tau in axons, dendrites and spines at three stages of maturation using live-cell imaging, photo-conversion and FRAP assays. Tau-mEOS2 cultures were compared with those over-expressing EGFP-tagged forms of human wild-type (hWT-Tau) and P301L mutant Tau (hP301L-Tau), modelling Tau accumulation in AD and FTLD-Tau, respectively. In developing neurons, Tau-mEOS2 followed a proximo-distal gradient in axons and a subcellular distribution similar to that of endogenous Tau in neurons obtained from wild-type mice, which were abolished, when either hWT-Tau or hP301L-Tau was over-expressed. For the three conditions, FRAP analysis revealed a similar mobility in dendrites compared with axons; however, Tau-mEOS2 was less mobile than hWT-Tau and hP301L-Tau and the mobile fraction was smaller, possibly reflecting less efficient microtubule binding of Tau when over-expressed. Together, our study presents Tau-mEOS2 mice as a novel tool for the study of Tau in a physiological and a pathological context. PMID:27378256

  19. Identification of a B lymphoid disorder defined by specific morphologic features, a del(11)(q13) and a same breakpoint far from CCND1

    SciTech Connect

    Morel, D.; Callet, E.; Reynaud, S.

    1994-09-01

    Chromosome rearrangements in 11q13 have been shown to occur in a variety of diffuse small B-cell lymphomas/leukemias, including, beside mantle cell lymphomas (MCL), some cases of CLL/SLL, PLL, and SLVL. If t(11;14)(q13;q32) may be considered as a hallmark of MCL, less is known about deletions involving 11q13. A series of 13 patients with a diffuse small B-lymphoma/leukemia was examined for morphology (cytology and histology), immunology, cytogenetics and FISH for some of them. According to karyotype findings, 2 groups were identified: Group 1: 9 patients (6M,3F), median age 60 yrs. without 11q anomalies. Apart from trisomy 12 (3 cases), diverse anomalies were identified including chromosomes 1, 2, 7, 8, 12, 17. Cases were classified as CLL (2), SLL/SLP (5), blast-rich immunocytomas (2). Group 2: 4 patients, all males, median age 52 yrs. with breakpoints in 11q13; there were 3 deletions, and a t(11;14) was present in another case. 2 patients presented with refractory disease followed for 23 and 9 months, respectively, without any consistent morphologic change, the chromosomal anomaly being present at diagnosis in 1.3 cases. Cytologically, a nucleolated cell component was the constant and striking feature and FISH study by cos 14, pHS 11, cos 17, cos 105 revealed the same breakpoint located far from CCND1. The fourth case, bearing the t(11;14), was diagnosed as CLL/PLL in cytology, but was histologically consistent with MCL; the breakpoint was located by FISH into the BCL1 locus. Even if this study needs further confirmation, it points at the 11q13 deletion as a genetic event leading to a more aggressive disease, associated with distinct cytologic features differing from MCL and a molecular event probably not involving BCL1/CCND1.

  20. Progestin and antiprogestin responsiveness in breast cancer is driven by the PRA/PRB ratio via AIB1 or SMRT recruitment to the CCND1 and MYC promoters.

    PubMed

    Wargon, Victoria; Riggio, Marina; Giulianelli, Sebastián; Sequeira, Gonzalo R; Rojas, Paola; May, María; Polo, María L; Gorostiaga, María A; Jacobsen, Britta; Molinolo, Alfredo; Novaro, Virginia; Lanari, Claudia

    2015-06-01

    There is emerging interest in understanding the role of progesterone receptors (PRs) in breast cancer. The aim of this study was to investigate the proliferative effect of progestins and antiprogestins depending on the relative expression of the A (PRA) and B (PRB) isoforms of PR. In mifepristone (MFP)-resistant murine carcinomas antiprogestin responsiveness was restored by re-expressing PRA using demethylating agents and histone deacetylase inhibitors. Consistently, in two human breast cancer xenograft models, one manipulated to overexpress PRA or PRB (IBH-6 cells), and the other expressing only PRA (T47D-YA) or PRB (T47D-YB), MFP selectively inhibited the growth of PRA-overexpressing tumors and stimulated IBH-6-PRB xenograft growth. Furthermore, in cells with high or equimolar PRA/PRB ratios, which are stimulated to proliferate in vitro by progestins, and are inhibited by MFP, MPA increased the interaction between PR and the coactivator AIB1, and MFP favored the interaction between PR and the corepressor SMRT. In a PRB-dominant context in which MFP stimulates and MPA inhibits cell proliferation, the opposite interactions were observed. Chromatin immunoprecipitation assays in T47D cells in the presence of MPA or MFP confirmed the interactions between PR and the coregulators at the CCND1 and MYC promoters. SMRT downregulation by siRNA abolished the inhibitory effect of MFP on MYC expression and cell proliferation. Our results indicate that antiprogestins are therapeutic tools that selectively inhibit PRA-overexpressing tumors by increasing the SMRT/AIB1 balance at the CCND1 and MYC promoters.

  1. Early over expression of messenger RNA for multiple genes, including insulin, in the Pancreatic Lymph Nodes of NOD mice is associated with Islet Autoimmunity

    PubMed Central

    Regnault, Béatrice; Osorio y Fortea, José; Miao, Dongmei; Eisenbarth, George; Melanitou, Evie

    2009-01-01

    Background Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting β cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process. Methods Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed. Results The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D. Conclusion Our data strongly suggest that the immune related mechanisms

  2. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress. PMID:25910649

  3. Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

    PubMed

    Tiwari, Lalit Dev; Mittal, Dheeraj; Chandra Mishra, Ratnesh; Grover, Anil

    2015-07-01

    Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.

  4. Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea).

    PubMed

    Singh, Natwar; Mishra, Avinash; Jha, Bhavanath

    2014-08-15

    Peroxisomal ascorbate peroxidase gene (SbpAPX) of an extreme halophyte Salicornia brachiata imparts abiotic stress endurance and plays a key role in the protection against oxidative stress. The cloned SbpAPX gene was transformed to local variety of peanut and about 100 transgenic plants were developed using optimized in vitro regeneration and Agrobacterium mediated genetic transformation method. The T0 transgenic plants were confirmed for the gene integration; grown under controlled condition in containment green house facility; seeds were harvested and T1 plants were raised. Transgenic plants (T1) were further confirmed by PCR using gene specific primers and histochemical GUS assay. About 40 transgenic plants (T1) were selected randomly and subjected for salt stress tolerance study. Transgenic plants remained green however non-transgenic plants showed bleaching and yellowish leaves under salt stress conditions. Under stress condition, transgenic plants continued normal growth and completed their life cycle. Transgenic peanut plants exhibited adequate tolerance under salt stress condition and thus could be explored for the cultivation in salt affected areas for the sustainable agriculture.

  5. Amplification and over-expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells

    PubMed Central

    Fan, Yihui; Ge, Ningling; Wang, Xiaosong; Sun, Wenjing; Mao, Renfang; Bu, Wen; Creighton, Chad J; Zheng, Pingju; Vasudevan, Sanjeev; An, Lei; Yang, Jinshu; Zhao, Yi-Jue; Zhang, Huiyuan; Li, Xiao-Nan; Rao, Pulivarthi H; Leung, Eastwood; Lu, Yong-Jie; Gray, Joe W; Schiff, Rachel; Hilsenbeck, Susan G; Osborne, C Kent; Yang, Jianhua; Zhang, Hong

    2014-01-01

    Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers. An integrative bioinformatics analysis of this region nominated the MAP3K 3 gene as a potential therapeutic target in breast cancer. This gene encodes mitogen-activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), which has not yet been reported to be associated with cancer-causing genetic aberrations. We found that MAP3K3 was amplified in approximately 8–20% of breast cancers. Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 non-amplified breast cancer cells. Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFα and TRAIL, as well as doxorubicin, VP-16 and fluorouracil, three commonly used chemotherapeutic drugs for treating breast cancer. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells (MCF-10A). Combined, these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy. Therefore, MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers, and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy. PMID:24122835

  6. Over-expression of an endogenous milk protein gene in transgenic mice is associated with impaired mammary alveolar development and a milchlos phenotype.

    PubMed

    Burdon, T; Wall, R J; Shamay, A; Smith, G H; Hennighausen, L

    1991-12-01

    The whey acidic protein (WAP) gene is expressed in mammary epithelial cells at late pregnancy and throughout lactation. We have generated transgenic mice in which a mouse WAP transgene is expressed precociously in pregnancy. From 13 founder mice bearing WAP transgenes, two female founders and the daughters from a male founder failed to lactate and nurture their offspring. We named this phenotype milchlos. Mammary tissue from postpartum milchlos mice was underdeveloped, contained too few alveoli and resembled the glands of non-transgenic mid-pregnant mice. The hypothesis that alveolar development in milchlos mice was functionally arrested in a prelactational state is consistent with low levels of alpha-lactalbumin mRNA, and an unidentified keratin RNA in mammary tissue from postpartum mice. Defects in alveolar function in milchlos mice were detected at mid-pregnancy; in non-transgenic mice, WAP was secreted into the alveolar lumen but remained preferentially in the cytoplasm of the alveolar epithelial cells in the milchlos mice. Since deregulated WAP expression resulted in impaired mammary development, it is possible that WAP plays a regulatory role in the terminal differentiation and development of mammary alveolar cells. PMID:1782141

  7. Over-expression of Brassica napus phosphatidylinositol-phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation.

    PubMed

    Georges, Fawzy; DAS, Shankar; Ray, Heather; Bock, Cheryl; Nokhrina, Kateryna; Kolla, Venkat Apparao; Keller, Wilf

    2009-12-01

    Phosphatidylinositol-specific phospholipase C (PtdIns-PLC2) plays a central role in the phosphatidylinositol-specific signal transduction pathway. It catalyses the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to produce two second messengers, sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate. The former is a membrane activator of protein kinase C in mammalian systems, and the latter is a Ca(2+) modulator which induces distinctive oscillating bursts of cytosolic Ca(2+), resulting in regulation of gene expression and activation of proteins. Sustained over-expression of BnPtdIns-PLC2 in transgenic Brassica napus lines brought about an early shift from vegetative to reproductive phases, and shorter maturation periods, accompanied by notable alterations in hormonal distribution patterns in various tissues. The photosynthetic rate increased, while stomata were partly closed. Numerous gene expression changes that included induction of stress-related genes such as glutathione S-transferase, hormone-regulated and regulatory genes, in addition to a number of kinases, calcium-regulated factors and transcription factors, were observed. Other changes included increased phytic acid levels and phytohormone organization patterns. These results suggest the importance of PtdIns-PLC2 as an elicitor of a battery of events that systematically control hormone regulation, and plant growth and development in what may be a preprogrammed mode.

  8. Characterization of Rice Homeobox Genes, OsHOX22 and OsHOX24, and Over-expression of OsHOX24 in Transgenic Arabidopsis Suggest Their Role in Abiotic Stress Response

    PubMed Central

    Bhattacharjee, Annapurna; Khurana, Jitendra P.; Jain, Mukesh

    2016-01-01

    Homeobox transcription factors are well known regulators of plant growth and development. In this study, we carried out functional analysis of two candidate stress-responsive HD-ZIP I class homeobox genes from rice, OsHOX22, and OsHOX24. These genes were highly up-regulated under various abiotic stress conditions at different stages of rice development, including seedling, mature and reproductive stages. The transcript levels of these genes were enhanced significantly in the presence of plant hormones, including abscisic acid (ABA), auxin, salicylic acid, and gibberellic acid. The recombinant full-length and truncated homeobox proteins were found to be localized in the nucleus. Electrophoretic mobility shift assay established the binding of these homeobox proteins with specific DNA sequences, AH1 (CAAT(A/T)ATTG) and AH2 (CAAT(C/G)ATTG). Transactivation assays in yeast revealed the transcriptional activation potential of full-length OsHOX22 and OsHOX24 proteins. Homo- and hetero-dimerization capabilities of these proteins have also been demonstrated. Further, we identified putative novel interacting proteins of OsHOX22 and OsHOX24 via yeast-two hybrid analysis. Over-expression of OsHOX24 imparted higher sensitivity to stress hormone, ABA, and abiotic stresses in the transgenic Arabidopsis plants as revealed by various physiological and phenotypic assays. Microarray analysis revealed differential expression of several stress-responsive genes in transgenic lines as compared to wild-type. Many of these genes were found to be involved in transcriptional regulation and various metabolic pathways. Altogether, our results suggest the possible role of OsHOX22/OsHOX24 homeobox proteins as negative regulators in abiotic stress responses. PMID:27242831

  9. Detection of Critical Genes Associated with Overall Survival (OS) and Progression-Free Survival (PFS) in Reconstructed Canine B-Cell Lymphoma Gene Regulatory Network (GRN).

    PubMed

    Zamani-Ahmadmahmudi, Mohamad; Najafi, Ali; Nassiri, Seyed Mahdi

    2016-01-01

    Canine B-cell lymphoma GRN was reconstructed from gene expression data in the STRING and MiMI databases. Critical genes of networks were identified and correlations of critical genes with overall survival (OS) and progression-free survival (PFS) were evaluated. Significant changes were detected in the expressions of GLUL, CD44, CD79A, ARF3, FOS, BLOC1S1, FYN, GZMB, GALNT3, IFI44, CD3G, GNG2, ESRP1, and CCND1 in the STRING network and of PECAM1, GLUL, CD44, GDI1, E2F4, TLE1, CD79A, UCP2, CCND1, FYN, RHOQ, BIN1, and A2M in the MiMI network. Final survival analysis highlighted CCND1 and FOS as genes with significant correlations with OS and PFS.

  10. DEK over-expression promotes mitotic defects and micronucleus formation

    PubMed Central

    Matrka, Marie C; Hennigan, Robert F; Kappes, Ferdinand; DeLay, Monica L; Lambert, Paul F; Aronow, Bruce J; Wells, Susanne I

    2015-01-01

    The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation PMID:25945971

  11. DEK over-expression promotes mitotic defects and micronucleus formation.

    PubMed

    Matrka, Marie C; Hennigan, Robert F; Kappes, Ferdinand; DeLay, Monica L; Lambert, Paul F; Aronow, Bruce J; Wells, Susanne I

    2015-01-01

    The DEK gene encodes a nuclear protein that binds chromatin and is involved in various fundamental nuclear processes including transcription, RNA splicing, DNA replication and DNA repair. Several cancer types characteristically over-express DEK at the earliest stages of transformation. In order to explore relevant mechanisms whereby DEK supports oncogenicity, we utilized cancer databases to identify gene transcripts whose expression patterns are tightly correlated with that of DEK. We identified an enrichment of genes involved in mitosis and thus investigated the regulation and possible function of DEK in cell division. Immunofluorescence analyses revealed that DEK dissociates from DNA in early prophase and re-associates with DNA during telophase in human keratinocytes. Mitotic cell populations displayed a sharp reduction in DEK protein levels compared to the corresponding interphase population, suggesting DEK may be degraded or otherwise removed from the cell prior to mitosis. Interestingly, DEK overexpression stimulated its own aberrant association with chromatin throughout mitosis. Furthermore, DEK co-localized with anaphase bridges, chromosome fragments, and micronuclei, suggesting a specific association with mitotically defective chromosomes. We found that DEK over-expression in both non-transformed and transformed cells is sufficient to stimulate micronucleus formation. These data support a model wherein normal chromosomal clearance of DEK is required for maintenance of high fidelity cell division and chromosomal integrity. Therefore, the overexpression of DEK and its incomplete removal from mitotic chromosomes promotes genomic instability through the generation of genetically abnormal daughter cells. Consequently, DEK over-expression may be involved in the initial steps of developing oncogenic mutations in cells leading to cancer initiation.

  12. Differential PAX5 levels promote malignant B cell infiltration, progression and drug resistance and predict a poor prognosis in MCL patients independent of CCND1

    PubMed Central

    Teo, Albert E.; Chen, Zheng; Miranda, Roberto N.; McDonnell, Timothy; Medeiros, L. Jeffrey; McCarty, Nami

    2015-01-01

    Reduced PAX5 levels play important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5− MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to bone marrow stromal cells, and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb and to PI3 kinase/mTOR and pSTAT3 pathway hyperactivation. Upon PAX5 silencing, the MCL cells displayed upregulated IL-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5−MCL cells are highly drug-resistant compared to PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B cell cancer and encourage future efforts to design effective therapies against MCL. PMID:26073757

  13. In-depth analysis of the critical genes and pathways in colorectal cancer

    PubMed Central

    LIU, FUGUO; JI, FENGZHI; JI, YULING; JIANG, YUEPING; SUN, XUEGUO; LU, YANYAN; ZHANG, LINGYUN; HAN, YUE; LIU, XISHUANG

    2015-01-01

    The present study aimed to investigate the molecular targets for colorectal cancer (CRC). Differentially expressed genes (DEGs) were screened between CRC and matched adjacent noncancerous samples. GENETIC_ASSOIATION_DB_DISEASE analysis was performed to identify CRC genes from the identified DEGs using the Database for Annotation, Visualization and Integrated Discovery, followed by Gene Οntology (GO) and Kyoto Encyclopedia of Genes and Genomes analysis for the CRC genes. A protein-protein interaction (PPI) network was constructed for the CRC genes, followed by determination and analysis of the hub genes, in terms of the protein domains and spatial structure. In total, 35 CRC genes were determined, including 19 upregulated and 16 downregulated genes. Downregulated N-acetyltransferase (NAT)1 and NAT2 were enriched in the caffeine metabolism pathway. The down-regulated and upregulated genes were enriched in a number of GO terms and pathways, respectively. Cyclin D1 (CCND1) and proliferating cell nuclear antigen (PCNA) were identified as the hub genes in the PPI network. The C-terminal and N-terminal domains were similar in PCNA, but different in CCND1. The results suggested PCNA, CCND1, NAT1 and NAT2 for use as biomarkers to enable early diagnosis and monitoring of CRC. These results form a basis for developing therapies, which target the unique protein domains of PCNA and CCND1. PMID:26239303

  14. Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

    PubMed

    Cheng, Lei; Yin, Sheng; Chen, Min; Sun, Baoguo; Hao, Shuai; Wang, Chengtao

    2016-08-01

    As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13 mg L(-1) after 24 h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas. PMID:27154464

  15. Transcriptomic profiling comparison of YAP over-expression and conditional knockout mouse tooth germs

    PubMed Central

    Liu, Ming; Wang, Xiu-Ping

    2015-01-01

    To identify the downstream target genes of YAP, we used RNA-Seq technology to compare the transcriptomic profilings of Yap conditional knockout (Yap CKO) and YAP over-expression mouse tooth germs. Our results showed that some Hox, Wnt and Laminin family genes had concurrent changes with YAP transcripts, indicating that the expression of these genes may be regulated by YAP. Here, we provide the detailed experimental procedure for the transcriptomic profiling results (NCBI GEO accession number GSE65524). The associated study on the regulation of Hoxa1 and Hoxc13 genes by YAP was published in Molecular Cellular Biology in 2015 [Liu et al., 2015]. PMID:26484260

  16. DAPIT Over-Expression Modulates Glucose Metabolism and Cell Behaviour in HEK293T Cells

    PubMed Central

    Kontro, Heidi; Cannino, Giuseppe; Rustin, Pierre; Dufour, Eric; Kainulainen, Heikki

    2015-01-01

    Introduction Diabetes Associated Protein in Insulin-sensitive Tissues (DAPIT) is a subunit of mitochondrial ATP synthase and has also been found to associate with the vacuolar H+-ATPase. Its expression is particularly high in cells with elevated aerobic metabolism and in epithelial cells that actively transport nutrients and ions. Deletion of DAPIT is known to induce loss of mitochondrial ATP synthase but the effects of its over-expression are obscure. Results In order to study the consequences of high expression of DAPIT, we constructed a transgenic cell line that constitutively expressed DAPIT in human embryonal kidney cells, HEK293T. Enhanced DAPIT expression decreased mtDNA content and mitochondrial mass, and saturated respiratory chain by decreasing H+-ATP synthase activity. DAPIT over-expression also increased mitochondrial membrane potential and superoxide level, and translocated the transcription factors hypoxia inducible factor 1α (Hif1α) and β-catenin to the nucleus. Accordingly, cells over-expressing DAPIT used more glucose and generated a larger amount of lactate compared to control cells. Interestingly, these changes were associated with an epithelial to mesenchymal (EMT)-like transition by changing E-cadherin to N-cadherin and up-regulating several key junction/adhesion proteins. At physiological level, DAPIT over-expression slowed down cell growth by G1 arrest and migration, and enhanced cell detachment. Several cancers also showed an increase in genomic copy number of Usmg5 (gene encoding DAPIT), thereby providing strong correlative evidence for DAPIT possibly having oncogenic function in cancers. Conclusions DAPIT over-expression thus appears to modulate mitochondrial functions and alter cellular regulations, promote anaerobic metabolism and induce EMT-like transition. We propose that DAPIT over-expression couples the changes in mitochondrial metabolism to physiological and pathophysiological regulations, and suggest it could play a critical

  17. TERT and AURKA gene copy number gains enhance the detection of acral lentiginous melanomas by fluorescence in situ hybridization.

    PubMed

    Diaz, Alba; Puig-Butillé, Joan Anton; Valera, Alexandra; Muñoz, Concha; Costa, Dolors; Garcia-Herrera, Adriana; Carrera, Cristina; Sole, Francesc; Malvehy, Josep; Puig, Susana; Alos, Llucia

    2014-03-01

    The study of specific chromosomal loci through fluorescence in situ hybridization (FISH) is useful in differential diagnosis of melanocytic tumors. However, sensitivity rates vary, probably because of molecular heterogeneity. Acral lentiginous melanomas are characterized by copy number gains of small genomic regions, including CCND1, TERT, and AURKA. In a series of 58 acral melanocytic lesions, we explored the value of a four-color FISH probe, used in addition to determining MYC gene status, and assessed the potential diagnostic usefulness of newly developed probes targeting TERT and AURKA. Moreover, we tested CCND1, TERT, and AURKA protein expression by immunohistochemistry. The four-color FISH probe detected 85.3% of melanomas and 29.4% of TERT and AURKA copy number gains. Sensitivity was 97% (confidence interval 95%, 82.9% to 99.8%) for the combined results of all probes. No MYC copy number gains were detected. No nevi showed aberrations. Immunohistochemistry revealed a higher percentage of cells positive for CCND1, TERT, and AURKA protein in melanomas than in nevi (P ≤ 0.001). A significant correlation between gene copy number gain and protein expression was found for CCND1 (P = 0.015). Our results indicate that addition of specific FISH probes to the current probe could improve sensitivity for the diagnosis of acral melanomas. Further studies in larger numbers of cases are needed to validate these results.

  18. Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco.

    PubMed

    John, Riffat; Ganeshan, Uma; Singh, Badri N; Kaul, Tanushri; Reddy, Malireddy K; Sopory, Sudhir K; Rajam, Manchikatla V

    2016-01-01

    Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants. PMID:27630644

  19. Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

    PubMed Central

    John, Riffat; Ganeshan, Uma; Singh, Badri N.; Kaul, Tanushri; Reddy, Malireddy K.; Sopory, Sudhir K.; Rajam, Manchikatla V.

    2016-01-01

    Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants.

  20. Over-expression of Topoisomerase II Enhances Salt Stress Tolerance in Tobacco

    PubMed Central

    John, Riffat; Ganeshan, Uma; Singh, Badri N.; Kaul, Tanushri; Reddy, Malireddy K.; Sopory, Sudhir K.; Rajam, Manchikatla V.

    2016-01-01

    Topoisomerases are unique enzymes having an ability to remove or add DNA supercoils and untangle the snarled DNA. They can cut, shuffle, and religate DNA strands and remove the torsional stress during DNA replication, transcription or recombination events. In the present study, we over-expressed topoisomerase II (TopoII) in tobacco (Nicotiana tabaccum) and examined its role in growth and development as well as salt (NaCl) stress tolerance. Several putative transgenic plants were generated and the transgene integration and expression was confirmed by PCR and Southern blot analyses, and RT-PCR analysis respectively. Percent seed germination, shoot growth, and chlorophyll content revealed that transgenic lines over-expressing the NtTopoIIα-1 gene exhibited enhanced tolerance to salt (150 and 200 mM NaCl) stress. Moreover, over-expression of TopoII lead to the elevation in proline and glycine betaine levels in response to both concentrations of NaCl as compared to wild-type. In response to NaCl stress, TopoII over-expressing lines showed reduced lipid peroxidation derived malondialdehyde (MDA) generation. These results suggest that TopoII plays a pivotal role in salt stress tolerance in plants. PMID:27630644

  1. Effects of resistin-like molecule β over-expression on gastric cancer cells in vitro

    PubMed Central

    Zheng, Li-Duan; Yang, Chun-Lei; Qi, Teng; Qi, Meng; Tong, Ling; Tong, Qiang-Song

    2012-01-01

    AIM: To investigate the effects of resistin-like molecule β (RELMβ) over-expression on the invasion, metastasis and angiogenesis of gastric cancer cells. METHODS: Human RELMβ encoding expression vector was constructed and transfected into the RELMβ lowly-expressed gastric cancer cell lines SGC-7901 and MKN-45. Gene expression was measured by Western blotting, reverse transcription polymerase chain reaction (PCR) and real-time quantitative PCR. Cell proliferation was measured by 2-(4,5-dimethyltriazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation and 5-ethynyl-20-deoxyuridine incorporation assays. The in vitro migration, invasion and metastasis of cancer cells were measured by cell adhesion assay, scratch assay and matrigel invasion assay. The angiogenic capabilities of cancer cells were measured by tube formation of endothelial cells. RESULTS: Transfection of RELMβ vector into SGC-7901 and MKN-45 cells resulted in over-expression of RELMβ, which did not influence the cellular proliferation. However, over-expression of RELMβ suppressed the in vitro adhesion, invasion and metastasis of cancer cells, accompanied by decreased expression of matrix metalloproteinase-2 (MMP-2) and MMP-9. Moreover, transfection of RELMβ attenuated the expression of vascular endothelial growth factor and in vitro angiogenic capabilities of cancer cells. CONCLUSION: Over-expression of RELMβ abolishes the invasion, metastasis and angiogenesis of gastric cancer cells in vitro, suggesting its potentials as a novel therapeutic target for gastric cancer. PMID:22371635

  2. Generation of a cre recombinase-conditional Nos1ap over-expression transgenic mouse

    PubMed Central

    Auer, Dallas R.; Sysa-Shah, Polina; Bedja, Djahida; Simmers, Jessica L.; Pak, Evgenia; Dutra, Amalia; Cohn, Ronald; Gabrielson, Kathleen L.

    2016-01-01

    Polymorphic non-coding variants at the NOS1AP locus have been associated with the common cardiac, metabolic and neurological traits and diseases. Although, in vitro gene targeting-based cellular and biochemical studies have shed some light on NOS1AP function in cardiac and neuronal tissue, to enhance our understanding of NOS1AP function in mammalian physiology and disease, we report the generation of cre recombinase-conditional Nos1ap over-expression transgenic mice (Nos1apTg). Conditional transgenic mice were generated by the pronuclear injection method and three independent, single-site, multiple copies integration event-based founder lines were selected. For heart-restricted over-expression, Nos1apTg mice were crossed with Mlc2v-cre and Nos1ap transcript over-expression was observed in left ventricles from Nos1apTg; Mlc2v-cre F1 mice. We believe that with the potential of conditional over-expression, Nos1apTg mice will be a useful resource in studying NOS1AP function in various tissues under physiological and disease states. PMID:24563304

  3. Over Expression of NANOS3 and DAZL in Human Embryonic Stem Cells

    PubMed Central

    Panula, Sarita; Reda, Ahmed; Stukenborg, Jan-Bernd; Ramathal, Cyril; Sukhwani, Meena; Albalushi, Halima; Edsgärd, Daniel; Nakamura, Michiko; Söder, Olle; Orwig, Kyle E.; Yamanaka, Shinya; Reijo Pera, Renee A.; Hovatta, Outi

    2016-01-01

    The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo. PMID:27768780

  4. Alteration of wing size through over-expression of scribbler isoforms.

    PubMed

    LaJeuensse, Dennis

    2010-01-01

    Scribbler was identified as a genetic modifier of the Drosophila tumor suppressor gene Merlin. Loss of Merlin results in hyperplasia in a number of different epithelial tissues including the wing and eye imaginal discs, however loss of scribbler does not. The lack of an overt proliferation sbb phenotype has complicated the mechanistic link between sbb and Merlin. Scribbler encodes two novel transcriptional repressors which function in numerous processes including axon guidance and pattern formation within the wing. While the two sbb isoforms have some redundant functions over-expression of two sbb isoforms within the wing show distinct and opposite effects. Over-expression of the smaller SbbA isoform results in a larger wing, while over-expression of larger SbbB isoform results in a smaller wing with defects in venation. Co-expression of sbb isoforms ameliorates the effects of expression of either isoform alone, suggesting that a balance between the express of each scribbler isoform is required to ensure proper size of the wing.

  5. Sequence analysis, cloning and over-expression of an endoxylanase from the alkaliphilic Bacillus halodurans.

    PubMed

    Martínez, M Alejandra; Delgado, Osvaldo D; Baigorí, Mario D; Siñeriz, Faustino

    2005-04-01

    The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-beta-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg-1. PMID:15973487

  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. Over-expression of OsHsfA7 enhanced salt and drought tolerance in transgenic rice.

    PubMed

    Liu, Ai-Ling; Zou, Jie; Liu, Cui-Fang; Zhou, Xiao-Yun; Zhang, Xian-Wen; Luo, Guang-Yu; Chen, Xin-Bo

    2013-01-01

    Heat shock proteins play an important role in plant stress tolerance and are mainly regulated by heat shock transcription factors (Hsfs). In this study, we generated transgenic rice over-expressing OsHsfA7 and carried out morphological observation and stress tolerance assays. Transgenic plants exhibited less, shorter lateral roots and root hair. Under salt treatment, over-expressing OsHsfA7 rice showed alleviative appearance of damage symptoms and higher survival rate, leaf electrical conductivity and malondialdehyde content of transgenic plants were lower than those of wild type plants. Meanwhile, transgenic rice seedlings restored normal growth but wild type plants could not be rescued after drought and re-watering treatment. These findings indicate that over-expression of OsHsfA7 gene can increase tolerance to salt and drought stresses in rice seedlings.

  8. Lentiviral vector-mediated over-expression of Sox9 protected chondrocytes from IL-1β induced degeneration and apoptosis

    PubMed Central

    Lu, Huading; Zeng, Chun; Chen, Mingwei; Lian, Liyi; Dai, Yuhu; Zhao, Huiqing

    2015-01-01

    To explore whether the over-expression of Sry-related HMG box (Sox9) in degenerative chondrocytes is able to improve cell regeneration and protects cells from inflammation induced apoptosis, we generated a Sox9 over-expressing vector delivery system in which the Sox9 gene was inserted into a lentiviral vector. After infecting mouse chondrocytes with the Sox9-encoding vector, we observed a high level of gene transduction efficiency and achieved a high level of Sox9 expression in the infected chondrocytes. To explore whether over-expression of Sox9 is able to induce cell regeneration and improve cell survival, we induced Sox9 over-expression by lentiviral vector infection 48 hours before IL-1β treatment. The cells were infected with the reporter gene GFP-encoded lentiviral vector as a negative control or left uninfected. 48-hours after IL-1β treatment, the chrondrocytes treated with IL-1β alone, underwent a degenerative process, with elevated expression of MMP-3, MMP-13, ADAMTS-5 and ALP, but the cell specific anabolic proteins collagen II and aggrecan were significantly suppressed. The cells infected with the GFP reporter vector had no increased regeneration after IL-1β treatment. The results indicated that Sox9 is an important chondrocyte transcription factor, promoting chondrocyte regeneration and cell survival, which were mediated through affecting multiple cell differentiation as well as anti-apoptotic signaling pathways. PMID:26617711

  9. Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads

    PubMed Central

    Lambeth, Luke; Raymond, Christopher S.; Roeszler, Kelly N.; Kuroiwa, Asato; Nakata, Tomohiro; Zarkower, David; Smith, Craig A.

    2014-01-01

    DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 is located on the Z sex chromosome and is currently the best candidate master regulator of avian gonadal sex differentiation. We previously showed that knockdown of DMRT1 expression during the period of sexual differentiation induces feminisation of male embryonic chicken gonads. This gene is therefore necessary for proper testis development in the chicken. However, whether it is sufficient to induce testicular differentiation has remained unresolved. We show here that over-expression of DMRT1 induces male pathway genes and antagonises the female pathway in embryonic chicken gonads. Ectopic DMRT1 expression in female gonads induces localised SOX9 and AMH expression. It also induces expression of the recently identified Z-linked male factor, Hemogen (HEMGN). Masculinised gonads show evidence of cord-like structures and retarded female-type cortical development. Furthermore, expression of the critical feminizing enzyme, aromatase, is reduced in the presence of over-expressed DMRT1. These data indicate that DMRT1 is an essential sex-linked regulator of gonadal differentiation in avians, and that it likely acts via a dosage mechanism established through the lack of global Z dosage compensation in birds. PMID:24576538

  10. Over-Expression of Meteorin Drives Gliogenesis Following Striatal Injury

    PubMed Central

    Wright, Jordan L.; Ermine, Charlotte M.; Jørgensen, Jesper R.; Parish, Clare L.; Thompson, Lachlan H.

    2016-01-01

    A number of studies have shown that damage to brain structures adjacent to neurogenic regions can result in migration of new neurons from neurogenic zones into the damaged tissue. The number of differentiated neurons that survive is low, however, and this has led to the idea that the introduction of extrinsic signaling factors, particularly neurotrophic proteins, may augment the neurogenic response to a level that would be therapeutically relevant. Here we report on the impact of the relatively newly described neurotrophic factor, Meteorin, when over-expressed in the striatum following excitotoxic injury. Birth-dating studies using bromo-deoxy-uridine (BrdU) showed that Meteorin did not enhance injury-induced striatal neurogenesis but significantly increased the proportion of new cells with astroglial and oligodendroglial features. As a basis for comparison we found under the same conditions, glial derived neurotrophic factor significantly enhanced neurogenesis but did not effect gliogenesis. The results highlight the specificity of action of different neurotrophic factors in modulating the proliferative response to injury. Meteorin may be an interesting candidate in pathological settings involving damage to white matter, for example after stroke or neonatal brain injury. PMID:27458346

  11. Reducing acetate excretion from E. coli K-12 by over-expressing the small RNA SgrS.

    PubMed

    Negrete, Alejandro; Majdalani, Nadim; Phue, Je-Nie; Shiloach, Joseph

    2013-01-25

    When exposed to the nonmetabolized glucose derivative alpha methyl glucoside (αMG), both Escherichia coli K-12 (JM109 and MG1655) and E. coli B (BL21) respond by reducing the concentration of the mRNA of the ptsG gene which is responsible for the biosynthesis of the glucose transporter EIICB(glu). This occurs through the over-expression of the noncoding small RNA SgrS, which interacts specifically with the mRNA of the ptsG gene and prevents its translation. However, when these bacteria are exposed to a glucose concentration of 40 g/L, over-expression of SgrS is observed only in E. coli B (BL21). Unlike E. coli K-12 (JM109 and MG1655), which are affected by high glucose concentration and produce higher levels of acetate, E. coli B (BL21) is not affected. Based on this information, it was assumed that over-expression of SgrS enables E. coli B (BL21) to reduce its acetate excretion by controlling the glucose transport. When SgrS was over-expressed in both E. coli K-12 strains from a multicopy plasmid, it was possible to reduce their acetate excretion levels to those seen in E. coli B. This observation opens a new approach towards controlling bacterial metabolism through the use of noncoding RNA.

  12. The formation of brown adipose tissue induced by transgenic over-expression of PPARγ2.

    PubMed

    Zhou, Ying; Yang, Jinzeng; Huang, Jinliang; Li, Ting; Xu, Dequan; Zuo, Bo; Hou, Liming; Wu, Wangjun; Zhang, Lin; Xia, Xiaoliang; Ma, Zhiyuan; Ren, Zhuqing; Xiong, Yuanzhu

    2014-04-18

    Brown adipose tissue (BAT) is specialized to dissipate energy as heat, therefore reducing fat deposition and counteracting obesity. Brown adipocytes arise from myoblastic progenitors during embryonic development by the action of transcription regulator PRDM16 binding to PPARγ, which promotes BAT-like phenotype in white adipose tissue. To investigate the capability of converting white adipose tissue to BAT or browning by PPARγ in vivo, we generated transgenic mice with over-expressed PPARγ2. The transgenic mice showed strong brown fat features in subcutaneous fat in morphology and histology. To provide molecular evidences on browning characteristics of the adipose tissue, we employed quantitative real-time PCR to determine BAT-specific gene expressions. The transgenic mice had remarkably elevated mRNA level of UCP1, Elovl3, PGC1α and Cebpα in subcutaneous fat. Compared with wild-type mice, UCP1 protein levels were increased significantly in transgenic mice. ATP concentration was slightly decreased in the subcutaneous fat of transgenic mice. Western blotting analysis also confirmed that phosphorylated AMPK and ACC proteins were significantly (P<0.01) increased in the transgenic mice. Therefore, this study demonstrated that over-expression of PPARγ2 in skeletal muscle can promote conversion of subcutaneous fat to brown fat formation, which can have beneficial effects on increasing energy metabolisms and combating obesity.

  13. DEK over expression as an independent biomarker for poor prognosis in colorectal cancer

    PubMed Central

    2013-01-01

    Background The DEK protein is related to chromatin reconstruction and gene transcription, and plays an important role in cell apoptosis. High expression levels of the human DEK gene have been correlated with numerous human malignancies. This study explores the roles of DEK in tumor progression and as a prognostic determinant of colorectal cancer. Methods Colorectal cancer specimens from 109 patients with strict follow-up, and colorectal adenomas from 52 patients were selected for analysis of DEK protein by immunohistochemistry. The correlations between DEK over expression and the clinicopathological features of colorectal cancers were evaluated by Chi-square test and Fisher’s exact tests. The survival rates were calculated by the Kaplan-Meier method, and the relationship between prognostic factors and patient survival was also analyzed by the Cox proportional hazard models. Results DEK protein showed a nuclear immunohistochemical staining pattern in colorectal cancers. The strongly positive rate of DEK protein was 48.62% (53/109) in colorectal cancers, which was significantly higher than that in either adjacent normal colon mucosa (9.17%, 10/109) or colorectal adenomas (13.46%, 7/52). DEK over expression in colorectal cancers was positively correlated with tumor size, grade, lymph node metastasis, serosal invasion, late stage, and disease-free survival- and 5-year survival rates. Further analysis showed that patients with late stage colorectal cancer and high DEK expression had worse survival rates than those with low DEK expression. Moreover, multivariate analysis showed high DEK expression, serosal invasion, and late stage are significant independent risk factors for mortality in colorectal cancer. Conclusions DEK plays an important role in the progression of colorectal cancers and it is an independent poor prognostic factor of colorectal cancers. PMID:23902796

  14. Over-Expression of a Cytochrome P450 Is Associated with Resistance to Pyriproxyfen in the Greenhouse Whitefly Trialeurodes vaporariorum

    PubMed Central

    Karatolos, Nikos; Williamson, Martin S.; Denholm, Ian; Gorman, Kevin; ffrench-Constant, Richard H.; Bass, Chris

    2012-01-01

    Background The juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood) which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s) are poorly understood. Results Bioassays against eggs of a German (TV8) population of T. vaporariorum revealed a moderate level (21-fold) of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel) displaying a much higher resistance ratio (>4000-fold). The enzyme inhibitor piperonyl butoxide (PBO) suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s). Quantitative PCR highlighted a single P450 gene (CYP4G61) that was highly over-expressed (81.7-fold) in TV8pyrsel. Conclusion Over-expression of a single cytochrome P450 gene (CYP4G61) has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen. PMID:22347432

  15. Pleiotropic effect of sigE over-expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803.

    PubMed

    Osanai, Takashi; Kuwahara, Ayuko; Iijima, Hiroko; Toyooka, Kiminori; Sato, Mayuko; Tanaka, Kan; Ikeuchi, Masahiko; Saito, Kazuki; Hirai, Masami Yokota

    2013-11-01

    Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium. PMID:23941239

  16. Effect of the over-expression of PII and PZ proteins on the nitrogenase activity of Azospirillum brasilense.

    PubMed

    Huergo, Luciano F; Filipaki, Angela; Chubatsu, Leda S; Yates, M Geoffrey; Steffens, Maria Berenice; Pedrosa, Fabio O; Souza, Emanuel M

    2005-12-01

    The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

  17. Induced over-expression of AtDREB2A CA improves drought tolerance in sugarcane.

    PubMed

    Reis, Rafaela Ribeiro; da Cunha, Bárbara Andrade Dias Brito; Martins, Polyana Kelly; Martins, Maria Thereza Bazzo; Alekcevetch, Jean Carlos; Chalfun, Antônio; Andrade, Alan Carvalho; Ribeiro, Ana Paula; Qin, Feng; Mizoi, Junya; Yamaguchi-Shinozaki, Kazuko; Nakashima, Kazuo; Carvalho, Josirley de Fátima Corrêa; de Sousa, Carlos Antônio Ferreira; Nepomuceno, Alexandre Lima; Kobayashi, Adilson Kenji; Molinari, Hugo Bruno Correa

    2014-05-01

    Drought is one of the most challenging agricultural issues limiting sustainable sugarcane production and, in some cases, yield losses caused by drought are nearly 50%. DREB proteins play vital regulatory roles in abiotic stress responses in plants. The transcription factor DREB2A interacts with a cis-acting DRE sequence to activate the expression of downstream genes that are involved in drought-, salt- and heat-stress response in Arabidopsis thaliana. In the present study, we evaluated the effects of stress-inducible over-expression of AtDREB2A CA on gene expression, leaf water potential (ΨL), relative water content (RWC), sucrose content and gas exchanges of sugarcane plants submitted to a four-days water deficit treatment in a rhizotron-grown root system. The plants were also phenotyped by scanning the roots and measuring morphological parameters of the shoot. The stress-inducible expression of AtDREB2A CA in transgenic sugarcane led to the up-regulation of genes involved in plant response to drought stress. The transgenic plants maintained higher RWC and ΨL over 4 days after withholding water and had higher photosynthetic rates until the 3rd day of water-deficit. Induced expression of AtDREB2A CA in sugarcane increased sucrose levels and improved bud sprouting of the transgenic plants. Our results indicate that induced expression of AtDREB2A CA in sugarcane enhanced its drought tolerance without biomass penalty.

  18. Over-expression of AtEXLA2 alters etiolated arabidopsis hypocotyl growth

    PubMed Central

    Boron, Agnieszka Karolina; Van Loock, Bram; Suslov, Dmitry; Markakis, Marios Nektarios; Verbelen, Jean-Pierre; Vissenberg, Kris

    2015-01-01

    Background and Aims Plant stature and shape are largely determined by cell elongation, a process that is strongly controlled at the level of the cell wall. This is associated with the presence of many cell wall proteins implicated in the elongation process. Several proteins and enzyme families have been suggested to be involved in the controlled weakening of the cell wall, and these include xyloglucan endotransglucosylases/hydrolases (XTHs), yieldins, lipid transfer proteins and expansins. Although expansins have been the subject of much research, the role and involvement of expansin-like genes/proteins remain mostly unclear. This study investigates the expression and function of AtEXLA2 (At4g38400), a member of the expansin-like A (EXLA) family in arabidposis, and considers its possible role in cell wall metabolism and growth. Methods Transgenic plants of Arabidopsis thaliana were grown, and lines over-expressing AtEXLA2 were identified. Plants were grown in the dark, on media containing growth hormones or precursors, or were gravistimulated. Hypocotyls were studied using transmission electron microscopy and extensiometry. Histochemical GUS (β-glucuronidase) stainings were performed. Key Results AtEXLA2 is one of the three EXLA members in arabidopsis. The protein lacks the typical domain responsible for expansin activity, but contains a presumed cellulose-interacting domain. Using promoter::GUS lines, the expression of AtEXLA2 was seen in germinating seedlings, hypocotyls, lateral root cap cells, columella cells and the central cylinder basally to the elongation zone of the root, and during different stages of lateral root development. Furthermore, promoter activity was detected in petioles, veins of leaves and filaments, and also in the peduncle of the flowers and in a zone just beneath the papillae. Over-expression of AtEXLA2 resulted in an increase of >10 % in the length of dark-grown hypocotyls and in slightly thicker walls in non-rapidly elongating etiolated

  19. Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

    PubMed

    Wriessnegger, Tamara; Moser, Sandra; Emmerstorfer-Augustin, Anita; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Pichler, Harald

    2016-04-01

    Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6. PMID:26898115

  20. Enhancing lignan biosynthesis by over-expressing pinoresinol lariciresinol reductase in transgenic wheat.

    PubMed

    Ayella, Allan K; Trick, Harold N; Wang, Weiqun

    2007-12-01

    Lignans are phenylpropane dimers that are biosynthesized via the phenylpropanoid pathway, in which pinoresinol lariciresinol reductase (PLR) catalyzes the last steps of lignan production. Our previous studies demonstrated that the contents of lignans in various wheat cultivars were significantly associated with anti-tumor activities in APC(Min) mice. To enhance lignan biosynthesis, this study was conducted to transform wheat cultivars ('Bobwhite', 'Madison', and 'Fielder', respectively) with the Forsythia intermedia PLR gene under the regulatory control of maize ubiquitin promoter. Of 24 putative transgenic wheat lines, we successfully obtained 3 transformants with the inserted ubiquitin-PLR gene as screened by PCR. Southern blot analysis further demonstrated that different copies of the PLR gene up to 5 were carried out in their genomes. Furthermore, a real-time PCR indicated approximately 17% increase of PLR gene expression over the control in 2 of the 3 positive transformants at T(0) generation. The levels of secoisolariciresinol diglucoside, a prominent lignan in wheat as determined by HPLC-MS, were found to be 2.2-times higher in one of the three positive transgenic sub-lines at T(2 )than that in the wild-type (117.9 +/- 4.5 vs. 52.9 +/- 19.8 mug/g, p <0.005). To the best of our knowledge, this is the first study that elevated lignan levels in a transgenic wheat line has been successfully achieved through genetic engineering of over-expressed PLR gene. Although future studies are needed for a stably expression and more efficient transformants, the new wheat line with significantly higher SDG contents obtained from this study may have potential application in providing additive health benefits for cancer prevention.

  1. Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

    NASA Technical Reports Server (NTRS)

    Hong, Y. S.; Ciszak, Ewa; Patel, Mulchand

    2000-01-01

    Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For

  2. IL-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells.

    PubMed

    Long, Jun; Zhang, Xulong; Wen, Mingjie; Kong, Qingli; Lv, Zhe; An, Yunqing; Wei, Xiao-Qing

    2013-01-01

    Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

  3. Effects of growth hormone over-expression on reproduction in the common carp Cyprinus carpio L.

    PubMed

    Cao, Mengxi; Chen, Ji; Peng, Wei; Wang, Yaping; Liao, Lanjie; Li, Yongming; Trudeau, Vance L; Zhu, Zuoyan; Hu, Wei

    2014-01-01

    To study the complex interaction between growth and reproduction we have established lines of transgenic common carp (Cyprinus carpio) carrying a grass carp (Ctenopharyngodon idellus) growth hormone (GH) transgene. The GH-transgenic fish showed delayed gonadal development compared with non-transgenic common carp. To gain a better understanding of the phenomenon, we studied body growth, gonad development, changes of reproduction related genes and hormones of GH-transgenic common carp for 2years. Over-expression of GH elevated peripheral gh transcription, serum GH levels, and inhibited endogenous GH expression in the pituitary. Hormone analyses indicated that GH-transgenic common carp had reduced pituitary and serum level of luteinizing hormone (LH). Among the tested genes, pituitary lhβ was inhibited in GH-transgenic fish. Further analyses in vitro showed that GH inhibited lhβ expression. Localization of ghr with LH indicates the possibility of direct regulation of GH on gonadotrophs. We also found that GH-transgenic common carp had reduced pituitary sensitivity to stimulation by co-treatments with a salmon gonadotropin-releasing hormone (GnRH) agonist and a dopamine antagonist. Together these results suggest that the main cause of delayed reproductive development in GH transgenic common carp is reduced LH production and release.

  4. Over-expression of sly-miR156a in tomato results in multiple vegetative and reproductive trait alterations and partial phenocopy of the sft mutant.

    PubMed

    Zhang, Xiaohui; Zou, Zhe; Zhang, Junhong; Zhang, Yuyang; Han, Qinqin; Hu, Tixu; Xu, Xiaoguang; Liu, Hui; Li, Hanxia; Ye, Zhibiao

    2011-01-21

    Plant microRNAs (miRNAs) are vital components of the translation control system that regulates plant development and reproduction. The biological function of sly-miR156 was investigated by over-expression in tomato plants. Transgenic tomato plants exhibited a drastically altered phenotype, with reduced height, smaller but more numerous leaves, and smaller fruit. The inflorescence structure of sly-miR156 over-expressing plants phenocopied the sft mutant. The putative targets of sly-miR156 were identified by data base search and included six SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box transcription factor genes. Their expression patterns were then determined in 35S-miR156a and wild type tomato plants. These target genes, as well as the tomato FLOWERING LOCUS T (FT) ortholog SFT, were significantly down-regulated in sly-miR156 over-expressing plants. These studies reveal novel phenotypes regulated by miR156.

  5. Heterogeneity of microRNAs expression in cervical cancer cells: over-expression of miR-196a

    PubMed Central

    Villegas-Ruiz, Vanessa; Juárez-Méndez, Sergio; Pérez-González, Oscar A; Arreola, Hugo; Paniagua-García, Lucero; Parra-Melquiadez, Miriam; Peralta-Rodríguez, Raúl; López-Romero, Ricardo; Monroy-García, Alberto; Mantilla-Morales, Alejandra; Gómez-Gutiérrez, Guillermo; Román-Bassaure, Edgar; Salcedo, Mauricio

    2014-01-01

    In recent years, the study of microRNAs associated with neoplastic processes has increased. Patterns of microRNA expression in different cell lines and different kinds of tumors have been identified; however, little is known about the alterations in regulatory pathways and genes involved in aberrant set of microRNAs. The identification of these altered microRNAs in several cervical cancer cells and potentially deregulated pathways involved constitute the principal goals of the present study. In the present work, the expression profiles of cellular microRNAs in Cervical Cancer tissues and cell lines were explored using microRNA microarray, Affymetrix. The most over-expressed was miR-196a, which was evaluated by real time PCR, and HOXC8 protein as potential target by immunohistochemistry assay. One hundred and twenty three human microRNAs differentially expressed in the cell tumor, 64 (52%) over-expressed and 59 (48%) under-expressed were observed. Among the microRNAs over-expressed, we focused on miR-196a; at present this microRNA is poorly studied in CC. The expression of this microRNA was evaluated by qRT-PCR, and HOXC8 by immunohistochemistry assay. There is not a specific microRNA expression profile in the CC cells, neither a microRNA related to HPV presence. Furthermore, the miR-196a was over-expressed, while an absence of HOXC8 expression was observed. We suggest that miR-196a could be played as oncomiR in CC. PMID:24817935

  6. Over-expression of OsPTR6 in rice increased plant growth at different nitrogen supplies but decreased nitrogen use efficiency at high ammonium supply.

    PubMed

    Fan, Xiaorong; Xie, Dan; Chen, Jingguang; Lu, Haiyan; Xu, Yanling; Ma, Cui; Xu, Guohua

    2014-10-01

    Nitrogen (N) plays a critical role in plant growth and productivity and PTR/NRT1 transporters are critical for rice growth. In this study, OsPTR6, a PTR/NRT1 transporter, was over-expressed in the Nipponbare rice cultivar by Agrobacterium tumefaciens transformation using the ubiquitin (Ubi) promoter. Three single-copy T2 generation transgenic lines, named OE1, OE5 and OE6, were produced and subjected to hydroponic growth experiments in different nitrogen treatments. The results showed the plant height and biomass of the over-expression lines were increased, and plant N accumulation and glutamine synthetase (GS) activities were enhanced at 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. The expression of OsATM1 genes in over-expression lines showed that the OsPTR6 over expression increased OsAMT1.1, OsATM1.2 and OsAMT1.3 expression at 0.2 and 5.0mmol/L NH4(+) and 2.5mmol/L NH4NO3. However, nitrogen utilisation efficiency (NUE) was decreased at 5.0mmol/LNH4(+). These data suggest that over-expression of the OsPTR6 gene could increase rice growth through increasing ammonium transporter expression and glutamine synthetase activity (GSA), but decreases nitrogen use efficiency under conditions of high ammonium supply.

  7. IGF2 over-expression in solitary fibrous tumours is independent of anatomical location and is related to loss of imprinting.

    PubMed

    Hajdu, Mihai; Singer, Samuel; Maki, Robert G; Schwartz, Gary K; Keohan, Mary Lou; Antonescu, Cristina R

    2010-07-01

    Solitary fibrous tumour (SFT) is a mesenchymal neoplasm composed of CD34-positive fibroblastic cells. The pathogenesis driving this neoplasm remains unclear, with no recurrent genetic aberrations described to date. Previous reports suggest a role for IGF2 over-expression in the pathogenesis of these tumours, implicated in triggering hypoglycaemia in some patients. The expression profiling of 23 SFTs was investigated using an Affymetrix U133A platform. The transcriptional signature was compared to a set of 34 soft tissue sarcomas spanning seven subtypes. Potential candidate genes were then further investigated for activating mutations or loss of imprinting (LOI). SFT had a distinct expression signature and clustered in a tight genomic cluster, separate from all other sarcoma subtypes. A number of over-expressed receptor tyrosine kinase (RTK) genes were identified in SFT, including DDR1, ERBB2 and FGFR1; however, no mutations were identified by cDNA sequencing. Over-expression of IGF2 was uniformly detected in SFT, regardless of anatomical location, and was related to LOI. In contrast, IGF1 and JUN over-expression was seen in pleural, but not meningeal, locations. SFT shows a distinctive expression signature, with over-expression of DDR1, ERBB2 and FGFR1. Despite of lack of activating mutations in these RTKs, therapy with specific inhibitors targeting these kinases might be considered in advanced/metastatic cases. Our results confirm LOI in several tumours expressing high levels of IGF2, which may explain the observed paraneoplastic hypoglycaemia.

  8. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    SciTech Connect

    Xu, H. Howard . E-mail: hxu3@calstatela.edu; Real, Lilian; Bailey, Melissa Wu

    2006-11-03

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

  9. Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus) Over-Expressing MicroRNA394.

    PubMed

    Song, Jian Bo; Shu, Xia Xia; Shen, Qi; Li, Bo Wen; Song, Jun; Yang, Zhi Min

    2015-01-01

    Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus) miR394 with its target gene Brassica napus leaf curling responsiveness (BnLCR) to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS) and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including leafy cotyledon1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development.

  10. Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus) Over-Expressing MicroRNA394.

    PubMed

    Song, Jian Bo; Shu, Xia Xia; Shen, Qi; Li, Bo Wen; Song, Jun; Yang, Zhi Min

    2015-01-01

    Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus) miR394 with its target gene Brassica napus leaf curling responsiveness (BnLCR) to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS) and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including leafy cotyledon1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development. PMID:25978066

  11. Altered Fruit and Seed Development of Transgenic Rapeseed (Brassica napus) Over-Expressing MicroRNA394

    PubMed Central

    Song, Jian Bo; Shu, Xia Xia; Shen, Qi; Li, Bo Wen; Song, Jun; Yang, Zhi Min

    2015-01-01

    Fruit and seed development in plants is a complex biological process mainly involved in input and biosynthesis of many storage compounds such as proteins and oils. Although the basic biochemical pathways for production of the storage metabolites in plants are well characterized, their regulatory mechanisms are not fully understood. In this study, we functionally identified rapeseed (Brassica napus) miR394 with its target gene Brassica napus LEAF CURLING RESPONSIVENESS (BnLCR) to dissect a role of miR394 during the fruit and seed development. Transgenic rapeseed plants over-expressing miR394 under the control of the cauliflower mosaic virus 35S promoter were generated. miR394 over-expression plants exhibited a delayed flowering time and enlarged size of plants, leaf blade, pods and seed body, but developed seeds with higher contents of protein and glucosinolates (GLS) and lower levels of oil accumulation as compared to wild-type. Over-expression of miR394 altered the fatty acid (FA) composition by increasing several FA species such as C16:0 and C18:0 and unsaturated species of C20:1 and C22:1 but lowering C18:3. This change was accompanied by induction of genes coding for transcription factors of FA synthesis including LEAFY COTYLEDON1 (BnLEC1), BnLEC2, and FUSCA3 (FUS3). Because the phytohormone auxin plays a crucial role in fruit development and seed patterning, the DR5-GUS reporter was used for monitoring the auxin response in Arabidopsis siliques and demonstrated that the DR5 gene was strongly expressed. These results suggest that BnmiR394 is involved in rapeseed fruit and seed development. PMID:25978066

  12. Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana.

    PubMed

    Tong, Xiaoling; Hrycaj, Steven; Podlaha, Ondrej; Popadic, Aleksandar; Monteiro, Antónia

    2014-10-15

    In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain - in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly׳s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies. PMID:25169193

  13. Over-expression of Ultrabithorax alters embryonic body plan and wing patterns in the butterfly Bicyclus anynana.

    PubMed

    Tong, Xiaoling; Hrycaj, Steven; Podlaha, Ondrej; Popadic, Aleksandar; Monteiro, Antónia

    2014-10-15

    In insects, forewings and hindwings usually have different shapes, sizes, and color patterns. A variety of RNAi experiments across insect species have shown that the hox gene Ultrabithorax (Ubx) is necessary to promote hindwing identity. However, it remains unclear whether Ubx is sufficient to confer hindwing fate to forewings across insects. Here, we address this question by over-expressing Ubx in the butterfly Bicyclus anynana using a heat-shock promoter. Ubx whole-body over-expression during embryonic and larvae development led to body plan changes in larvae but to mere quantitative changes to adult morphology, respectively. Embryonic heat-shocks led to fused segments, loss of thoracic and abdominal limbs, and transformation of head limbs to larger appendages. Larval heat-shocks led to reduced eyespot size in the expected homeotic direction, but neither additional eyespots nor wing shape changes were observed in forewings as expected of a homeotic transformation. Interestingly, Ubx was found to be expressed in a novel, non-characteristic domain - in the hindwing eyespot centers. Furthermore, ectopic expression of Ubx on the pupal wing activated the eyespot-associated genes spalt and Distal-less, known to be directly repressed by Ubx in the fly׳s haltere and leg primordia, respectively, and led to the differentiation of black wing scales. These results suggest that Ubx has been co-opted into a novel eyespot gene regulatory network, and that it is capable of activating black pigmentation in butterflies.

  14. Network analysis of microRNAs, transcription factors, target genes and host genes in nasopharyngeal carcinoma

    PubMed Central

    WANG, HAO; XU, ZHIWEN; MA, MENGYAO; WANG, NING; WANG, KUNHAO

    2016-01-01

    Numerous studies on the morbidity of nasopharyngeal carcinoma (NPC) have identified several genes, microRNAs (miRNAs or miRs) and transcription factors (TFs) that influence the pathogenesis of NPC. However, summarizing all the regulatory networks involved in NPC is challenging. In the present study, the genes, miRNAs and TFs involved in NPC were considered as the nodes of the so-called regulatory network, and the associations between them were investigated. To clearly represent these associations, three regulatory networks were built seperately, namely, the differentially expressed network, the associated network and the global network. The differentially expressed network is the most important one of these three networks, since its nodes are differentially expressed genes whose mutations may lead to the development of NPC. Therefore, by modifying the aberrant expression of those genes that are differentially expressed in this network, their dysregulation may be corrected and the tumorigenesis of NPC may thus be prevented. Analysis of the aforementioned three networks highlighted the importance of certain pathways, such as self-adaptation pathways, in the development of NPC. For example, cyclin D1 (CCND1) was observed to regulate Homo sapiens-miR-20a, which in turn targeted CCND1. The present study conducted a systematic analysis of the pathogenesis of NPC through the three aforementioned regulatory networks, and provided a theoretical model for biologists. Future studies are required to evaluate the influence of the highlighted pathways in NPC. PMID:27313701

  15. Aberrant over-expression of COX-1 intersects multiple pro-tumorigenic pathways in high-grade serous ovarian cancer

    PubMed Central

    Wilson, Andrew J.; Fadare, Oluwole; Beeghly-Fadiel, Alicia; Son, Deok-Soo; Liu, Qi; Zhao, Shilin; Saskowski, Jeanette; Uddin, Md. Jashim; Daniel, Cristina; Crews, Brenda; Lehmann, Brian D.; Pietenpol, Jennifer A.; Crispens, Marta A.; Marnett, Lawrence J.; Khabele, Dineo

    2015-01-01

    Cyclooxygenase-1 (COX-1) is implicated in ovarian cancer. However, patterns of COX expression and function have been unclear and controversial. In this report, patterns of COX-1 and COX-2 gene expression were obtained from RNA-seq data through The Cancer Genome Atlas. Our analysis revealed markedly higher COX-1 mRNA expression than COX-2 in high-grade serous ovarian cancers (HGSOC) and higher COX-1 expression in HGSOC tumors than 10 other tumor types. High expression of COX-1 in HGSOC tumors was confirmed in an independent tissue microarray. In contrast, lower or similar expression of COX-1 compared to COX-2 was observed in endometrioid, mucinous and clear cell tumors. Stable COX-1 knockdown in HGSOC-representative OVCAR-3 ovarian cancer cells reduced gene expression in multiple pro-tumorigenic pathways. Functional cell viability, clonogenicity, and migration/invasion assays were consistent with transcriptomic changes. These effects were reversed by stable over-expression of COX-1 in SKOV-3 cells. Our results demonstrate a distinct pattern of COX-1 over-expression in HGSOC tumors and strong association of COX-1 with multiple pro-tumorigenic pathways in ovarian cancer cells. These findings provide additional insight into the role of COX-1 in human ovarian cancer and support further development of methods to selectively target COX-1 in the management of HGSOC tumors. PMID:25972361

  16. Over-expression of mango (Mangifera indica L.) MiARF2 inhibits root and hypocotyl growth of Arabidopsis.

    PubMed

    Wu, Bei; Li, Yun-He; Wu, Jian-Yong; Chen, Qi-Zhu; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

    2011-06-01

    An auxin response factor 2 gene, MiARF2, was cloned in our previous study [1] from the cotyledon section of mango (Mangifera indica L. cv. Zihua) during adventitious root formation, which shares an 84% amino acid sequence similarity to Arabidopsis ARF2. This study was to examine the effects of over-expression of the full-length MiARF2 open reading frame on the root and hypocotyl growth in Arabidopsis. Phenotype analysis showed that the T(3) transgenic lines had about 20-30% reduction in the length of hypocotyls and roots of the seedlings in comparison with the wild-type. The transcription levels of ANT and ARGOS genes which play a role in controlling organ size and cell proliferation in the transgenic seedlings also decreased. Therefore, the inhibited root and hypocotyl growth in the transgenic seedlings may be associated with the down-regulated transcription of ANT and ARGOS by the over-expression of MiARF2. This study also suggests that although MiARF2 only has a single DNA-binding domain (DBD), it can function as other ARF-like proteins containing complete DBD, middle region (MR) and carboxy-terminal dimerization domain (CTD).

  17. The over-expression of a chrysanthemum WRKY transcription factor enhances aphid resistance.

    PubMed

    Li, Peiling; Song, Aiping; Gao, Chunyan; Jiang, Jiafu; Chen, Sumei; Fang, Weimin; Zhang, Fei; Chen, Fadi

    2015-10-01

    Members of the large WRKY transcription factor family are responsible for the regulation of plant growth, development and the stress response. Here, five WRKY members were isolated from chrysanthemum. They each contained a single WRKY domain and a C2H2 zinc finger motif, so were classified into group II. Transient expression experiments demonstrated that all five were expressed in the nucleus, although CmWRKY42 was also expressed in the cytoplasm. When expressed heterologously in yeast, the products of CmWRKY22 and CmWRKY48 exhibited transactivation activity, while those of CmWRKY21, CmWRKY40 and CmWRKY42 did not. The transcription of the five CmWRKY genes was profiled when the plants were challenged with a variety of abiotic and biotic stress agents, as well as being treated with various phytohormones. CmWRKY21 proved to be markedly induced by salinity stress, and suppressed by high temperature exposure; CmWRKY22 was induced by high temperature exposure; CmWRKY40 was highly induced by salinity stress, and treatment with either abscisic acid (ABA) or methyl jasmonate (MeJA); CmWRKY42 was up-regulated by salinity stress, low temperature, ABA and MeJA treatment and aphid infestation; CmWRKY48 was induced by drought stress, ABA and MeJA treatment and aphid infestation. The function of CmWRKY48 was further investigated by over-expressing it transgenically. The constitutive expression of this transcription factor inhibited the aphids' population growth capacity, suggesting that it may represent an important component of the plant's defense machinery against aphids. PMID:26184088

  18. Enhanced disease resistance in transgenic carrot (Daucus carota L.) plants over-expressing a rice cationic peroxidase.

    PubMed

    Wally, O; Punja, Z K

    2010-10-01

    Plant class III peroxidases are involved in numerous responses related to pathogen resistance including controlling hydrogen peroxide (H(2)O(2)) levels and lignin formation. Peroxidases catalyze the oxidation of organic compounds using H(2)O(2) as an oxidant. We examined the mechanisms of disease resistance in a transgenic carrot line (P23) which constitutively over-expresses the rice cationic peroxidase OsPrx114 (previously known as PO-C1) and which exhibits enhanced resistance to necrotrophic foliar pathogens. OsPrx114 over-expression led to a slight enhancement of constitutive transcript levels of pathogenesis-related (PR) genes. These transcript levels were dramatically increased in line P23 compared to controls [GUS construct under the control of 35S promoter (35S::GUS)] when tissues were treated with cell wall fragments of the fungal pathogen Sclerotinia sclerotiorum (SS-walls), and to a lesser extent with 2,6-dichloroisonicotinic acid. There was no basal increase in basal H(2)O(2) levels in tissues of the line P23. However, during an oxidative burst response elicited by SS-walls, H(2)O(2) accumulation was reduced in line P23 despite, typical media alkalinization associated with oxidative burst responses was observed, suggesting that OsPrx114 was involved in rapid H(2)O(2) consumption during the oxidative burst response. Tap roots of line P23 had increased lignin formation in the outer periderm tissues, which was further increased during challenge inoculation with Alternaria radicina. Plant susceptibility to a biotrophic pathogen, Erysiphe heraclei, was not affected. Disease resistance to necrotrophic pathogens in carrot as a result of OsPrx114 over-expression is manifested through increased PR transcript accumulation, rapid removal of H(2)O(2) during oxidative burst response and enhanced lignin formation.

  19. Metabolic engineering of morphinan alkaloids by over-expression of codeinone reductase in transgenic hairy roots of Papaver bracteatum, the Iranian poppy.

    PubMed

    Sharafi, Ali; Sohi, Haleh Hashemi; Mousavi, Amir; Azadi, Pejman; Khalifani, Bahman Hosseini; Razavi, Khadijeh

    2013-03-01

    Papaver bracteatum has a high content of thebaine. It is used as an alternative to P. somniferum for the production of benzylisoquinoline alkaloid. Papaver bracteatum was genetically engineered to over-express codeinone reductase gene in hairy root cultures. Transcript level of the codeinone reductase gene in transgenic hairy root lines increased up to ten- and 24-fold in comparison with hairy roots without CodR over-expression and wild type roots, respectively. Codeine was produced at (0.04 % dry wt) and morphine was at (0.28 % dry wt) in the transgenic hairy root lines. Papaver bracteatum hairy roots expressing CodR gene thus have a high potential to produce morphinan alkaloids.

  20. HER3 over-expression and overall survival in gastrointestinal cancers.

    PubMed

    Wang, Yadong; Yang, Haiyan; Duan, Guangcai

    2015-12-15

    Published studies on the association between human epidermal factor receptor 3 (HER3) expression and overall survival (OS) in gastrointestinal cancers have yielded conflicting results. The aim of this study was to explore the association of HER3 over-expression with OS in gastrointestinal cancers. A systematic search was performed through Medline/PubMed, Embase, Science Direct and Elsevier. The summary odds ratio (OR) with 95% confidence interval (CI) was calculated to estimate the strength of the association. Overall, we observed that HER3 over-expression was associated with worse OS at five years (OR = 1.38, 95% CI: 1.04-1.82); however, HER3 over-expression was not associated with worse OS at three years (OR = 1.33, 95% CI: 0.97-1.84). The cumulative meta-analysis showed similar results. In subgroup analyses by tumor type, HER3 over-expression in gastric cancers was associated with worse OS at both three years (OR = 1.69, 95% CI: 1.28-2.25) and five years (OR = 1.74, 95% CI: 1.26-2.41). In conclusion, our results suggest that HER3 over-expression may be associated with worse overall survival in gastric cancers. Well-designed studies with a large sample size are required to further confirm our findings.

  1. Over-expression of polyphenol oxidase gene in strawberry fruit delays the fungus infection process

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polyphenols are secondary metabolites widely present in plants and beneficial to human health. In this study, the changes of polyphenol contents during strawberry fruit development as well as changes of polyphenol oxidase (PPO) was analyzed. The polyphenol content showed declining trend during fruit...

  2. Over-Expression of Cysteine Leucine Rich Protein Is Related to SAG Resistance in Clinical Isolates of Leishmania donovani

    PubMed Central

    Das, Sanchita; Shah, Priyanka; Tandon, Rati; Yadav, Narendra Kumar; Sahasrabuddhe, Amogh A.; Sundar, Shyam; Siddiqi, Mohammad Imran; Dube, Anuradha

    2015-01-01

    Background Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche

  3. Serotonin accumulation in transgenic rice by over-expressing tryptophan decarboxylase results in a dark brown phenotype and stunted growth.

    PubMed

    Kanjanaphachoat, Parawee; Wei, Bi-Yin; Lo, Shuen-Fang; Wang, I-Wen; Wang, Chang-Sheng; Yu, Su-May; Yen, Ming-Liang; Chiu, Sheng-Hsien; Lai, Chien-Chen; Chen, Liang-Jwu

    2012-04-01

    A mutant M47286 with a stunted growth, low fertility and dark-brown phenotype was identified from a T-DNA-tagged rice mutant library. This mutant contained a copy of the T-DNA tag inserted at the location where the expression of two putative tryptophan decarboxylase genes, TDC-1 and TDC-3, were activated. Enzymatic assays of both recombinant proteins showed tryptophan decarboxylase activities that converted tryptophan to tryptamine, which could be converted to serotonin by a constitutively expressed tryptamine 5' hydroxylase (T5H) in rice plants. Over-expression of TDC-1 and TDC-3 in transgenic rice recapitulated the stunted growth, darkbrown phenotype and resulted in a low fertility similar to M47286. The degree of stunted growth and dark-brown color was proportional to the expression levels of TDC-1 and TDC-3. The levels of tryptamine and serotonin accumulation in these transgenic rice lines were also directly correlated with the expression levels of TDC-1 and TDC-3. A mass spectrometry assay demonstrated that the darkbrown leaves and hulls in the TDC-overexpressing transgenic rice were caused by the accumulation of serotonin dimer and that the stunted growth and low fertility were also caused by the accumulation of serotonin and serotonin dimer, but not tryptamine. These results represent the first evidence that over-expression of TDC results in stunted growth, low fertility and the accumulation of serotonin, which when converted to serotonin dimer, leads to a dark brown plant color.

  4. Polymorphisms in cell cycle regulatory genes, urinary arsenic profile and urothelial carcinoma

    SciTech Connect

    Chung, C.-J.; Huang, C.-J.; Pu, Y.-S.; Su, C.-T.; Huang, Y.-K.; Chen, Y.-T.; Hsueh, Y.-M.

    2008-10-15

    Introduction: Polymorphisms in p53, p21 and CCND1 could regulate the progression of the cell cycle and might increase the susceptibility to inorganic arsenic-related cancer risk. The goal of our study was to evaluate the roles of cell cycle regulatory gene polymorphisms in the carcinogenesis of arsenic-related urothelial carcinoma (UC). Methods: A hospital-based case-controlled study was conducted to explore the relationships among the urinary arsenic profile, 8-hydroxydeoxyguanosine (8-OHdG) levels, p53 codon 72, p21 codon 31 and CCND1 G870A polymorphisms and UC risk. The urinary arsenic profile was determined using high-performance liquid chromatography (HPLC) and hydride generator-atomic absorption spectrometry (HG-AAS). 8-OHdG levels were measured by high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP). Results: Subjects carrying the p21 Arg/Arg genotype had an increased UC risk (age and gender adjusted OR = 1.53; 95% CI, 1.02-2.29). However, there was no association of p53 or CCND1 polymorphisms with UC risk. Significant effects were observed in terms of a combination of the three gene polymorphisms and a cumulative exposure of cigarette smoking, along with the urinary arsenic profile on the UC risk. The higher total arsenic concentration, monomethylarsonic acid percentage (MMA%) and lower dimethylarsinic acid percentage (DMA%), possessed greater gene variant numbers, had a higher UC risk and revealed significant dose-response relationships. However, effects of urinary 8-OHdG levels combined with three gene polymorphisms did not seem to be important for UC risk. Conclusions: The results showed that the variant genotype of p21 might be a predictor of inorganic arsenic-related UC risk.

  5. Genome-wide screening of indicator genes for assessing the potential carcinogenic risk of Nanjing city drinking water.

    PubMed

    Zhang, Rui; Cheng, Shupei; Li, Aimin; Sun, Jie; Zhang, Yan; Zhang, Xuxiang

    2011-07-01

    Effects of all pollutants existing in the Nanjing city drinking water (DWNC) on mouse gene transcription levels were measured to assess the DWNC carcinogenic risks and to identify candidate indicator genes for assessing and early warning the cancer risks. Transcriptional expression levels of 14,000 hepatic genes for the treatment group mice (Mus musculus, ICR) fed with DWNC for 90 days were detected using the GeneChip(®) Mouse Genome 430A 2.0 array. The analysis indicated that the transcriptional levels of 294 genes were up-regulated and 542 ones were down-regulated. Of these genes, 12 ones identified to be involved in at least five different types of cancers were further analyzed. An interrogation by Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that three (including ITGAV, CCND1 and SMAD2) of the 12 genes were mapped to pathway in cancer. Gene Ontology (GO) function annotation also showed that they were associated with the functional categories of cell cycle regulation, adhesion, apoptosis, signal transduction and so on which are closely implicated in tumorigenesis and progression. The correlations between the aberrant expressions of them and the genesis and progression of cancers have been further documented by a number of scientific researches. These results might demonstrate that the potential toxicity and carcinogenic risks were associated with DWNC. Moreover, ITGAV, CCND1 and SMAD2 were identified as the most likely candidate indicator genes for the assessment of the combined carcinogenic risk of all pollutants existing in DWNC.

  6. Enhancement of Chlorogenic Acid Production in Hairy Roots of Platycodon grandiflorum by Over-Expression of An Arabidopsis thaliana Transcription Factor AtPAP1

    PubMed Central

    Tuan, Pham Anh; Kwon, Do Yeon; Lee, Sanghyun; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Nam Il; Park, Sang Un

    2014-01-01

    To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA. PMID:25153629

  7. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    PubMed

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  8. Alternative Sigma Factor Over-Expression Enables Heterologous Expression of a Type II Polyketide Biosynthetic Pathway in Escherichia coli

    PubMed Central

    Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.

    2013-01-01

    Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102

  9. Over-Expression of Rice CBS Domain Containing Protein, OsCBSX3, Confers Rice Resistance to Magnaporthe oryzae Inoculation.

    PubMed

    Mou, Shaoliang; Shi, Lanping; Lin, Wei; Liu, Yanyan; Shen, Lei; Guan, Deyi; He, Shuilin

    2015-01-01

    Cystathionine β-synthase (CBS) domain containing proteins (CDCPs) constitute a big family in plants and some members in this family have been implicated in a variety of biological processes, but the precise functions and the underlying mechanism of the majority of this family in plant immunity remain to be elucidated. In the present study, a CBS domain containing protein gene, OsCBSX3, is functionally characterized in rice resistance against Magnaporthe oryzae (M. oryzae). By quantitative real-time PCR, transcripts of OsCBSX3 are up-regulated significantly by inoculation of M. oryzae and the exogenously applied salicylic acid (SA) and methyl jasmonate (MeJA). OsCBSX3 is exclusively localized to the plasma membrane by transient expression of OsCBSX3 fused to green fluorescent protein (GFP) through approach of Agrobacterium infiltration in Nicotiana benthamiana leaves. The plants of homozygous T3 transgenic rice lines of over-expressing OsCBSX3 exhibit significant enhanced resistance to M. oryzae inoculation, manifested by decreased disease symptoms, and inhibition of pathogen growth detected in DNA. Consistently, the over-expression of OsCBSX3 enhances the transcript levels of immunity associated marker genes including PR1a, PR1b, PR5, AOS2, PAL, NH1, and OsWRKY13 in plants inoculated with M. oryzae. These results suggest that OsCBSX3 acts as a positive regulator in resistance of rice to M. oryzae regulated by SA and JA-mediated signaling pathways synergistically. PMID:26184180

  10. Enhanced tolerance to low temperature in tobacco by over-expression of a new maize protein phosphatase 2C, ZmPP2C2.

    PubMed

    Hu, Xiaoli; Liu, Lixia; Xiao, Beilei; Li, Dapeng; Xing, Xin; Kong, Xiangpei; Li, Dequan

    2010-10-15

    Low temperature is one of the most common environmental stresses affecting plant growth and agricultural production. Serine/threonine protein phosphatases 2C (PP2Cs) have been suggested to play an important role in stress signaling. To identify potential new member of the PP2C proteins in maize and investigate its functions for stress responses, the ZmPP2C2 gene, encoding a new PP2C protein from maize roots, was cloned by RT-PCR and RACE-PCR. Its constitutive expression in roots, stems and leaves of maize seedlings was detected by RNA gel blot, and its regulation in response to cold stress was also examined. To further evaluate its function in the cold stress response, we over-expressed the ZmPP2C2 gene in tobacco under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, and assessed a series of physiological changes in wild type and transgenic plants under low temperatures. Compared with wild type tobacco under cold stress, plants that over-expressed ZmPP2C2 displayed higher germination speed and rate, higher antioxidant enzyme (SOD, POD, CAT) activities, with lower cold-induced electrolyte leakage and malondialdehyde (MDA) contents. These results show that over-expression of ZmPP2C2 in tobacco enhanced tolerance to cold stress, suggesting that this new gene, ZmPP2C2, may act as a positive regulator of cold resistance in plants.

  11. MicroRNA 218 Mediates the Effects of Tbx5a Over-Expression on Zebrafish Heart Development

    PubMed Central

    Chiavacci, Elena; Dolfi, Luca; Verduci, Lorena; Meghini, Francesco; Gestri, Gaia; Evangelista, Alberto Mercatanti Monica; Wilson, Stephen W.; Cremisi, Federico; Pitto, Letizia

    2012-01-01

    tbx5, a member of the T-box gene family, encodes one of the key transcription factors mediating vertebrate heart development. Tbx5 function in heart development appears to be exquisitely sensitive to gene dosage, since both haploinsufficiency and gene duplication generate the cardiac abnormalities associated with Holt−Oram syndrome (HOS), a highly penetrant autosomal dominant disease characterized by congenital heart defects of varying severity and upper limb malformation. It is suggested that tight integration of microRNAs and transcription factors into the cardiac genetic circuitry provides a rich and robust array of regulatory interactions to control cardiac gene expression. Based on these considerations, we performed an in silico screening to identify microRNAs embedded in genes highly sensitive to Tbx5 dosage. Among the identified microRNAs, we focused our attention on miR-218-1 that, together with its host gene, slit2, is involved in heart development. We found correlated expression of tbx5 and miR-218 during cardiomyocyte differentiation of mouse P19CL6 cells. In zebrafish embryos, we show that both Tbx5 and miR-218 dysregulation have a severe impact on heart development, affecting early heart morphogenesis. Interestingly, down-regulation of miR-218 is able to rescue the heart defects generated by tbx5 over-expression supporting the notion that miR-218 is a crucial mediator of Tbx5 in heart development and suggesting its possible involvement in the onset of heart malformations. PMID:23226307

  12. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

    PubMed

    Sinha, Deepak K; Chandran, Predeesh; Timm, Alicia E; Aguirre-Rojas, Lina; Smith, C Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion.

  13. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants.

    PubMed

    Sinha, Deepak K; Chandran, Predeesh; Timm, Alicia E; Aguirre-Rojas, Lina; Smith, C Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

  14. Over-expression of the cercosporin facilitator protein, CFP, in Cercospora kikuchii up-regulates production and secretion of cercosporin.

    PubMed

    Upchurch, R G; Rose, M S; Eweida, M

    2001-10-16

    CFP (cercosporin facilitator protein), a light-regulated gene from the soybean fungal pathogen Cercospora kikuchii, encodes the putative major facilitator transporter of the fungal polyketide cercosporin. Gene disruption of CFP in C. kikuchii strain Gus-3 resulted in dramatically reduced cercosporin production and virulence, and increased sensitivity to the toxin. Two C. kikuchii transformant strains (10-1 and 10-11) that over-produce cercosporin were recovered from the complementation of CFP gene-disrupted strain Gus-3. Southern analysis revealed that these strains contained multiple genomic copies of CFP and over-expressed CFP transcript and protein. Although 10-1 and 10-11 produce and secrete significantly elevated levels of cercosporin, they exhibit wild-type resistance to cercosporin, and maintain a wild-type pattern of light-regulated toxin accumulation. Restoration of wild-type cercosporin resistance in 10-1 and 10-11 suggests that CFP does contribute substantially to cercosporin resistance via toxin secretion. The three-fold increase in toxin accumulation, predominantly associated with the mycelium fraction of these CFP multi-copy strains, suggests that CFP may also have a significant, but unknown, role in regulating toxin production.

  15. Mapping MRI/MRS Parameters with Genetic Over-expression Profiles In Human Prostate Cancer: Demonstrating the Potential

    PubMed Central

    Lenkinski, Robert E.; Bloch, B. Nicholas; Liu, Fangbing; Frangioni, John V.; Perner, Sven; Rubin, Mark A.; Genega, Elizabeth; Rofsky, Neil M.; Gaston, Sandra M.

    2009-01-01

    Magnetic resonance imaging (MRI) and MR spectroscopy can probe a variety of physiological (e.g. blood vessel permeability) and metabolic characteristics of prostate cancer. However, little is known about the changes in gene expression that underlie the spectral and imaging features observed in prostate cancer. Tumor induced changes in vascular permeability and angiogenesis are thought to contribute to patterns of dynamic contrast enhanced (DCE) MRI images of prostate cancer even though the genetic basis of tumor vasculogenesis is complex and the specific mechanisms underlying these DCEMRI features have not yet been determined. In order to identify the changes in gene expression that correspond to MRS and DCEMRI patterns in human prostate cancers, we have utilized tissue print micropeel techniques to generate “whole mount” molecular maps of radical prostatectomy specimens that correspond to pre-surgical MRI/MRS studies. These molecular maps include RNA expression profiles from both Affymetrix GeneChip microarrays and quantitative reverse transcriptase PCR (qrt-PCR) analysis, as well as immunohistochemical studies. Using these methods on patients with prostate cancer, we found robust over-expression of choline kinase a in the majority of primary tumors. We also observed overexpression of neuropeptide Y (NPY), a newly identified angiogenic factor, in a subset of DCEMRI positive prostate cancers. These studies set the stage for establishing MRI/MRS parameters as validated biomarkers for human prostate cancer. PMID:18752015

  16. Virulent Diuraphis noxia Aphids Over-Express Calcium Signaling Proteins to Overcome Defenses of Aphid-Resistant Wheat Plants

    PubMed Central

    Sinha, Deepak K.; Chandran, Predeesh; Timm, Alicia E.; Aguirre-Rojas, Lina; Smith, C. Michael

    2016-01-01

    The Russian wheat aphid, Diuraphis noxia, an invasive phytotoxic pest of wheat, Triticum aestivum, and barley, Hordeum vulgare, causes huge economic losses in Africa, South America, and North America. Most acceptable and ecologically beneficial aphid management strategies include selection and breeding of D. noxia-resistant varieties, and numerous D. noxia resistance genes have been identified in T. aestivum and H. vulgare. North American D. noxia biotype 1 is avirulent to T. aestivum varieties possessing Dn4 or Dn7 genes, while biotype 2 is virulent to Dn4 and avirulent to Dn7. The current investigation utilized next-generation RNAseq technology to reveal that biotype 2 over expresses proteins involved in calcium signaling, which activates phosphoinositide (PI) metabolism. Calcium signaling proteins comprised 36% of all transcripts identified in the two D. noxia biotypes. Depending on plant resistance gene-aphid biotype interaction, additional transcript groups included those involved in tissue growth; defense and stress response; zinc ion and related cofactor binding; and apoptosis. Activation of enzymes involved in PI metabolism by D. noxia biotype 2 aphids allows depletion of plant calcium that normally blocks aphid feeding sites in phloem sieve elements and enables successful, continuous feeding on plants resistant to avirulent biotype 1. Inhibition of the key enzyme phospholipase C significantly reduced biotype 2 salivation into phloem and phloem sap ingestion. PMID:26815857

  17. Largescale Transcriptomics Analysis Suggests Over-Expression of BGH3, MMP9 and PDIA3 in Oral Squamous Cell Carcinoma.

    PubMed

    He, Yuan; Shao, Fangyang; Pi, Weidong; Shi, Cong; Chen, Yujia; Gong, Diping; Wang, Bingjie; Cao, Zhiwei; Tang, Kailin

    2016-01-01

    Oral squamous cell carcinoma (OSCC) has been reported as the most prevalent cancer of the head and neck region, while early diagnosis remains challenging. Here we took a comprehensive bioinformatics study on microarray data of 326 OSCC clinical samples with control of 165 normal tissues. The cell interaction pathways of ECM-receptor interaction and focal adhesion were found to be significantly regulated in OSCC samples. Further analysis of the topological properties and expression consistency identified that three hub genes in the gene interaction network, MMP9, PDIA3 and BGH3, were consistently up-expressed in OSCC samples. When being validated on additional microarray datasets of 41 OSCC samples, the validation rate of over-expressed BGH3, MMP9, and PDIA3 reached 90%, 90% and 84% respectively. At last, immuno-histochemical assays were done to test the protein expression of the three genes on newly collected clinical samples of 35 OSCC, 20 samples of pre-OSCC stage, and 12 normal oral mucosa specimens. Their protein expression levels were also found to progressively increase from normal mucosa to pre-OSCC stage and further to OSCC (ANOVA p = 0.000), suggesting their key roles in OSCC pathogenesis. Based on above solid validation, we propose BGH3, MMP9 and PDIA3 might be further explored as potential biomarkers to aid OSCC diagnosis.

  18. Pleiotrophin over-expression provides trophic support to dopaminergic neurons in parkinsonian rats

    PubMed Central

    2011-01-01

    Background Pleiotrophin is known to promote the survival and differentiation of dopaminergic neurons in vitro and is up-regulated in the substantia nigra of Parkinson's disease patients. To establish whether pleiotrophin has a trophic effect on nigrostriatal dopaminergic neurons in vivo, we injected a recombinant adenovirus expressing pleiotrophin in the substantia nigra of 6-hydroxydopamine lesioned rats. Results The viral vector induced pleiotrophin over-expression by astrocytes in the substantia nigra pars compacta, without modifying endogenous neuronal expression. The percentage of tyrosine hydroxylase-immunoreactive cells as well as the area of their projections in the lesioned striatum was higher in pleiotrophin-treated animals than in controls. Conclusions These results indicate that pleiotrophin over-expression partially rescues tyrosine hydroxylase-immunoreactive cell bodies and terminals of dopaminergic neurons undergoing 6-hydroxydopamine-induced degeneration. PMID:21649894

  19. Transgenic over-expression of mammalian heparanase delays prion disease onset and progression

    PubMed Central

    Kovalchuk Ben-Zaken, O; Nissan, I; Tzaban, S; Taraboulos, A; Zcharia, E; Matzger, S; Shafat, I; Vlodavsky, I; Tal, Y.

    2015-01-01

    Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression. PMID:26168721

  20. Anosmin-1 over-expression regulates oligodendrocyte precursor cell proliferation, migration and myelin sheath thickness.

    PubMed

    Murcia-Belmonte, Verónica; Esteban, Pedro F; Martínez-Hernández, José; Gruart, Agnès; Luján, Rafael; Delgado-García, José María; de Castro, Fernando

    2016-04-01

    During development of the central nervous system, anosmin-1 (A1) works as a chemotropic cue contributing to axonal outgrowth and collateralization, as well as modulating the migration of different cell types, fibroblast growth factor receptor 1 (FGFR1) being the main receptor involved in all these events. To further understand the role of A1 during development, we have analysed the over-expression of human A1 in a transgenic mouse line. Compared with control mice during development and in early adulthood, A1 over-expressing transgenic mice showed an enhanced oligodendrocyte precursor cell (OPC) proliferation and a higher number of OPCs in the subventricular zone and in the corpus callosum (CC). The migratory capacity of OPCs from the transgenic mice is increased in vitro due to a higher basal activation of ERK1/2 mediated through FGFR1 and they also produced more myelin basic protein (MBP). In vivo, the over-expression of A1 resulted in an elevated number of mature oligodendrocytes with higher levels of MBP mRNA and protein, as well as increased levels of activation of the ERK1/2 proteins, while electron microscopy revealed thicker myelin sheaths around the axons of the CC in adulthood. Also in the mature CC, the nodes of Ranvier were significantly longer and the conduction velocity of the nerve impulse in vivo was significantly increased in the CC of A1 over-expressing transgenic mice. Altogether, these data confirmed the involvement of A1 in oligodendrogliogenesis and its relevance for myelination.

  1. Over-expression of catalase in myeloid cells confers acute protection following myocardial infarction.

    PubMed

    Cabigas, E Bernadette; Somasuntharam, Inthirai; Brown, Milton E; Che, Pao Lin; Pendergrass, Karl D; Chiang, Bryce; Taylor, W Robert; Davis, Michael E

    2014-05-21

    Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.

  2. Over-expression of thymosin beta 4 promotes abnormal tooth development and stimulation of hair growth.

    PubMed

    Cha, Hee-Jae; Philp, Deborah; Lee, Soo-Hyun; Moon, Hye-Sung; Kleinman, Hynda K; Nakamura, Takashi

    2010-01-01

    Thymosin beta 4 has multi-functional roles in cell physiology. It accelerates wound healing, hair growth and angiogenesis, and increases laminin-5 expression in corneal epithelium. Furthermore, thymosin beta 4 stimulates tumor growth and metastasis by induction of cell migration and vascular endothelial growth factor-mediated angiogenesis. Using a construct on the skin-specific keratin-5 promoter, we have developed thymosin beta 4 over-expressing transgenic mice to further study its functional roles. Thymosin beta 4 in adult skin and in embryonic stages of the transgenic mouse was analyzed by both Western blot and immunohistochemistry. The over-expression of thymosin beta 4 was observed especially around hair follicles and in the teeth in the transgenic mice. We examined the phenotype of the thymosin beta 4 over-expressing mice. Hair growth was accelerated. In addition, the transgenic mice had abnormally-shaped white teeth and dull incisors. We found that the expression of laminin-5 was up-regulated in the skin of the transgenic mice. We conclude that thymosin beta 4 has an important physiological role in hair growth and in tooth development.

  3. Reduced sensitivity to both positive and negative reinforcement in mice over-expressing the 5-hydroxytryptamine transporter.

    PubMed

    Line, Samantha J; Barkus, Chris; Rawlings, Nancy; Jennings, Katie; McHugh, Stephen; Sharp, Trevor; Bannerman, David M

    2014-12-01

    The 5-hydroxytryptamine (5-HT) transporter (5-HTT) is believed to play a key role in both normal and pathological psychological states. Much previous data suggest that the s allele of the polymorphic regulatory region of the 5-HTT gene promoter is associated with reduced 5-HTT expression and vulnerability to psychiatric disorders, including anxiety and depression. In comparison, the l allele, which increases 5-HTT expression, is generally considered protective. However, recent data link this allele to both abnormal 5-HT signalling and psychopathic traits. Here, we studied the processing of aversive and rewarding cues in transgenic mice that over-express the 5-HTT (5-HTTOE mice). Compared with wild-type mice, 5-HTTOE mice froze less in response to both a tone that had previously been paired with footshock, and the conditioning context. In addition, on a decision-making T-maze task, 5-HTTOE mice displayed reduced preference for a larger, delayed reward and increased preference for a smaller, immediate reward, suggesting increased impulsiveness compared with wild-type mice. However, further inspection of the data revealed that 5-HTTOE mice displayed a relative insensitivity to reward magnitude, irrespective of delay. In contrast, 5-HTTOE mice appeared normal on tests of spatial working and reference memory, which required an absolute choice between options associated with either reward or no reward. Overall, the present findings suggest that 5-HTT over-expression results in a reduced sensitivity to both positive and negative reinforcers. Thus, these data show that increased 5-HTT expression has some maladaptive effects, supporting recent suggestions that l allele homozygosity may be a potential risk factor for disabling psychiatric traits.

  4. LIM domain only 2 over-expression in prostate stromal cells facilitates prostate cancer progression through paracrine of Interleukin-11

    PubMed Central

    Ruan, Yuan; Wang, Xiao-Hai; Zhao, Wei; Wang, Xing-Jie; Zhu, Yi-Ping; Gao, Yuan; Hao, Kui-Yuan; Chen, Lei; Han, Bang-Min; Xia, Shu-Jie; Zhao, Fu-Jun

    2016-01-01

    Mechanisms of stromal-epithelial crosstalk are essential for Prostate cancer (PCa) tumorigenesis and progression. Peripheral zone of the prostate gland possesses a stronger inclination for PCa than transition zone. We previously found a variety of genes that differently expressed among different prostate stromal cells, including LIM domain only 2 (LMO2) which highly expressed in peripheral zone derived stromal cells (PZSCs) and PCa associated fibroblasts (CAFs) compared to transition zone derived stromal cells (TZSCs). Studies on its role in tumors have highlighted LMO2 as an oncogene. Herein, we aim to study the potential mechanisms of stromal LMO2 in promoting PCa progression. The in vitro cells co-culture and in vivo cells recombination revealed that LMO2 over-expressed prostate stromal cells could promote the proliferation and invasiveness of either prostate epithelial or cancer cells. Further protein array screening confirmed that stromal LMO2 stimulated the secretion of Interleukin-11 (IL-11), which could promote proliferation and invasiveness of PCa cells via IL-11 receptor α (IL11Rα) – STAT3 signaling. Moreover, stromal LMO2 over-expression could suppress miR-204-5p which was proven to be a negative regulator of IL-11 expression. Taken together, results of our study demonstrate that prostate stromal LMO2 is capable of stimulating IL-11 secretion and by which activates IL11Rα – STAT3 signaling in PCa cells and then facilitates PCa progression. These results may make stromal LMO2 responsible for zonal characteristic of PCa and as a target for PCa microenvironment-targeted therapy. PMID:27028859

  5. Pro-migratory actions of the prostacyclin receptor in human breast cancer cells that over-express cyclooxygenase-2.

    PubMed

    Allison, Sarah E; Petrovic, Nenad; Mackenzie, Peter I; Murray, Michael

    2015-08-15

    Metastasis is the major cause of death in cancer patients. Elevated expression of cyclooxygenase-2 (COX-2) is observed in many human cancers and over-production of downstream prostaglandins (PGs) has been shown to stimulate metastasis. A role for increased PGE2 production has been proposed, but whether other PGs contribute is currently unclear. In this study the pro-migratory actions of individual PGs were evaluated in MDA-MB-468 breast cancer cells that stably over-expressed COX-2 (MDA-COX-2 cells); cell migration was quantified using 3D-matrigel droplet assays. Inhibition of the prostacyclin and PGE synthases, but not alternate prostanoid synthases, prevented the increase in MDA-COX-2 cell migration produced by arachidonic acid (AA); direct treatment of cells with the stable prostacyclin analogue cicaprost also promoted migration. Pharmacological antagonism and knockdown of the IP receptor decreased cell migration, while antagonists of the alternate DP, EP2, FP, and TP prostanoid receptors were inactive. In support of these findings, activation of the IP receptor also enhanced migration in the MDA-MB-468, MDA-MB-231 and A549 cell lines, and IP receptor knock-down in MDA-COX-2 cells decreased the expression of a number of pro-migratory genes. In further studies, the prostacyclin/IP receptor and PGE2/EP4 receptor pathways were found to be functionally independent and the inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) selectively impaired the IP-receptor-dependent migration in MDA-COX-2 cells. Taken together, the prostacyclin/IP/PI3K-p38 MAPK axis has emerged as a novel pro-migratory pathway in breast cancer cells that over-express COX-2. This information could be utilized in novel treatment strategies to minimize tumor metastasis.

  6. Reduced sensitivity to both positive and negative reinforcement in mice over-expressing the 5-hydroxytryptamine transporter.

    PubMed

    Line, Samantha J; Barkus, Chris; Rawlings, Nancy; Jennings, Katie; McHugh, Stephen; Sharp, Trevor; Bannerman, David M

    2014-12-01

    The 5-hydroxytryptamine (5-HT) transporter (5-HTT) is believed to play a key role in both normal and pathological psychological states. Much previous data suggest that the s allele of the polymorphic regulatory region of the 5-HTT gene promoter is associated with reduced 5-HTT expression and vulnerability to psychiatric disorders, including anxiety and depression. In comparison, the l allele, which increases 5-HTT expression, is generally considered protective. However, recent data link this allele to both abnormal 5-HT signalling and psychopathic traits. Here, we studied the processing of aversive and rewarding cues in transgenic mice that over-express the 5-HTT (5-HTTOE mice). Compared with wild-type mice, 5-HTTOE mice froze less in response to both a tone that had previously been paired with footshock, and the conditioning context. In addition, on a decision-making T-maze task, 5-HTTOE mice displayed reduced preference for a larger, delayed reward and increased preference for a smaller, immediate reward, suggesting increased impulsiveness compared with wild-type mice. However, further inspection of the data revealed that 5-HTTOE mice displayed a relative insensitivity to reward magnitude, irrespective of delay. In contrast, 5-HTTOE mice appeared normal on tests of spatial working and reference memory, which required an absolute choice between options associated with either reward or no reward. Overall, the present findings suggest that 5-HTT over-expression results in a reduced sensitivity to both positive and negative reinforcers. Thus, these data show that increased 5-HTT expression has some maladaptive effects, supporting recent suggestions that l allele homozygosity may be a potential risk factor for disabling psychiatric traits. PMID:25283165

  7. Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

    PubMed

    Rajasekaran, Parthiban; Surendran, Naveen; Seleem, Mohamed N; Sriranganathan, Nammalwar; Schurig, Gerhardt G; Boyle, Stephen M

    2011-04-12

    Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.

  8. Cell-Cycle Gene Alterations in 4,864 Tumors Analyzed by Next-Generation Sequencing: Implications for Targeted Therapeutics.

    PubMed

    Helsten, Teresa; Kato, Shumei; Schwaederle, Maria; Tomson, Brett N; Buys, Timon P H; Elkin, Sheryl K; Carter, Jennifer L; Kurzrock, Razelle

    2016-07-01

    Alterations in the cyclin-dependent kinase (CDK)-retinoblastoma (RB) machinery disrupt cell-cycle regulation and are being targeted in drug development. To understand the cancer types impacted by this pathway, we analyzed frequency of abnormalities in key cell-cycle genes across 4,864 tumors using next-generation sequencing (182 or 236 genes; Clinical Laboratory Improvement Amendments laboratory). Aberrations in the cell-cycle pathway were identified in 39% of cancers, making this pathway one of the most commonly altered in cancer. The frequency of aberrations was as follows: CDKN2A/B (20.1% of all patients), RB1 (7.6%), CCND1 (6.1%), CCNE1 (3.6%), CDK4 (3.2%), CCND3 (1.8%), CCND2 (1.7%), and CDK6 (1.7%). Rates and types of aberrant cell-cycle pathway genes differed between cancer types and within histologies. Analysis of coexisting and mutually exclusive genetic aberrations showed that CCND1, CCND2, and CCND3 aberrations were all positively associated with CDK6 aberrations [OR and P values, multivariate analysis: CCND1 and CDK6 (OR = 3.5; P < 0.0001), CCND2 and CDK6 (OR = 4.3; P = 0.003), CCND3 and CDK6 (OR = 3.6; P = 0.007)]. In contrast, RB1 alterations were negatively associated with multiple gene anomalies in the cell-cycle pathway, including CCND1 (OR = 0.25; P = 0.003), CKD4 (OR = 0.10; P = 0.001), and CDKN2A/B (OR = 0.21; P < 0.0001). In conclusion, aberrations in the cell-cycle pathway were very common in diverse cancers (39% of 4,864 neoplasms). The frequencies and types of alterations differed between and within tumor types and will be informative for drug development strategies. Mol Cancer Ther; 15(7); 1682-90. ©2016 AACR.

  9. Cell-Cycle Gene Alterations in 4,864 Tumors Analyzed by Next-Generation Sequencing: Implications for Targeted Therapeutics.

    PubMed

    Helsten, Teresa; Kato, Shumei; Schwaederle, Maria; Tomson, Brett N; Buys, Timon P H; Elkin, Sheryl K; Carter, Jennifer L; Kurzrock, Razelle

    2016-07-01

    Alterations in the cyclin-dependent kinase (CDK)-retinoblastoma (RB) machinery disrupt cell-cycle regulation and are being targeted in drug development. To understand the cancer types impacted by this pathway, we analyzed frequency of abnormalities in key cell-cycle genes across 4,864 tumors using next-generation sequencing (182 or 236 genes; Clinical Laboratory Improvement Amendments laboratory). Aberrations in the cell-cycle pathway were identified in 39% of cancers, making this pathway one of the most commonly altered in cancer. The frequency of aberrations was as follows: CDKN2A/B (20.1% of all patients), RB1 (7.6%), CCND1 (6.1%), CCNE1 (3.6%), CDK4 (3.2%), CCND3 (1.8%), CCND2 (1.7%), and CDK6 (1.7%). Rates and types of aberrant cell-cycle pathway genes differed between cancer types and within histologies. Analysis of coexisting and mutually exclusive genetic aberrations showed that CCND1, CCND2, and CCND3 aberrations were all positively associated with CDK6 aberrations [OR and P values, multivariate analysis: CCND1 and CDK6 (OR = 3.5; P < 0.0001), CCND2 and CDK6 (OR = 4.3; P = 0.003), CCND3 and CDK6 (OR = 3.6; P = 0.007)]. In contrast, RB1 alterations were negatively associated with multiple gene anomalies in the cell-cycle pathway, including CCND1 (OR = 0.25; P = 0.003), CKD4 (OR = 0.10; P = 0.001), and CDKN2A/B (OR = 0.21; P < 0.0001). In conclusion, aberrations in the cell-cycle pathway were very common in diverse cancers (39% of 4,864 neoplasms). The frequencies and types of alterations differed between and within tumor types and will be informative for drug development strategies. Mol Cancer Ther; 15(7); 1682-90. ©2016 AACR. PMID:27196769

  10. Enhanced Tolerance of Transgenic Potato Plants Over-Expressing Non-specific Lipid Transfer Protein-1 (StnsLTP1) against Multiple Abiotic Stresses.

    PubMed

    Gangadhar, Baniekal H; Sajeesh, Kappachery; Venkatesh, Jelli; Baskar, Venkidasamy; Abhinandan, Kumar; Yu, Jae W; Prasad, Ram; Mishra, Raghvendra K

    2016-01-01

    Abiotic stresses such as heat, drought, and salinity are major environmental constraints that limit potato (Solanum tuberosum L.) production worldwide. Previously, we found a potential thermo-tolerance gene, named StnsLTP1 from potato using yeast functional screening. Here, we report the functional characterization of StnsLTP1 and its role in multiple abiotic stresses in potato plants. Computational analysis of StnsLTP1 with other plant LTPs showed eight conserved cysteine residues, and four α-helices stabilized by four disulfide bridges. Expression analysis of StnsLTP1 gene showed differential expression under heat, water-deficit and salt stresses. Transgenic potato lines over-expressing StnsLTP1 gene displayed enhanced cell membrane integrity under stress conditions, as indicated by reduced membrane lipid per-oxidation, and hydrogen peroxide content relative to untransformed (UT) control plants. In addition, transgenic lines over-expressing StLTP1 also exhibited increased antioxidant enzyme activity with enhanced accumulation of ascorbates, and up-regulation of stress-related genes including StAPX, StCAT, StSOD, StHsfA3, StHSP70, and StsHSP20 compared with the UT plants. These results suggests that StnsLTP1 transgenic plants acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of reactive oxygen species and regulated expression of stress-related genes. PMID:27597854

  11. Enhanced Tolerance of Transgenic Potato Plants Over-Expressing Non-specific Lipid Transfer Protein-1 (StnsLTP1) against Multiple Abiotic Stresses

    PubMed Central

    Gangadhar, Baniekal H.; Sajeesh, Kappachery; Venkatesh, Jelli; Baskar, Venkidasamy; Abhinandan, Kumar; Yu, Jae W.; Prasad, Ram; Mishra, Raghvendra K.

    2016-01-01

    Abiotic stresses such as heat, drought, and salinity are major environmental constraints that limit potato (Solanum tuberosum L.) production worldwide. Previously, we found a potential thermo-tolerance gene, named StnsLTP1 from potato using yeast functional screening. Here, we report the functional characterization of StnsLTP1 and its role in multiple abiotic stresses in potato plants. Computational analysis of StnsLTP1 with other plant LTPs showed eight conserved cysteine residues, and four α-helices stabilized by four disulfide bridges. Expression analysis of StnsLTP1 gene showed differential expression under heat, water-deficit and salt stresses. Transgenic potato lines over-expressing StnsLTP1 gene displayed enhanced cell membrane integrity under stress conditions, as indicated by reduced membrane lipid per-oxidation, and hydrogen peroxide content relative to untransformed (UT) control plants. In addition, transgenic lines over-expressing StLTP1 also exhibited increased antioxidant enzyme activity with enhanced accumulation of ascorbates, and up-regulation of stress-related genes including StAPX, StCAT, StSOD, StHsfA3, StHSP70, and StsHSP20 compared with the UT plants. These results suggests that StnsLTP1 transgenic plants acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of reactive oxygen species and regulated expression of stress-related genes. PMID:27597854

  12. Enhanced Tolerance of Transgenic Potato Plants Over-Expressing Non-specific Lipid Transfer Protein-1 (StnsLTP1) against Multiple Abiotic Stresses

    PubMed Central

    Gangadhar, Baniekal H.; Sajeesh, Kappachery; Venkatesh, Jelli; Baskar, Venkidasamy; Abhinandan, Kumar; Yu, Jae W.; Prasad, Ram; Mishra, Raghvendra K.

    2016-01-01

    Abiotic stresses such as heat, drought, and salinity are major environmental constraints that limit potato (Solanum tuberosum L.) production worldwide. Previously, we found a potential thermo-tolerance gene, named StnsLTP1 from potato using yeast functional screening. Here, we report the functional characterization of StnsLTP1 and its role in multiple abiotic stresses in potato plants. Computational analysis of StnsLTP1 with other plant LTPs showed eight conserved cysteine residues, and four α-helices stabilized by four disulfide bridges. Expression analysis of StnsLTP1 gene showed differential expression under heat, water-deficit and salt stresses. Transgenic potato lines over-expressing StnsLTP1 gene displayed enhanced cell membrane integrity under stress conditions, as indicated by reduced membrane lipid per-oxidation, and hydrogen peroxide content relative to untransformed (UT) control plants. In addition, transgenic lines over-expressing StLTP1 also exhibited increased antioxidant enzyme activity with enhanced accumulation of ascorbates, and up-regulation of stress-related genes including StAPX, StCAT, StSOD, StHsfA3, StHSP70, and StsHSP20 compared with the UT plants. These results suggests that StnsLTP1 transgenic plants acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of reactive oxygen species and regulated expression of stress-related genes.

  13. Enhanced Tolerance of Transgenic Potato Plants Over-Expressing Non-specific Lipid Transfer Protein-1 (StnsLTP1) against Multiple Abiotic Stresses.

    PubMed

    Gangadhar, Baniekal H; Sajeesh, Kappachery; Venkatesh, Jelli; Baskar, Venkidasamy; Abhinandan, Kumar; Yu, Jae W; Prasad, Ram; Mishra, Raghvendra K

    2016-01-01

    Abiotic stresses such as heat, drought, and salinity are major environmental constraints that limit potato (Solanum tuberosum L.) production worldwide. Previously, we found a potential thermo-tolerance gene, named StnsLTP1 from potato using yeast functional screening. Here, we report the functional characterization of StnsLTP1 and its role in multiple abiotic stresses in potato plants. Computational analysis of StnsLTP1 with other plant LTPs showed eight conserved cysteine residues, and four α-helices stabilized by four disulfide bridges. Expression analysis of StnsLTP1 gene showed differential expression under heat, water-deficit and salt stresses. Transgenic potato lines over-expressing StnsLTP1 gene displayed enhanced cell membrane integrity under stress conditions, as indicated by reduced membrane lipid per-oxidation, and hydrogen peroxide content relative to untransformed (UT) control plants. In addition, transgenic lines over-expressing StLTP1 also exhibited increased antioxidant enzyme activity with enhanced accumulation of ascorbates, and up-regulation of stress-related genes including StAPX, StCAT, StSOD, StHsfA3, StHSP70, and StsHSP20 compared with the UT plants. These results suggests that StnsLTP1 transgenic plants acquired improved tolerance to multiple abiotic stresses through enhanced activation of antioxidative defense mechanisms via cyclic scavenging of reactive oxygen species and regulated expression of stress-related genes.

  14. Enhancement of β-carotene production by over-expression of HMG-CoA reductase coupled with addition of ergosterol biosynthesis inhibitors in recombinant Saccharomyces cerevisiae.

    PubMed

    Yan, Guo-liang; Wen, Ke-rui; Duan, Chang-qing

    2012-02-01

    In this study, the synergistic effect of overexpressing the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene and adding ergosterol synthesis inhibitor, ketoconazole, on β-carotene production in the recombinant Saccharomyces cerevisiae was investigated. The results showed that the over-expression of HMG-CoA reductase gene and adding 100 mg/l ketoconazole alone can result in 135.1 and 15.6% increment of β-carotene concentration compared with that of the control (2.05 mg/g dry weight of cells), respectively. However, the combination of overexpressing HMG-CoA reductase gene and adding ketoconazole can achieve a 206.8% increment of pigment content (6.29 mg/g dry weight of cells) compared with that of the control. Due to the fact that over-expression of the HMG-CoA reductase gene can simultaneously improve the flux of the sterol and carotenoid biosynthetic pathway, it can be concluded that under the circumstances of blocking sterol biosynthesis, increasing the activity of HMG-CoA reductase can result in more precursors FPP fluxing into carotenoid branch and obtain a high increment of β-carotene production. The results of this study collectively suggest that the combination of overexpressing HMG-CoA reductase gene and supplying ergosterol synthesis inhibitor is an effective strategy to improve the production of desirable isoprenoid compounds such as carotenoids. PMID:22086347

  15. Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

    PubMed

    Fuentes, S I; Allen, D J; Ortiz-Lopez, A; Hernández, G

    2001-05-01

    Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution. PMID:11432923

  16. VCC-1 over-expression inhibits cisplatin-induced apoptosis in HepG2 cells

    SciTech Connect

    Zhou, Zhitao; Lu, Xiao; Zhu, Ping; Zhu, Wei; Mu, Xia; Qu, Rongmei; Li, Ming

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer VCC-1 is hypothesized to be associated with carcinogenesis. Black-Right-Pointing-Pointer Levels of VCC-1 are increased significantly in HCC. Black-Right-Pointing-Pointer Over-expression of VCC-1 could promotes cellular proliferation rate. Black-Right-Pointing-Pointer Over-expression of VCC-1 inhibit the cisplatin-provoked apoptosis in HepG2 cells. Black-Right-Pointing-Pointer VCC-1 plays an important role in control the tumor growth and apoptosis. -- Abstract: Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy.

  17. Expressed gene sequences from larval genes over-expressed upon Babesia bovis infection of Rhipecephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Boophilus ticks are vectors of Babesia bovis, the protozoan causative agent of cattle fever, a disease which is responsible for significant production losses to cattle producers in much of Africa, Central and South America and Australia. We utilized subtractive cDNA library synthesis techniques to o...

  18. Tobacco plants over-expressing the sweet orange tau glutathione transferases (CsGSTUs) acquire tolerance to the diphenyl ether herbicide fluorodifen and to salt and drought stresses.

    PubMed

    Lo Cicero, Luca; Madesis, Panagiotis; Tsaftaris, Athanasios; Lo Piero, Angela Roberta

    2015-08-01

    The glutathione transferases (GSTs) are members of a superfamily of enzymes with pivotal role in the detoxification of both xenobiotic and endogenous compounds. In this work, the generation and characterization of transgenic tobacco plants over-expressing tau glutathione transferases from Citrus sinensis (CsGSTU1 and CsGSTU2) and several cross-mutate forms of these genes are reported. Putative transformed plants were verified for the presence of the transgenes and the relative quantification of transgene copy number was evaluated by Taqman real time PCR. The analysis of gene expression revealed that transformed plants exhibit high levels of CsGSTU transcription suggesting that the insertion of the transgenes occurred in transcriptional active regions of the tobacco genome. In planta studies demonstrate that transformed tobacco plants gain tolerance against fluorodifen. Simultaneously, the wild type CsGSTU genes were in vitro expressed and their kinetic properties were determined using fluorodifen as substrate. The results show that CsGSTU2 follows a Michaelis-Menten hyperbolic kinetic, whereas CsGSTU1 generates a sigmoid plot typical of the regulatory enzymes, thus suggesting that when working at sub-lethal fluorodifen concentrations CsGSTU2 can counteract the herbicide injury more efficiently than the CsGSTU1. Moreover, the transgenic tobacco plant over-expressing CsGSTs exhibited both drought and salinity stress tolerance. However, as we show that CsGSTUs do not function as glutathione peroxidase in vitro, the protective effect against salt and drought stress is not due to a direct scavenging activity of the oxidative stress byproducts. The transgenic tobacco plants, which are described in the present study, can be helpful for phytoremediation of residual xenobiotics in the environment and overall the over-expression of CsGSTUs can be helpful to develop genetically modified crops with high resistance to abiotic stresses.

  19. Over-Expression of PUMA Correlates with the Apoptosis of Spinal Cord Cells in Rat Neuropathic Intermittent Claudication Model

    PubMed Central

    Ma, Bin; Shi, Jiangang; Jia, Lianshun; Yuan, Wen; Wu, Jianfeng; Fu, Zhiyi; Wang, Yuan; Liu, Ning; Guan, Zhengmao

    2013-01-01

    Background Neuropathic intermittent claudication (NIC) is a typical clinical symptom of lumbar spinal stenosis and the apoptosis of neurons caused by cauda equina compression (CEC) has been proposed as an important reason. Whereas, the factors and the mechanism involved in the process of apoptosis induced by CEC remain unclear. Methodology and Results In our modified rat model of NIC, a trapezoid-shaped silicon rubber was inserted into the epidural space under the L5 and L6 vertebral plate. Obvious apoptosis was observed in spinal cord cells after compression by TUNEL assay. Simultaneously, qRT-PCR and immunohistochemistry showed that the expression levels of PUMA (p53 up-regulated modulator of apoptosis) and p53 were upregulated significantly in spinal cord under compression, while the expression of p53 inhibitor MDM2 and SirT2 decreased in the same region. Furthermore, CEC also resulted in the upregulation of Bcl-2 pro-apoptotic genes expression and caspase-3 activation. With the protection of Methylprednisolone, the upregulation of PUMA and p53 expression as well as the decrease of MDM2 and SirT2 in spinal cord were partially rescued in western bolt analysis. Conclusions These results suggest that over-expression of PUMA correlates with CEC caused apoptosis of spinal cord cells, which is characterized by the increase of p53, Bax and Bad expression. PUMA upregulation might be crucial to induce apoptosis of spinal cord cells through p53-dependent pathway in CEC. PMID:23658678

  20. The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression

    PubMed Central

    de Jonge, Nick; Björkholm, Magnus; Xu, Dawei

    2015-01-01

    DNA methyltransferase inhibitors (DNMTIs) such as 5-azacytidine (5-AZA) have been used for treatment of acute myeloid leukemia (AML) and other malignancies. Although inhibiting global/gene-specific DNA methylation is widely accepted as a key mechanism behind DNMTI anti-tumor activity, other mechanisms are likely involved in DNMTI's action. Because telomerase reverse transcriptase (TERT) plays key roles in cancer through telomere elongation and telomere lengthening-independent activities, and TERT has been shown to confer chemo- or radio-resistance to cancer cells, we determine whether DNMTIs affect telomere function and whether TERT/telomerase interferes with their anti-cancer efficacy. We showed that 5-AZA induced DNA damage and telomere dysfunction in AML cell lines by demonstrating the presence of 53-BP1 foci and the co-localization of 53-BP1 foci with telomere signals, respectively. Telomere dysfunction was coupled with diminished TERT expression, shorter telomere and apoptosis in 5-AZA-treated cells. However, 5-AZA treatment did not lead to changes in the methylation status of subtelomere regions. Down-regulation of TERT expression similarly occurred in primary leukemic cells derived from AML patients exposed to 5-AZA. TERT over-expression significantly attenuated 5-AZA-mediated DNA damage, telomere dysfunction and apoptosis of AML cells. Collectively, 5-AZA mediates the down-regulation of TERT expression, and induces telomere dysfunction, which consequently exerts an anti-tumor activity. PMID:25682873

  1. Perhexiline maleate enhances antitumor efficacy of cisplatin in neuroblastoma by inducing over-expression of NDM29 ncRNA

    PubMed Central

    Vella, Serena; Penna, Ilaria; Longo, Luca; Pioggia, Giulia; Garbati, Patrizia; Florio, Tullio; Rossi, Fabio; Pagano, Aldo

    2015-01-01

    High Risk Neuroblastoma (HR-NB) is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumour nodules. In a recent study, we demonstrated that in vitro and in vivo over-expression of the non-coding RNA NDM29 (neuroblastoma differentiation marker 29) induces NB cell differentiation, dramatically reducing their malignancy. Among gene expression changes, differentiated phenotype induced by NDM29 is characterized by decrease of the expression of ABC transporters responsible for anticancer drug resistance. Thus, the pharmacological induction of NDM29, in principle, might represent a possible novel strategy to increase cytotoxic drug responses. In this work, we identify a small molecule able to induce the expression of NDM29 in NB cells, conferring to malignant cells increased susceptibility to cisplatin cytotoxic effects. We demonstrate that the pharmacological induction of NDM29 expression in vivo enhances the antitumoral effects of chemotherapy specifically on tumour initiating/cancer stem cells sub-population, usually refractory to therapies and responsible for tumour relapse. In summary, we suggest a novel therapeutical approach possibly useful to treat very aggressive NB cases with poor prognosis. This novel pharmacological strategy aims to promote differentiation of “stem-like” cells to render them more susceptible to the killing action of cytotoxic anticancer drugs. PMID:26674674

  2. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance.

    PubMed

    Rallos, Lynn Esther E; Baudoin, Anton B

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  3. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

    PubMed Central

    Rallos, Lynn Esther E.; Baudoin, Anton B.

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  4. Transgenic mice over-expressing human beta-amyloid have functional nicotinic alpha 7 receptors.

    PubMed

    Spencer, J P; Weil, A; Hill, K; Hussain, I; Richardson, J C; Cusdin, F S; Chen, Y H; Randall, A D

    2006-02-01

    A potentially major factor in the development of Alzheimer's disease is the enhanced production of soluble beta-amyloid peptide fragments amyloid beta peptide(1-40) and amyloid beta peptide(1-42). These amyloid peptides are generated by cleavage of the amyloid-precursor protein and aggregate spontaneously to form amyloid plaques, which are a classical pathological hallmark in Alzheimer's disease. Although the precise mechanisms are unknown, it is widely believed that amyloid peptides initiate the degenerative process, resulting in subsequent cognitive decline. One interaction of amyloid beta peptide that may contribute to an impairment of cognition is its high affinity binding to the alpha 7 nicotinic receptor; a receptor shown to be important for cognition in a number of studies. There is some controversy, however, whether amyloid beta peptide inhibits or activates this receptor. We have cloned and stably expressed the human alpha 7 receptor and investigated its interaction with amyloid beta peptide using patch clamp electrophysiology. Human alpha 7 was activated in a concentration-dependent fashion by nicotine, acetylcholine and choline and potently inhibited by methyllycaconitine citrate. The responses were inwardly rectifying and exhibited rapid activation, desensitization and deactivation. Amyloid beta peptide(1-42) antagonized human alpha7 responses in a partially reversible fashion; no agonist effects of amyloid beta peptide(1-42) were detected. A similar inhibition of mouse alpha 7 was also observed. In addition, we have assessed the function of native alpha 7 receptors in hippocampal slices prepared from transgenic mice that over-express human amyloid. Despite this clear inhibition of recombinant receptors, hippocampal GABAergic interneurones in slices from beta-amyloid over-expressing mice still possess alpha 7 receptor-mediated currents.

  5. Over-expression of tetraspanin 8 in malignant glioma regulates tumor cell progression

    SciTech Connect

    Pan, Si-Jian; Wu, Yue-Bing; Cai, Shang; Pan, Yi-Xin; Liu, Wei; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang

    2015-03-13

    Tumor cell invasion and proliferation remain the overwhelming causes of death for malignant glioma patients. To establish effective therapeutic methods, new targets implied in these processes have to be identified. Tetraspanin 8 (Tspn8) forms complexes with a large variety of trans-membrane and/or cytosolic proteins to regulate several important cellular functions. In the current study, we found that Tspn8 was over-expressed in multiple clinical malignant glioma tissues, and its expression level correlated with the grade of tumors. Tspn8 expression in malignant glioma cells (U251MG and U87MG lines) is important for cell proliferation and migration. siRNA-mediated knockdown of Tspn8 markedly reduced in vitro proliferation and migration of U251MG and U87MG cells. Meanwhile, Tspn8 silencing also increased the sensitivity of temozolomide (TMZ), and significantly increased U251MG or U87MG cell death and apoptosis by TMZ were achieved with Tspn8 knockdown. We observed that Tspn8 formed a complex with activated focal adhesion kinase (FAK) in both human malignant glioma tissues and in above glioma cells. This complexation appeared required for FAK activation, since Tspn8 knockdown inhibited FAK activation in U251MG and U87MG cells. These results provide evidence that Tspn8 contributes to the pathogenesis of glioblastoma probably by promoting proliferation, migration and TMZ-resistance of glioma cells. Therefore, targeting Tspn8 may provide a potential therapeutic intervention for malignant glioma. - Highlights: • Tspn8 is over-expressed in multiple clinical malignant glioma tissues. • Tspn8 expression is correlated with the grade of malignant gliomas. • Tspn8 knockdown suppresses U251MG/U87MG proliferation and in vitro migration. • Tspn8 knockdown significantly increases TMZ sensitivity in U251MG/U87MG cells. • Tspn8 forms a complex with FAK, required for FAK activation.

  6. Lincomycin-induced over-expression of mature recombinant cholera toxin B subunit and the holotoxin in Escherichia coli.

    PubMed

    Arimitsu, Hideyuki; Tsukamoto, Kentaro; Ochi, Sadayuki; Sasaki, Keiko; Kato, Michio; Taniguchi, Koki; Oguma, Keiji; Tsuji, Takao

    2009-10-01

    Cholera toxin (CT) B subunit (CTB) was overproduced using a novel expression system in Escherichia coli. An expression plasmid was constructed by inserting the gene encoding the full-length CTB and the Shine-Dalgarno (SD) sequence derived from CTB or from the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic E. coli into the lacZalpha gene fragment in the pBluescript SK(+) vector. The E. coli strain MV1184 was transformed with each plasmid and then cultured in CAYE broth containing lincomycin. Recombinant CTB (rCTB) was purified from each cell extract. rCTB was overproduced in both transformants without obvious toxicity and was structurally and biologically identical to that of CT purified from Vibrio cholerae, indicating that the original SD and CTB signal sequences were also sufficient to express rCTB in E. coli. Lincomycin-induced rCTB expression was inhibited by mutating the lac promoter, suggesting that lincomycin affects the lactose operon. Based on these findings, we constructed a plasmid that contained the wild-type CT operon and successfully overproduced CT (rCT) using the same procedure for rCTB. Although rCT had an intact A subunit, the amino-terminal modifications and biological properties of the A and B subunits of rCT were identical to those of CT. These results suggest that this novel rCTB over-expression system would also be useful to generate both wild-type and mutant CT proteins that will facilitate further studies on the characteristics of CT, such as mucosal adjuvant activity. PMID:19410003

  7. Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil.

    PubMed

    Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

    2011-07-01

    Recent studies of transgenic poplars over-expressing the genes gsh1 and gsh2 encoding γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO(2) diffusion, chlorophyll and carbohydrate content in wild-type poplar and transgenic plants over-expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal-contaminated soil in the field. Over-expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6-fold leaf area per leaf compared to wild-type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over-expression of γ-ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3-fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild-type plants but not in transformants. Biomass accumulation of wild-type poplars decreased in contaminated soil by more than 30-fold, whereas transformants showed a twofold decrease

  8. Over-expression of TSPO in the hippocampal CA1 area alleviates cognitive dysfunction caused by lipopolysaccharide in mice.

    PubMed

    Zhang, Hui; Ma, Li; Yin, Yan-Ling; Dong, Lian-Qiang; Cheng, Gang-Ge; Ma, Ya-Qun; Li, Yun-Feng; Xu, Bai-Nan

    2016-09-01

    The translocator protein 18kDa (TSPO) is closely related to regulation of immune/inflammatory response. However, the putative role and signaling mechanisms of TSPO in regulation of neuroinflammation remain unclear. GV287 lentiviral vectors mediating TSPO over-expression were injected into bilateral hippocampal CA1 areas to test whether TSPO over-expression was neuroprotective in lipopolysaccharide (LPS)-induced mice model. Finasteride, a blocker of allopregnanolone production, was used to test whether the protective effects were related to steroideogenesis. The results demonstrated that TSPO over-expression increased progesterone and allopregnanolone synthesis. TSPO over-expression in CA1 area improved LPS-induced cognitive deficiency in mice and this cognitive improvement was reversed by finasteride administration. These data suggest that up-regulation of TSPO level during neuroinflammation may be an adaptive response mechanism, a way to provide more neurosteroids. We confer that TSPO could be an attractive drug target for controlling neuroinflammation in the future.

  9. Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy

    PubMed Central

    Peng, Chang; Zhu, Jing; Sun, Hui-Chao; Huang, Xu-Pei; Zhao, Wei-An; Zheng, Min; Liu, Ling-Juan; Tian, Jie

    2014-01-01

    Background Cardiovascular malformations can be caused by abnormalities in Gata4 expression during fetal development. In a previous study, we demonstrated that ethanol exposure could lead to histone hyperacetylation and Gata4 over-expression in fetal mouse hearts. However, the potential mechanisms of histone hyperacetylation and Gata4 over-expression induced by ethanol remain unclear. Methods and Results Pregnant mice were gavaged with ethanol or saline. Fetal mouse hearts were collected for analysis. The results of ethanol fed groups showed that global HAT activity was unusually high in the hearts of fetal mice while global HDAC activity remained unchanged. Binding of P300, CBP, PCAF, SRC1, but not GCN5, were increased on the Gata4 promoter relative to the saline treated group. Increased acetylation of H3K9 and increased mRNA expression of Gata4, α-MHC, cTnT were observed in these hearts. Treatment with the pan-histone acetylase inhibitor, anacardic acid, reduced the binding of P300, PCAF to the Gata4 promoter and reversed H3K9 hyperacetylation in the presence of ethanol. Interestingly, anacardic acid attenuated over-expression of Gata4, α-MHC and cTnT in fetal mouse hearts exposed to ethanol. Conclusions Our results suggest that P300 and PCAF may be critical regulatory factors that mediate Gata4 over-expression induced by ethanol exposure. Alternatively, P300, PCAF and Gata4 may coordinate over-expression of cardiac downstream genes in mouse hearts exposed to ethanol. Anacardic acid may thus protect against ethanol-induced Gata4, α-MHC, cTnT over-expression by inhibiting the binding of P300 and PCAF to the promoter region of these genes. PMID:25101666

  10. Entamoeba histolytica: the over expression of a mutated EhRabB protein produces a decrease of in vitro and in vivo virulence.

    PubMed

    Juárez-Hernández, L J; García-Pérez, R M; Salas-Casas, A; García-Rivera, G; Orozco, E; Rodríguez, M A

    2013-03-01

    Vesicular trafficking, which is implicated in secretion of cytolytic molecules as well as in phagocytosis, plays an important role in the pathogenic mechanism of Entamoeba histolytica, the protozoan parasite causative of human amoebiasis. Thus, Rab GTPases, that are key regulators of vesicle trafficking, should be considered as molecules involved in the parasite virulence. EhRabB is a Rab protein located in cytoplasmic vesicles that are translocated to phagocytic mouths during ingestion of target cells, suggesting that this Rab protein is involved in phagocytosis. To prove this hypothesis, we over expressed the wild type EhrabB gene and a mutant gene encoding for a protein (RabBN118I) unable to bind guanine nucleotides and therefore constitutively inactive. The over expression of the mutated protein in E. histolytica trophozoites provoked a dominant negative effect, reflected in a significant decrease of both phagocytosis and cytopathic effect as well as in a failure to produce hepatic abscesses in hamsters. These results confirm that EhRabB is involved in phagocytosis and virulence of E. histolytica.

  11. Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo

    SciTech Connect

    Guseva, Daria; Rizvanov, Albert A.; Salafutdinov, Ilnur I.; Kudryashova, Nezhdana V.; Palotás, András; Islamov, Rustem R.

    2014-09-05

    Highlights: • Gene and cell-based therapies comprise innovative aspects of regenerative medicine. • Genetically modified hUCB-MCs enhanced differentiation of cells in a mouse model of ALS. • Stem cells successfully transformed into micro-glial and endothelial lines in spinal cords. • Over-expressing oct4 and sox2 also induced production of neural marker PGP9.5. • Formation of new nerve cells, secreting trophic factors and neo-vascularisation could improve symptoms in ALS. - Abstract: Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.

  12. Differential effect of over-expressing UGT1A1 and CYP1A1 on xenobiotic assault in MCF-7 cells.

    PubMed

    Leung, Hau Y; Wang, Yun; Leung, Lai K

    2007-12-01

    Gene mutation has been considered as a major step of carcinogenesis. Some defective genes may induce spontaneous tumorigenesis, while others are required to interact with the environment to induce cancer. CYP1A1 and UGT1A1 are encoded for the respective phase I and II drug-metabolizing enzymes. Their expressions have been associated with breast cancer incidence in women, and some xenobiotics are substrates of these two enzymes. In the current study, cytochrome P450 (CYP) 1A1 and UDP-glucuronosyltransferase (UGT) 1A1 were over-expressed in the breast cancer MCF-7 cells, and potential interactions between these enzymes and estrogen or polycyclic aromatic hydrocarbon were evaluated. Compared with control cells (MCF-7(VEC)), reduced cell proliferation was seen in cells expressing UGT1A1 (MCF-7(UGT1A1)) under estradiol treatment. 7,12-Dimethylbenz[a]anthracene (DMBA) is an established breast cancer initiator in animal model. Over-expressing UGT1A1 reduced the binding of DMBA to DNA, and increased MCF-7(UGT1A1) intact cells under DMBA treatment was verified by comet assay. On the other hand, intensified DMBA binding and damages were observed in MCF-7(CYP1A1) cells. This study supported that UGT1A1 but not CYP1A1 expression could protect against xenobiotic assault. PMID:17981384

  13. Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit development under limiting phosphorus conditions.

    PubMed

    Yang, Haibing; Zhang, Xiao; Gaxiola, Roberto A; Xu, Guohua; Peer, Wendy Ann; Murphy, Angus S

    2014-07-01

    Phosphorus (P), an element required for plant growth, fruit set, fruit development, and fruit ripening, can be deficient or unavailable in agricultural soils. Previously, it was shown that over-expression of a proton-pyrophosphatase gene AVP1/AVP1D (AVP1DOX) in Arabidopsis, rice, and tomato resulted in the enhancement of root branching and overall mass with the result of increased mineral P acquisition. However, although AVP1 over-expression also increased shoot biomass in Arabidopsis, this effect was not observed in tomato under phosphate-sufficient conditions. AVP1DOX tomato plants exhibited increased rootward auxin transport and root acidification compared with control plants. AVP1DOX tomato plants were analysed in detail under limiting P conditions in greenhouse and field trials. AVP1DOX plants produced 25% (P=0.001) more marketable ripened fruit per plant under P-deficient conditions compared with the controls. Further, under low phosphate conditions, AVP1DOX plants displayed increased phosphate transport from leaf (source) to fruit (sink) compared to controls. AVP1DOX plants also showed an 11% increase in transplant survival (P<0.01) in both greenhouse and field trials compared with the control plants. These results suggest that selection of tomato cultivars for increased proton pyrophosphatase gene expression could be useful when selecting for cultivars to be grown on marginal soils.

  14. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

    SciTech Connect

    Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H.

    2010-11-05

    Research highlights: {yields} COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}, a synthetic diffusible ubiquinone. {yields} The significance that purified Coq10p contains bound Q{sub 6} was examined by testing over-expression of Coq10p on respiration. {yields} Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. {yields} Respiratory deficiency caused by more Coq10p was specific and restored by Q{sub 2} in mitochondria or by Coq8p in cells. {yields} Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}. Rescue of respiration by Q{sub 2} is a characteristic of mutants blocked in coenzyme Q{sub 6} synthesis. Unlike Q{sub 6} deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q{sub 6}. The physiological significance of earlier observations that purified Coq10p contains bound Q{sub 6} was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q{sub 2}. This suggests that in vivo binding of Q{sub 6} by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains

  15. Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

    SciTech Connect

    Hals, Ingrid K.; Ogata, Hirotaka; Pettersen, Elin; Ma, Zuheng; Bjoerklund, Anneli; Skorpen, Frank; Egeberg, Kjartan Wollo; Grill, Valdemar

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positive findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the

  16. Diet-Induced Over-Expression of Flightless-I Protein and Its Relation to Flightlessness in Mediterranean Fruit Fly, Ceratitis capitata

    PubMed Central

    Cho, Il Kyu; Chang, Chiou Ling; Li, Qing X.

    2013-01-01

    The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation. PMID:24312525

  17. Unregulated brain iron deposition in transgenic mice over-expressing HMOX1 in the astrocytic compartment.

    PubMed

    Song, Wei; Zukor, Hillel; Lin, Shih-Hsiung; Liberman, Adrienne; Tavitian, Ayda; Mui, Jeannie; Vali, Hojatollah; Fillebeen, Carine; Pantopoulos, Kostas; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Schipper, Hyman M

    2012-10-01

    The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO⁻ signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

  18. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.

    PubMed

    Zampol, Mariana A; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H

    2010-11-01

    COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6). The physiological significance of earlier observations that purified Coq10p contains bound Q(6) was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q(2). This suggests that in vivo binding of Q(6) by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.

  19. Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer

    PubMed Central

    McFadyen, M C E; Cruickshank, M E; Miller, I D; McLeod, H L; Melvin, W T; Haites, N E; Parkin, D; Murray, G I

    2001-01-01

    Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P= 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11461084

  20. Pulmonary hypertension in smoking mice over-expressing protease-activated receptor-2.

    PubMed

    De Cunto, G; Cardini, S; Cirino, G; Geppetti, P; Lungarella, G; Lucattelli, M

    2011-04-01

    The mechanism(s) involved in the development of pulmonary hypertension (PH) in COPD is still the object of investigation. Cigarette smoke (CS) may lead to remodelling of intrapulmonary vessels and dynamic changes in vascular function, at least in some smokers. A role for proteases in PH has been recently put forward. We investigated, in smoking mice, the role of protease-activated receptor (PAR)-2 in the pathogenesis of PH associated with emphysema. We demonstrated that CS exposure can modulate PAR-2 expression in mouse lung. Acute CS exposure induces in wildtype (WT) and in transgenic mice over-expressing PAR-2 (FVB(PAR-2-TgN)) a similar degree of neutrophil influx in bronchoalveolar lavage fluids. After chronic CS exposure WT and FVB(PAR-2-TgN) mice show emphysema, but only transgenic mice develop muscularisation of small intrapulmonary vessels that precedes the development of PH (~45% increase) and right ventricular hypertrophy. Smoking in FVB(PAR-2-TgN) mice results in an imbalance between vasoconstrictors (especially endothelin-1) and vasodilators (i.e. vascular endothelial growth factor, endothelial nitric oxide synthase and inducible nitric oxide synthase) and enhanced production of growth factors involved both in fibroblast-smooth muscle cell transaction (i.e. platelet-derived growth factor (PDGF) and transforming growth factor β) and vascular cell proliferation (PDGF). PAR-2 signalling can influence the production and release of many factors, which may play a role in the development of PH in smokers. PMID:20693251

  1. GPX4 and GPX7 Over-Expression in Human Hepatocellular Carcinoma Tissues

    PubMed Central

    Guerriero, E.; Capone, F.; Accardo, M.; Sorice, A.; Costantini, M.; Colonna, G.; Castello, G.

    2015-01-01

    Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. The selenium is an essential trace mineral implicated as a key factor in the early stage of cancer and exerts its biological function through the selenoproteins. In the last years our group has been studying the involvement of some selenoproteins in HCC. However, no many data are reported in literature about the correlation between HCC and the glutathione peroxidases (GPXs), both selenium and non selenium-containing GPXs. In this paper we have evaluated the GPX4 and GPX7 expression in some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC by immunohistochemistry and RT-qPCR analysis. Our results evidenced that i) GPX4 and GPX7 had a statistically significant over-expression in HCC tissues compared to cirrhotic counterparts used as non tumor tissues, and ii) their expression was higher in grade III HCC tissues with respect to grade I-II samples. Therefore, we propose to use GPX4 and GPX7 as possible markers for improving HCC diagnosis/prognosis. PMID:26708178

  2. An Over Expression APP Model for Anti-Alzheimer Disease Drug Screening Created by Zinc Finger Nuclease Technology

    PubMed Central

    Mao, Yiqing; Li, Zhixin; Wang, Rong; Guo, Tingting; Jin, Ling; Song, Rongjing; Xu, Wei; Zhou, Na; Zhang, Yizhuang; Hu, Ruobi; Wang, Xi; Huang, Huakang; Lei, Zhen; Niu, Gang; Irwin, David M.; Tan, Huanran

    2013-01-01

    Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer’s disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents. PMID:24223114

  3. An over expression APP model for anti-Alzheimer disease drug screening created by zinc finger nuclease technology.

    PubMed

    Zhang, Xiaojing; Li, Hui; Mao, Yiqing; Li, Zhixin; Wang, Rong; Guo, Tingting; Jin, Ling; Song, Rongjing; Xu, Wei; Zhou, Na; Zhang, Yizhuang; Hu, Ruobi; Wang, Xi; Huang, Huakang; Lei, Zhen; Niu, Gang; Irwin, David M; Tan, Huanran

    2013-01-01

    Zinc Finger Nucleases (ZFNs), famous for their ability to precisely and efficiently modify specific genomic loci, have been employed in numerous transgenic model organism and cell constructions. Here we employ the ZFNs technology, with homologous recombination (HR), to construct sequence-specific Amyloid Precursor Protein (APP) knock-in cells. With the use of ZFNs, we established APP knock in cell lines with gene-modification efficiencies of about 7%. We electroporated DNA fragment containing the promoter and the protein coding regions of the zinc finger nucleases into cells, instead of the plasmids, to avoid problems associated with off target homologous recombination, and adopted a pair of mutated FokI cleavage domains to reduce the toxic effects of the ZFNs on cell growth. Since over-expression of APP, or a subdomain of it, might lead to an immediately lethal effect, we used the Cre-LoxP System to regulate APP expression. Our genetically transformed cell lines, w5c1 and s12c8, showed detectable APP and Amyloid β (Aβ) production. The Swedish double mutation in the APP coding sequence enhanced APP and Aβ abundance. What is more, the activity of the three key secretases in Aβ formation could be modulated, indicating that these transgenic cells have potential for drug screening to modify amyloid metabolism in cells. Our transformed cells could readily be propagated in culture and should provide an excellent experimental medium for elucidating aspects of the molecular pathogenesis of Alzheimer's disease, especially those concerning the amyloidogenic pathways involving mutations in the APP coding sequence. The cellular models may also serve as a tool for deriving potentially useful therapeutic agents.

  4. Characterization of cytoplasmic cyclin D1 as a marker of invasiveness in cancer

    PubMed Central

    Santacana, Maria; Fernández-Hernández, Rita; Gatius, Sònia; Pedraza, Neus; Pallarés, Judit; Cemeli, Tània; Valls, Joan; Tarres, Marc; Ferrezuelo, Francisco; Dolcet, Xavier; Matias-Guiu, Xavier; Garí, Eloi

    2016-01-01

    Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome. PMID:27105504

  5. Effect of Over-Expression of Zinc-Finger Protein (ZFX) on Self-Renewal and Drug-Resistance of Hepatocellular Carcinoma.

    PubMed

    Zhang, Shuhong; Shu, Ronghua; Yue, Meng; Zhang, Shuhong

    2016-01-01

    BACKGROUND X-chromosome-coupled zinc finger protein (ZFX) in the Zfy protein family is abundantly expressed in both embryonic and hematopoietic stem cells (HSCs). ZFX exist in various tumor cells and is correlated with proliferation and survival of tumor cells. As a malignant tumor with high invasiveness, hepatocellular carcinoma (HCC) may present resistance against chemotherapy and features of stem cells. This study aimed to explore the expression of ZFX in HCC cells, in an attempt to illustrate the role of ZFX in tumorigenesis. MATERIAL AND METHODS The expression of ZFX in tumor tissues was quantified by RT-PCR. The ZFX expression was then silenced to evaluate the stem cell-like features of HCC cells, including self-renewal, colony formation, and cell cycle, along with the sensitivity to cisplatin. Xenograft of ZFX-overexpressed HCC on nude mice was performed to evaluate the in vivo effect of ZFX on tumor growth. RESULTS Quantitative RT-PCR showed over-expression of ZFX in 51.8% of HCC tumors. The silencing of ZFX gene inhibited the self-renewal, colony formation, and proliferation ability of HCC cells (p<0.05 in all cases) via the cell cycle arrest at G0/G1 phase, in addition to the elevated sensitivity of tumor cells to cisplatin (p<0.001). Further studies showed that binding between ZFX and promoter regions of Nanog or SOX-2 regulatory factor initiate their expression in HCC cells. The xenograft experiment indicated the potentiation of tumor growth by ZFX over-expression. CONCLUSIONS ZFX is over-expressed in HCC cells, and correlates with stem cell-like features and pleiotropic characteristics. PMID:27566731

  6. Effect of Over-Expression of Zinc-Finger Protein (ZFX) on Self-Renewal and Drug-Resistance of Hepatocellular Carcinoma

    PubMed Central

    Zhang, Shuhong; Shu, Ronghua; Yue, Meng; Zhang, Shuhong

    2016-01-01

    Background X-chromosome-coupled zinc finger protein (ZFX) in the Zfy protein family is abundantly expressed in both embryonic and hematopoietic stem cells (HSCs). ZFX exist in various tumor cells and is correlated with proliferation and survival of tumor cells. As a malignant tumor with high invasiveness, hepatocellular carcinoma (HCC) may present resistance against chemotherapy and features of stem cells. This study aimed to explore the expression of ZFX in HCC cells, in an attempt to illustrate the role of ZFX in tumorigenesis. Material/Methods The expression of ZFX in tumor tissues was quantified by RT-PCR. The ZFX expression was then silenced to evaluate the stem cell-like features of HCC cells, including self-renewal, colony formation, and cell cycle, along with the sensitivity to cisplatin. Xenograft of ZFX-overexpressed HCC on nude mice was performed to evaluate the in vivo effect of ZFX on tumor growth. Results Quantitative RT-PCR showed over-expression of ZFX in 51.8% of HCC tumors. The silencing of ZFX gene inhibited the self-renewal, colony formation, and proliferation ability of HCC cells (p<0.05 in all cases) via the cell cycle arrest at G0/G1 phase, in addition to the elevated sensitivity of tumor cells to cisplatin (p<0.001). Further studies showed that binding between ZFX and promoter regions of Nanog or SOX-2 regulatory factor initiate their expression in HCC cells. The xenograft experiment indicated the potentiation of tumor growth by ZFX over-expression. Conclusions ZFX is over-expressed in HCC cells, and correlates with stem cell-like features and pleiotropic characteristics. PMID:27566731

  7. Involvement of subtype 1 metabotropic glutamate receptors in apoptosis and caspase-7 over-expression in spinal cord of neuropathic rats

    PubMed Central

    Siniscalco, Dario; Giordano, Catia; Fuccio, Carlo; Luongo, Livio; Ferraraccio, Franca; Rossi, Francesca; de Novellis, Vito; Roth, Kevin A.; Maione, Sabatino

    2008-01-01

    The effect of the non-selective, 1-aminoindan-1,5-dicarboxylic acid (AIDA), and selective (3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4- methoxycyclohexyl) methanone (JNJ16259685), metabotropic glutamate subtype 1 (mGlu1) receptor antagonists, on rat sciatic nerve chronic constrictive injury (CCI)- induced hyperalgesia, allodynia, spinal dorsal horn apoptosis, and gliosis was examined at 3 and 7 days post-injury. RT-PCR analysis showed increased expression of bax, apoptotic protease-activating factor-1 (apaf-1), nestin, GFAP, and caspase-7 mRNA in the dorsal horn spinal cord by 3 days post-CCI. At 7 days post-CCI, only over-expression of bcl-2, nestin and GFAP mRNA was observed. Administration of AIDA reduced thermal hyperalgesia and mechanical allodynia at 3 and 7 days post-CCI; administration of JNJ16259685 reduced thermal hyperalgesia at 3 and 7 days post-CCI, but not mechanical allodynia. AIDA decreased the mRNA levels of bax, apaf-1, GFAP and caspase-7 genes. JNJ16259685 increased the mRNA levels of bcl- 2 and GFAP gene, and decreased APAF-1 and caspases-7 genes. Inhibiting mGlu1 receptors also reduced TUNEL-positive profiles and immunohistochemical reactivity for caspase-7. We report here that despite inhibiting CCI-induced over-expression of pro-apoptotic genes in the spinal cord dorsal horn, the selective mGlu1 receptor antagonist JNJ16259685 exerted only a slight and transient allodynic effect. Moreover, JNJ16259685, but not the non-selective AIDA, increased astrogliosis which may account for its decreased analgesic efficacy. This study provides evidence that the contemporary and partial blockade of group I and likely ionotropic glutamate receptors may be a more suitable therapy than selective blockade of mGlu1 subtype receptors condition to decrease neuropathic pain symptoms. PMID:18325779

  8. Phenotypic characterisation of lung macrophages in asthma: over-expression of CCL17

    PubMed Central

    Staples, Karl J; Hinks, Timothy SC; Ward, Jon A.; Gunn, Victoria; Smith, Caroline; Djukanovic, Ratko

    2013-01-01

    Background Studies using monocyte-derived macrophages (MDM) and animal models have suggested a role for alternatively-activated (M2) macrophages in asthmatic inflammation, but in vivo evidence for this phenotype in human asthma is lacking. Objective Phenotypically to characterize lung macrophages from asthmatic patients in relation to disease severity and treatment. Methods M2 biomarkers were first identified using MDM exposed to Th2 cytokines and then used to phenotype sputum and bronchoalveolar lavage (BAL) macrophages from 12 healthy controls, 12 mild and 14 moderate asthmatics and to assess the effects of corticosteroids and phosphatidylinositol-3-kinase (PI3K) inhibitors. Results Sputum macrophages from asthmatics expressed significantly more CCL17 mRNA but less CD163 than macrophages from healthy individuals. However, none of the other M2 biomarkers were differentially expressed in asthma and ex vivo BAL cells spontaneously produced similar amounts of M2 cytokine/chemokines (IL-10, CCL17 and CCL22). CCL17 mRNA over-expression correlated weakly but significantly with sputum eosinophilia (p=0.0252) and was also observed in macrophages from moderate asthmatics treated with inhaled steroids, suggesting relative insensitivity to inhibition by corticosteroids. The PI3Kinase inhibitor LY294002 inhibited basal CCL17 release from BAL cells and IL-4-stimulated release from MDM. Conclusions This study does not support the existence in human asthma of the full M2 phenotype described to date, but points to upregulation of CCL17 in both mild and moderate asthma, providing a further source for this ligand of CCR4+ cells that contribute to airways inflammation. CCL17 expression is corticosteroid resistant but is suppressed by PI3Kinase enzyme inhibitors. PMID:22981793

  9. Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modifications in potato.

    PubMed

    Pino, María-Teresa; Skinner, Jeffrey S; Jeknić, Zoran; Hayes, Patrick M; Soeldner, Alfred H; Thomashow, Michael F; Chen, Tony H H

    2008-04-01

    We studied the effect of ectopic AtCBF over-expression on physiological alterations that occur during cold exposure in frost-sensitive Solanum tuberosum and frost-tolerant Solanum commersonii. Relative to wild-type plants, ectopic AtCBF1 over-expression induced expression of COR genes without a cold stimulus in both species, and imparted a significant freezing tolerance gain in both species: 2 degrees C in S. tuberosum and up to 4 degrees C in S. commersonii. Transgenic S. commersonii displayed improved cold acclimation potential, whereas transgenic S. tuberosum was still incapable of cold acclimation. During cold treatment, leaves of wild-type S. commersonii showed significant thickening resulting from palisade cell lengthening and intercellular space enlargement, whereas those of S. tuberosum did not. Ectopic AtCBF1 activity induced these same leaf alterations in the absence of cold in both species. In transgenic S. commersonii, AtCBF1 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild type, transgenic S. commersonii leaves were darker green, had higher chlorophyll and lower anthocyanin levels, greater stomatal numbers, and displayed greater photosynthetic capacity, suggesting higher productivity potential. These results suggest an endogenous CBFpathway is involved in many of the structural, biochemical and physiological alterations associated with cold acclimation in these Solanum species.

  10. Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses.

    PubMed

    Sun, Wei-Hong; Duan, Ming; Shu, De-Feng; Yang, Sha; Meng, Qing-Wei

    2010-08-01

    Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.

  11. Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors

    PubMed Central

    Ferreira, Wallax Augusto Silva; Araújo, Mariana Diniz; de Oliveira, Edivaldo Herculano Correa; Brito, José Reginaldo Nascimento; Burbano, Rommel Rodriguez; Harada, Maria Lúcia; Borges, Bárbara do Nascimento

    2015-01-01

    Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression. PMID:26317630

  12. Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors.

    PubMed

    Ferreira, Wallax Augusto Silva; Araújo, Mariana Diniz; Anselmo, Nilson Praia; de Oliveira, Edivaldo Herculano Correa; Brito, José Reginaldo Nascimento; Burbano, Rommel Rodriguez; Harada, Maria Lúcia; Borges, Bárbara do Nascimento

    2015-01-01

    Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression. PMID:26317630

  13. Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

    PubMed

    Sasaki, Nobumitsu; Ogata, Takuya; Deguchi, Masakazu; Nagai, Shoko; Tamai, Atsushi; Meshi, Tetsuo; Kawakami, Shigeki; Watanabe, Yuichiro; Matsushita, Yasuhiko; Nyunoya, Hiroshi

    2009-03-01

    Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.

  14. Over-expression of TSPO in the hippocampal CA1 area alleviates cognitive dysfunction caused by lipopolysaccharide in mice.

    PubMed

    Zhang, Hui; Ma, Li; Yin, Yan-Ling; Dong, Lian-Qiang; Cheng, Gang-Ge; Ma, Ya-Qun; Li, Yun-Feng; Xu, Bai-Nan

    2016-09-01

    The translocator protein 18kDa (TSPO) is closely related to regulation of immune/inflammatory response. However, the putative role and signaling mechanisms of TSPO in regulation of neuroinflammation remain unclear. GV287 lentiviral vectors mediating TSPO over-expression were injected into bilateral hippocampal CA1 areas to test whether TSPO over-expression was neuroprotective in lipopolysaccharide (LPS)-induced mice model. Finasteride, a blocker of allopregnanolone production, was used to test whether the protective effects were related to steroideogenesis. The results demonstrated that TSPO over-expression increased progesterone and allopregnanolone synthesis. TSPO over-expression in CA1 area improved LPS-induced cognitive deficiency in mice and this cognitive improvement was reversed by finasteride administration. These data suggest that up-regulation of TSPO level during neuroinflammation may be an adaptive response mechanism, a way to provide more neurosteroids. We confer that TSPO could be an attractive drug target for controlling neuroinflammation in the future. PMID:27265418

  15. Over-expression of Oct4 and Sox2 transcription factors enhances differentiation of human umbilical cord blood cells in vivo.

    PubMed

    Guseva, Daria; Rizvanov, Albert A; Salafutdinov, Ilnur I; Kudryashova, Nezhdana V; Palotás, András; Islamov, Rustem R

    2014-09-01

    Gene and cell-based therapies comprise innovative aspects of regenerative medicine. Even though stem cells represent a highly potential therapeutic strategy, their wide-spread exploitation is marred by ethical concerns, potential for malignant transformation and a plethora of other technical issues, largely restricting their use to experimental studies. Utilizing genetically modified human umbilical cord blood mono-nuclear cells (hUCB-MCs), this communication reports enhanced differentiation of transplants in a mouse model of amyotrophic lateral sclerosis (ALS). Over-expressing Oct4 and Sox2 induced production of neural marker PGP9.5, as well as transformation of hUCB-MCs into micro-glial and endothelial lines in ALS spinal cords. In addition to producing new nerve cells, providing degenerated areas with trophic factors and neo-vascularisation might prevent and even reverse progressive loss of moto-neurons and skeletal muscle paralysis.

  16. Arbuscular mycorrhizal protein mRNA over-expression in bread wheat seedlings by Trichoderma harzianum Raifi (KRL-AG2) elicitation.

    PubMed

    Al-Asbahi, Adnan A S

    2012-02-25

    Association between arbuscular mycorrhizal fungi (AMF) and majority of terrestrial plant species provides many benefits to plants that range from stress alleviation and bioremediation in soils polluted with heavy metals to plant growth promotion and yield quantity. Some non-arbuscular mycorrhizal fungi such as, Trichoderma harzianum, are known to enhance the AMF symbiosis with vascular plants. However, information about their role in AMF symbiosis is still limited. Shoots of (Avocet S) wheat seedlings were sprayed with the fungal culture filtrate and gene expression patterns were analyzed in the treated tissues. An increase in the level of mRNA of arbuscular mycorrhizal protein comparing with control was found. The over-expression of this protein in wheat tissues might contribute in initiation of AMF colonization in wheat tissues. The result of this study can spark future researches to elucidate possible role of this protein in the symbiotic interaction mechanisms between soil AMF and various plant roots.

  17. [Enhanced biohydrogen production by homologous over-expression of fnr, pncB, fdhF in Klebsiella sp. HQ-3].

    PubMed

    Wang, Shuyu; Wang, Jun; Xu, Li; Pi, Jian; Zhang, Houjin; Yan, Yunjun

    2013-09-01

    To enhance biohydrogen production of Klebsiella sp. HQ-3, the global transcriptional factor (Fnr), formate dehydrogenase H (FDH1) and the pncB gene encoding the nicotinic acid phosphoribosyltransferase (NAPRTase) were for the first time over-expressed in Klebsiella sp. HQ-3. The fnr, fdhF, pncB genes were cloned from the genomic DNA of Klebsiella sp. HQ-3 by 3 pairs of universal primers, and introduced into the corresponding sites of the modified pET28a-Pkan, resulting in the plasmids pET28a-Pkan-fnr, pET28a-Pkan-fdhF and pET28a-Pkan-pncB. The 4 plasmids were then electroported into wild Klebsiella sp. HQ-3 to create HQ-3-fnr, HQ-3-fdhF, HQ-3-pncB and HQ-3-C, respectively. Hydrogen production was measured using a gas chromatograph and the metabolites were analyzed with a high-performance liquid chromatograph (HPLC). The results indicate that over-expression of fnr, fdhF and pncB significantly enhanced hydrogen production in the three recombinant strains. Hydrogen production per mol glucose for HQ-3 fnr, HQ-3 pncB, HQ-3 fdhF was 1.113, 1.106 and 1.063 mol of hydrogen/mol glucose, which was respectively increased by 12.26%, 11.62% and 7.28% compared with that of the control strain HQ-3-C (0.991 mol of hydrogen/mol glucose). Moreover, the analysis of HPLC showed that the concentrations of formate and lactate were markedly decreased, but succinate remained unchanged in culture media compared with those of the control strain HQ-3-C.

  18. Exaggerated myocardial oxLDL amount and LOX-1 receptor over-expression associated with coronary microvessel inflammation in unstable angina.

    PubMed

    Neri Serneri, Gian Gastone; Coppo, Mirella; Bandinelli, Manuela; Paoletti, Paoletto; Toscano, Thomas; Micalizzi, Ezio; Chiostri, Marco; Boddi, Maria

    2013-02-01

    The pathophysiological relationship between coronary atherosclerosis and coronary microvessels remains undefined and the specific causative role of oxidatively modified low density lipoprotein (oxLDL) in human atherosclerosis is debated. The purposes of this study are to investigate whether coronary microvessels are involved in coronary atherosclerosis and whether increased myocardial oxLDL amount can be associated with coronary microvessel inflammation. A combination of immunohistochemical, RT-PCR and real-time PCR studies performed on myocardial biopsy specimens from patients with mitral stenosis (control hearts, CHs) and from unstable and stable angina patients (UAP and SAP), demonstrated that myocardial oxLDL was associated with a chronic low-grade inflammation in SAP and with a severe high grade inflammation in UAP. oxLDL amount was notably higher in UAP than in SAP and in UAP the high grade of inflammation was correlated with the increased amount of oxLDL in endothelial cells and macrophages. The exaggerated amount of oxLDL in UAP and the interaction of oxLDL with lectin-like oxLDL (LOX-1) receptor are amplified by the activation of transcriptional factor octamere 1 (OCT-1) with consequent activation of a series of inflammatory endothelial feed-back mechanisms resulting in LOX-1 gene over-expression, endothelial inflammation as well as uncontrolled nuclear factor kappa B (NFkB) activation. Moreover, in UAP genes for signal transducer and activator transcriptional factor 1α (STAT1α), angiotensin converting enzyme (ACE) and numerous pro-inflammatory cytokines were over-expressed. The present results may have clinical relevance because they show that coronary atherosclerosis is a disease not confined to the large arteries but involving the whole coronary tree. In UAP the exaggerated amount of myocardial oxLDL is associated with widespread high grade microvessel inflammation. PMID:23237633

  19. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars. PMID:24737077

  20. Over-expression of PsGPD, a mushroom glyceraldehyde-3-phosphate dehydrogenase gene, enhances salt tolerance in rice plants.

    PubMed

    Cho, Jung-Il; Lim, Hye-Min; Siddiqui, Zamin Shaheed; Park, Sung-Han; Kim, A-Ram; Kwon, Taek-Ryoun; Lee, Seong-Kon; Park, Soo-Chul; Jeong, Mi-Jeong; Lee, Gang-Seob

    2014-08-01

    Transgenic potatoes expressing glyceraldehyde-3-phosphate dehydrogenase (GPD), isolated from the oyster mushroom, Pleurotus sajor-caju, had increased tolerance to salt stress (Jeong et al. Biochem Biophys Res Commun 278:192-196, 2000). To examine the physiological mechanisms enhancing salt tolerance in GPD-transgenic rice plants, the salt tolerance of five GPD transgenic rice lines (T1-T5) derived from Dongjin rice cultivar were evaluated in a fixed 150 mM saline environment in comparison to two known wild-type rice cultivars, Dongjin (salt sensitive) and Pokali (salt tolerant). Transgenic lines, T2, T3, and T5, had a substantial increase in biomass and relative water content compared to Dongjin. Stomatal conductance and osmotic potential were higher in the GPD transgenic lines and were similar to those in Pokali. The results are discussed based on the comparative physiological response of GPD transgenic lines with those of the salt-sensitive and salt-tolerant rice cultivars.

  1. Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

    PubMed Central

    Ortiz-Espín, Ana; Locato, Vittoria; Camejo, Daymi; Schiermeyer, Andreas; De Gara, Laura; Sevilla, Francisca; Jiménez, Ana

    2015-01-01

    Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H2O2. Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H2O2 and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H2O2. Analysis of different hallmarks of programmed cell death was also carried out. Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H2O2, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H2O2 and nitric oxide contents and maintenance of the glutathione redox state were observed. Conclusions A decreased content of endogenous H2O2 may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and

  2. Cloning and over-expression of an alkaline protease from Bacillus licheniformis.

    PubMed

    Tang, Xue-Ming; Shen, Wei; Lakay, F M; Shao, Wei-Lan; Wang, Zheng-Xiang; Prior, B A; Zhuge, Jian

    2004-06-01

    The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816. PMID:15269522

  3. Functional consequences of the over-expression of TRPC6 channels in HEK cells: impact on the homeostasis of zinc.

    PubMed

    Chevallet, Mireille; Jarvis, Louis; Harel, Amélie; Luche, Sylvie; Degot, Sébastien; Chapuis, Violaine; Boulay, Guylain; Rabilloud, Thierry; Bouron, Alexandre

    2014-07-01

    The canonical transient receptor potential 6 (TRPC6) protein is a non-selective cation channel able to transport essential trace elements like iron (Fe) and zinc (Zn) through the plasma membrane. Its over-expression in HEK-293 cells causes an intracellular accumulation of Zn, indicating that it could be involved in Zn transport. This finding prompted us to better understand the role played by TRPC6 in Zn homeostasis. Experiments done using the fluorescent probe FluoZin-3 showed that HEK cells possess an intracellular pool of mobilisable Zn present in compartments sensitive to the vesicular proton pump inhibitor Baf-A, which affects endo/lysosomes. TRPC6 over-expression facilitates the basal uptake of Zn and enhances the size of the pool of Zn sensitive to Baf-A. Quantitative RT-PCR experiments showed that TRPC6 over-expression does not affect the mRNA expression of Zn transporters (ZnT-1, ZnT-5, ZnT-6, ZnT-7, ZnT-9, Zip1, Zip6, Zip7, and Zip14); however it up-regulates the mRNA expression of metallothionein-I and -II. This alters the Zn buffering capacities of the cells as illustrated by the experiments done using the Zn ionophore Na pyrithione. In addition, HEK cells over-expressing TRPC6 grow slower than their parental HEK cells. This feature can be mimicked by growing HEK cells in a culture medium supplemented with 5 μM of Zn acetate. Finally, a proteomic analysis revealed that TRPC6 up-regulates the expression of the actin-associated proteins ezrin and cofilin-1, and changes the organisation of the actin cytoskeleton without changing the cellular actin content. Altogether, these data indicate that TRPC6 is participating in the transport of Zn and influences the Zn storage and buffering capacities of the cells. PMID:24733507

  4. Wnt/β-catenin pathway is required for epithelial to mesenchymal transition in CXCL12 over expressed breast cancer cells

    PubMed Central

    Shan, Shumei; Lv, Qiang; Zhao, Yiling; Liu, Chunfeng; Sun, Yingyan; Xi, Kemin; Xiao, Jiayi; Li, Caijuan

    2015-01-01

    CXCL12 is positively associated with the metastasis and prognosis of various human malignancies. Cancer-associated fibroblasts (CAFs), the main cells secreting CXCL12, are capable of inducing epithelial to mesenchymal transition (EMT) of breast cancer cells. However, it has not been completely understood whether CXCL12 is involved in EMT of breast cancer cells and the underlying mechanisms. The present study aimed to investigate the effects of CXCL12 on the EMT and cancer stem cell (CSC)-like phenotypes formation by transfecting pEGFP-N1-CXCL12 plasmid into MCF-7 cells. Real time-PCR and Western blot analysis demonstrated the successful over expression of CXCL12 in MCF-7 cells. Cell counting kit-8 assay, wound healing assay and Transwell invasion analysis confirmed that over expression of CXCL12 significantly promoted the proliferation, migration and invasion in MCF-7 cells (P<0.05). In addition, ALDH activity was dramatically enhanced compared with parental (P<0.001), accompanied by the notably elevated mRNA and protein levels of OCT-4, Nanog, and SOX2 in CXCL12 overexpressed-MCF-7 cells (P<0.001). Furthermore, we observed the down regulation of E-cadherin and up regulation of vimentin, N-cadherin, and α-SMA in CXCL12 overexpressed-MCF-7 cells (P<0.01). Meanwhile, western blot and immunofluorescence assay showed that over expression of CXCL12 activated Wnt/β-catenin pathway to induce EMT of MCF-7 cells, as evidenced by the increased expression of E-cadherin after silencing β-catenin by siRNA interference (P<0.001). Collectively, our findings suggested that over expression of CXCL12 could trigger EMT by activating Wnt/β-catenin pathway and induce CSC-like phenotypes formation to promote the proliferation and metastasis in MCF-7. Hence, CXCL12 may become a promising candidate for breast cancer therapy. PMID:26722422

  5. Functional response to SDF1α through over-expression of CXCR4 on adult subventricular zone progenitor cells

    PubMed Central

    Liu, Xian Shuang; Chopp, Michael; Santra, Manoranjan; Hozeska-Solgot, Ann; Zhang, Rui Lan; Wang, Lei; Teng, Hua; Liu, Mei; Zhang, Zheng Gang

    2008-01-01

    The chemokine receptor CXCR4 and its ligand, stromal cell derived factor-1α (SDF1α) regulate neuroblast migration towards the ischemic boundary after stroke. Using loss-and gain-function, we investigated the biological effect of CXCR4/SDF1α on neural progenitor cells. Neural progenitor cells, from the subventricular zone (SVZ) of the adult rat, were transfected with rat CXCR4-pLEGFP-C1 and pSIREN-RetroQ-CXCR4-siRNA retroviral vectors. Migration assay analysis showed that inhibition of CXCR4 by siRNA significantly reduced cell migration compared to the empty vector, indicating that CXCR4 mediated neural progenitor cell motility. When neural progenitor cells were cultured in growth medium containing bFGF (20 ng/ml), over-expression of CXCR4 significantly reduced the cell proliferation as measured by the number of bromodeoxyuridine+ (BrdU+) cells (26.4%) compared with the number in the control group (54.0%). Addition of a high concentration of SDF1α (500 ng/ml) into the progenitor cells with over-expression of CXCR4 reversed the cell proliferation back to the control levels (57.6%). Immunostaining analysis showed that neither over-expression nor inhibition of CXCR4 altered the population of neurons and astrocytes, when neural progenitor cells were cultured in differentiation medium. These in vitro results suggest that CXCR4/SDF1α primarily regulates adult neural progenitor cell motility but not differentiation, while over-expression of CXCR4 in the absence of SDF1α decreases neural progenitor cell proliferation. PMID:18598677

  6. EGFR gene copy number increase in vulvar carcinomas is linked with poor clinical outcome.

    PubMed

    Woelber, L; Hess, S; Bohlken, H; Tennstedt, P; Eulenburg, C; Simon, R; Gieseking, F; Jaenicke, F; Mahner, S; Choschzick, M

    2012-02-01

    EGFR copy number increases have been frequently reported in cancer including vulvar carcinomas. Co-amplification of cancer genes plays an important role in the development of many tumour types. To better understand the effect of EGFR aberrations on vulvar cancer phenotype and patient prognosis, the authors analysed EGFR copy number changes using fluorescence in situ hybridisation and EGFR expression by immunohistochemistry in a tissue microarray containing 183 squamous cell carcinomas of vulva. Furthermore, the authors analysed the co-amplification frequency of EGFR with HER2, CCND1, MYC and PIK3CA, respectively. EGFR copy number increase was found in 39.3% of the tumours. Seventeen per cent of vulvar carcinomas showed EGFR high polysomy including 9% with amplification of the EGFR gene. Copy number gain of the EGFR locus was associated with non-basaloid phenotype (p=0.03), high-tumour stage (p<0.001), human papillomaviruse negativity of tumours (p=0.04) and the number of lymph node metastases (p=0.02). EGFR protein expression was statistically correlated to EGFR copy number increase (p<0.05). The observed co-amplification rate of EGFR with all four additionally examined oncogenes was much higher than statistically expected. There was a highly significant association between EGFR copy number increase and CCND1 amplifications (p<0.001) as well as the total number of gene amplifications (p=0.04). EGFR copy number gains were significantly related to unfavourable patient outcome in univariate analysis and multivariate Cox regression analysis. In conclusion, EGFR copy number increases are detectable in a substantial proportion of vulvar carcinomas with relationships to advanced tumour stages and the development of lymph node metastases. EGFR copy number aberrations are connected to other gene amplifications and probably define an human papillomaviruses-independent pathway in the development of vulvar carcinomas. These data support the potential utility of EGFR inhibitors

  7. Over-expression of an S-domain receptor-like kinase extracellular domain improves panicle architecture and grain yield in rice

    PubMed Central

    Zou, Xiaohua; Qin, Zhengrui; Zhang, Chunyu; Liu, Bin; Liu, Jun; Zhang, Chengsheng; Lin, Chentao; Li, Hongyu; Zhao, Tao

    2015-01-01

    The S-domain receptor kinase (SRK) comprises a highly polymorphic subfamily of receptor-like kinases (RLKs) originally found to be involved in the self-incompatibility response in Brassica. Although several members have been identified to play roles in developmental control and disease responses, the correlation between SRKs and yield components in rice is still unclear. The utility of transgenic expression of a dominant negative form of SRK, OsLSK1 (Large spike S-domain receptor like Kinase 1), is reported here for the improvement of grain yield components in rice. OsLSK1 was highly expressed in nodes of rice and is a plasma membrane protein. The expression of OsLSK1 responded to the exogenous application of growth hormones, to abiotic stresses, and its extracellular domain could form homodimers or heterodimers with other related SRKs. Over-expression of a truncated version of OsLSK1 (including the extracellular and transmembrane domain of OsLSK1 without the intracellular kinase domain) increased plant height and improve yield components, including primary branches per panicle and grains per primary branch, resulting in about a 55.8% increase of the total grain yield per plot (10 plants). Transcriptional analysis indicated that several key genes involved in the GA biosynthetic and signalling pathway were up-regulated in transgenic plants. However, full-length cDNA over-expression and RNAi of OsLSK1 transgenic plants did not exhibit a detectable visual phenotype and possible reasons for this were discussed. These results indicate that OsLSK1 may act redundantly with its homologues to affect yield traits in rice and manipulation of OsLSK1 by the dominant negative method is a practicable strategy to improve grain yield in rice and other crops. PMID:26428067

  8. Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.

    PubMed

    Kudoh, Kai; Kawano, Yusuke; Hotta, Shingo; Sekine, Midori; Watanabe, Takafumi; Ihara, Masaki

    2014-07-01

    Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.

  9. Cloning, over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum.

    PubMed

    Shi, Hao; Zhang, Yu; Zhong, Hui; Huang, Yingjuan; Li, Xun; Wang, Fei

    2014-03-01

    The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K m , k cat and k cat /K m values of the xylanase for beechwood xylan were 1.8 mg ml(-1), 520 s(-1) and 289 ml mg(-1) s(-1), respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis. PMID:24170174

  10. An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in Escherichia coli

    PubMed Central

    2012-01-01

    Background A useful goal for metabolic engineering would be to generate non-growing but metabolically active quiescent cells which would divert the metabolic fluxes towards product formation rather than growth. However, for products like recombinant proteins, which are intricately coupled to the growth process it is difficult to identify the genes that need to be knocked-out/knocked-in to get this desired phenotype. To circumvent this we adopted an inverse metabolic engineering strategy which would screen for the desired phenotype and thus help in the identification of genetic targets which need to be modified to get overproducers of recombinant protein. Such quiescent cells would obviate the need for high cell density cultures and increase the operational life span of bioprocesses. Results A novel strategy for generating a library, consisting of randomly down regulated metabolic pathways in E. coli was designed by cloning small genomic DNA fragments in expression vectors. Some of these DNA fragments got inserted in the reverse orientation thereby generating anti-sense RNA upon induction. These anti-sense fragments would hybridize to the sense mRNA of specific genes leading to gene ‘silencing’. This library was first screened for slow growth phenotype and subsequently for enhanced over-expression ability. Using Green Fluorescent Protein (GFP) as a reporter protein on second plasmid, we were able to identify metabolic blocks which led to significant increase in expression levels. Thus down-regulating the ribB gene (3, 4 dihydroxy-2-butanone-4-phosphate synthase) led to a 7 fold increase in specific product yields while down regulating the gene kdpD (histidine kinase) led to 3.2 fold increase in specific yields. Conclusion We have designed a high throughput screening approach which is a useful tool in the repertoire of reverse metabolic engineering strategies for the generation of improved hosts for recombinant protein expression. PMID:22759404

  11. Cardiomyocyte-Specific miRNA-30c Over-Expression Causes Dilated Cardiomyopathy

    PubMed Central

    Wijnen, Wino J.; van der Made, Ingeborg; van den Oever, Stephanie; Hiller, Monika; de Boer, Bouke A.; Picavet, Daisy I.; Chatzispyrou, Iliana A.; Houtkooper, Riekelt H.; Tijsen, Anke J.; Hagoort, Jaco; van Veen, Henk; Everts, Vincent; Ruijter, Jan M.; Pinto, Yigal M.; Creemers, Esther E.

    2014-01-01

    MicroRNAs (miRNAs) regulate many aspects of cellular function and their deregulation has been implicated in heart disease. MiRNA-30c is differentially expressed in the heart during the progression towards heart failure and in vitro studies hint to its importance in cellular physiology. As little is known about the in vivo function of miRNA-30c in the heart, we generated transgenic mice that specifically overexpress miRNA-30c in cardiomyocytes. We show that these mice display no abnormalities until about 6 weeks of age, but subsequently develop a severely dilated cardiomyopathy. Gene expression analysis of the miRNA-30c transgenic hearts before onset of the phenotype indicated disturbed mitochondrial function. This was further evident by the downregulation of mitochondrial oxidative phosphorylation (OXPHOS) complexes III and IV at the protein level. Taken together these data indicate impaired mitochondrial function due to OXPHOS protein depletion as a potential cause for the observed dilated cardiomyopathic phenotype in miRNA-30c transgenic mice. We thus establish an in vivo role for miRNA-30c in cardiac physiology, particularly in mitochondrial function. PMID:24789369

  12. Over-Expression of CD200 Predicts Poor Prognosis in Cutaneous Squamous Cell Carcinoma

    PubMed Central

    Li, Li; Tian, YanLi; Shi, ChengFang; Zhang, Hua; Zhou, Zhi

    2016-01-01

    Background CD200 is reported to be involved in tumor progression and can serve as a prognostic marker in several cancers. The purpose of this study was to evaluate the prognostic significance of CD200 in cutaneous squamous cell carcinoma (CSCC). Material/Methods The relative mRNA and protein expression of CD200 in the tumor tissues and corresponding normal tissues of 102 CSCC patients were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis, respectively. The chi-square test was used to analyze the association between CD200 expression and clinical features of CSCC patients. In addition, the overall survival of the patients according to the expression level of CD200 was estimated by Kaplan-Meier analysis and the prognostic significance of the gene was analyzed by Cox regression analysis. Results Increased expression of CD200 was detected in the tumor tissues compared with the corresponding normal tissues both at mRNA and protein level. And CD200 expression level was associated with tumor differentiation grade (P=0.041) and clinical stage (P=0.004). Patients with high expression level of CD200 had a shorter overall survival than those with low expression (31.3 months vs. 41.9 months) and there was a significant difference between them (log-rank test, P<0.001). Cox regression analysis indicated that CD200 could be an independent marker for the prognosis of CSCC. Conclusions CD200 is up-regulated and may be a novel biomarker for the prognosis in CSCC, and it may be a potential therapeutic target for CSCC. PMID:27035797

  13. Construction and Characterization of Recombinant HEK Cell Over Expressing α4 Integrin

    PubMed Central

    Naderi Beni, Shamsi; Kouhpayeh, Shirin; Hejazi, Zahra; Heidari Hafshejani, Nahid; Khanahmad, Hossein

    2015-01-01

    Purpose: Integrins are heterodimeric membrane proteins, which are exposed to post translational modifications in eukaryotic cells in contrast to prokaryotic cells. These modifications provide advantages for production of proper nanobody, mono and polyclonal antibody against this surface protein and also in aptamer selection process. Since the majority of diagnostic and therapeutic antibodies, target the surface epitopes, eukaryotic membrane proteins provide an appropriate model for further investigation on therapeutic agents. Methods: Escherichia coli strain top 10, was used as host for ITGA-4 expression vector encoding the human integrin α4. The plasmid was extracted and consequently, ITGA-4 vector was digested to make a linear plasmid. Human Embryonic Kidney-293 (HEK-293) cell transfected with linear plasmid and subsequently screened for stable ITGA-4 expressing Cells. Three separated clones were isolated twenty one days after transfection. Chromosomal DNA was extracted from ITGA-4-transfected cells. The presence of ITGA-4 gene in HEK-293 genome was confirmed by PCR. The expression level of ITGA-4 on HEK-293 cells was also analyzed by Flow cytometry. Results: Flow cytometric analysis showed that HEK-293 cells have no expression of integrin α4 on their surface while 95% of transfected HEK-293 cells with ITGA4, expressed different levels of integrin α4 on their surfaces which correlates well with genomic DNA PCR amplification results. Conclusion: The results suggest that we have successfully constructed the integrin α4 expressing HEK293 cell, which will facilitate further research into the production of antibody, nanobody and aptamer against α4 integrin. PMID:26504766

  14. Over-expression of Chinese cabbage calreticulin 1, BrCRT1, enhances shoot and root regeneration, but retards plant growth in transgenic tobacco.

    PubMed

    Jin, Zheng-Lu; Hong, Joon Ki; Yang, Kyung Ae; Koo, Ja Choon; Choi, Young Ju; Chung, Woo Sik; Yun, Dae-Jin; Lee, Sang Yeol; Cho, Moo Je; Lim, Chae Oh

    2005-10-01

    Calreticulin (CRT) is a ubiquitously expressed, high capacity Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). A cDNA encoding a calreticulin, BrCRT1 (Brassica rapa Calreticulin 1), has been isolated from Chinese cabbage (B. rapa subsp. pekinensis) flower bud. Constitutive over-expression of the BrCRT1 gene promotes robust shoot production and root formation at sub-optimal concentrations of BA/NAA, which are important factors controlling plant regeneration in tissue culture. In contrast, the suppressed BrCRT1 line exhibited a slight reduction of shoot and root regeneration. In spite of enhanced regeneration in tissue culture, the seedling and plant growth rate was inhibited in soil. The steady state level of BrCRT1 transcripts was sensitive to exogenous auxins and cytokinins, and rapidly accumulated within 30 min, and this induction required de novo protein synthesis. Together with the results of transgenic tobacco plants and mRNA analysis in Chinese cabbage, our data suggest that BrCRT1 genes may up-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root regeneration processes.

  15. The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24).

    PubMed

    Varol, Nuray; Konac, Ece; Onen, Ilke Hacer; Gurocak, Serhat; Alp, Ebru; Yilmaz, Akin; Menevse, Sevda; Sozen, Sinan

    2014-07-01

    The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both β-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 μM), DAC (10 μM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3β, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of β-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/β-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3β mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/β-catenin pathway and reduced

  16. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse.

    PubMed

    Coelho, Paula A; Bury, Leah; Shahbazi, Marta N; Liakath-Ali, Kifayathullah; Tate, Peri H; Wormald, Sam; Hindley, Christopher J; Huch, Meritxell; Archer, Joy; Skarnes, William C; Zernicka-Goetz, Magdalena; Glover, David M

    2015-12-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  17. Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse

    PubMed Central

    Coelho, Paula A.; Bury, Leah; Shahbazi, Marta N.; Liakath-Ali, Kifayathullah; Tate, Peri H.; Wormald, Sam; Hindley, Christopher J.; Huch, Meritxell; Archer, Joy; Skarnes, William C.; Zernicka-Goetz, Magdalena; Glover, David M.

    2015-01-01

    To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development. PMID:26701933

  18. Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

    PubMed

    Belgaroui, Nibras; Zaidi, Ikram; Farhat, Ameny; Chouayekh, Hichem; Bouain, Nadia; Chay, Sandrine; Curie, Catherine; Mari, Stéphane; Masmoudi, Khaled; Davidian, Jean-Claude; Berthomieu, Pierre; Rouached, Hatem; Hanin, Moez

    2014-11-01

    Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling.

  19. Over-expression of the bacterial phytase US417 in Arabidopsis reduces the concentration of phytic acid and reveals its involvement in the regulation of sulfate and phosphate homeostasis and signaling.

    PubMed

    Belgaroui, Nibras; Zaidi, Ikram; Farhat, Ameny; Chouayekh, Hichem; Bouain, Nadia; Chay, Sandrine; Curie, Catherine; Mari, Stéphane; Masmoudi, Khaled; Davidian, Jean-Claude; Berthomieu, Pierre; Rouached, Hatem; Hanin, Moez

    2014-11-01

    Phytic acid (PA) is the main phosphorus storage form in plant seeds. It is recognized as an anti-nutrient for humans and non-ruminant animals, as well as one of the major sources of phosphorus that contributes to eutrophication. Therefore, engineering plants with low PA content without affecting plant growth capacity has become a major focus in plant breeding. Nevertheless, lack of knowledge on the role of PA seed reserves in regulating plant growth and in maintaining ion homeostasis hinders such an agronomical application. In this context, we report here that the over-expression of the bacterial phytase PHY-US417 in Arabidopsis leads to a significant decrease in seed PA, without any effect on the seed germination potential. Interestingly, this over-expression also induced a higher remobilization of free iron during germination. Moreover, the PHY-over-expressor lines show an increase in inorganic phosphate and sulfate contents, and a higher biomass production after phosphate starvation. Finally, phosphate sensing was altered because of the changes in the expression of genes induced by phosphate starvation or involved in phosphate or sulfate transport. Together, these results show that the over-expression of PHY-US417 reduces PA concentration, and provide the first evidence for the involvement of PA in the regulation of sulfate and phosphate homeostasis and signaling. PMID:25231959

  20. Anosmin-1 over-expression increases adult neurogenesis in the subventricular zone and neuroblast migration to the olfactory bulb.

    PubMed

    García-González, Diego; Murcia-Belmonte, Verónica; Esteban, Pedro F; Ortega, Felipe; Díaz, David; Sánchez-Vera, Irene; Lebrón-Galán, Rafael; Escobar-Castañondo, Laura; Martínez-Millán, Luis; Weruaga, Eduardo; García-Verdugo, José Manuel; Berninger, Benedikt; de Castro, Fernando

    2016-01-01

    New subventricular zone (SVZ)-derived neuroblasts that migrate via the rostral migratory stream are continuously added to the olfactory bulb (OB) of the adult rodent brain. Anosmin-1 (A1) is an extracellular matrix protein that binds to FGF receptor 1 (FGFR1) to exert its biological effects. When mutated as in Kallmann syndrome patients, A1 is associated with severe OB morphogenesis defects leading to anosmia and hypogonadotropic hypogonadism. Here, we show that A1 over-expression in adult mice strongly increases proliferation in the SVZ, mainly with symmetrical divisions, and produces substantial morphological changes in the normal SVZ architecture, where we also report the presence of FGFR1 in almost all SVZ cells. Interestingly, for the first time we show FGFR1 expression in the basal body of primary cilia in neural progenitor cells. Additionally, we have found that A1 over-expression also enhances neuroblast motility, mainly through FGFR1 activity. Together, these changes lead to a selective increase in several GABAergic interneuron populations in different OB layers. These specific alterations in the OB would be sufficient to disrupt the normal processing of sensory information and consequently alter olfactory memory. In summary, this work shows that FGFR1-mediated A1 activity plays a crucial role in the continuous remodelling of the adult OB. PMID:25300351

  1. Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase.

    PubMed

    Yang, Haibing; Knapp, Jane; Koirala, Pratistha; Rajagopal, Divya; Peer, Wendy Ann; Silbart, Lawrence K; Murphy, Angus; Gaxiola, Roberto A

    2007-11-01

    Plants challenged by limited phosphorus undergo dramatic morphological and architectural changes in their root systems in order to increase their absorptive surface area. In this paper, it is shown that phosphorus deficiency results in increased expression of the type I H+-pyrophosphatase AVP1 (AVP, Arabidopsis vacuolar pyrophosphatase), subsequent increased P-type adenosine triphosphatase (P-ATPase)-mediated rhizosphere acidification and root proliferation. Molecular genetic manipulation of AVP1 expression in Arabidopsis, tomato and rice results in plants that outperform controls when challenged with limited phosphorus. However, AVP1 over-expression and the resulting rhizosphere acidification do not result in increased sensitivity to AlPO4, apparently because of the enhancement of potassium uptake and the release of organic acids. Thus, the over-expression of type I H+-pyrophosphatases appears to be a generally applicable technology to help alleviate agricultural losses in low-phosphorus tropical/subtropical soils and to reduce phosphorus runoff pollution of aquatic and marine environments resulting from fertilizer application. PMID:17711412

  2. Dysregulating IRES-dependent translation contributes to over-expression of the Aurora A kinase onco-protein

    PubMed Central

    Dobson, Tara; Chen, Juan; Krushel, Les A.

    2014-01-01

    Over-expression of the oncoprotein, Aurora A kinase occurs in multiple types of carcinomas, often early during cell transformation. To identify mechanism(s) contributing to enhanced Aurora A protein expression, we examined normal human lung fibroblast and breast epithelial cells and compared them to non-tumorigenic breast (MCF10A and MCF12A) and tumorigenic breast and cervical epithelial cell lines (MCF-7 and HeLa S3, respectively). A subset of these immortalized lines (MCF10, MCF12A, and HeLa S3) exhibited increased levels of Aurora A protein, independent of tumorigenicity. The increase in Aurora A protein expression in these immortalized cells was not due to increased transcription/RNA stability, protein half-life or cap-dependent translation. Assays utilizing monocistronic and dicistronic RNA constructs revealed that the Aurora A 5′ leader contains an internal ribosomal entry site (IRES), which is regulated through the cell cycle, peaking in G2/M phase. Moreover, IRES activity was increased in the immortalized cell lines in which Aurora A protein expression was also enhanced. Additional assays indicated that the increased internal initiation is specific to the Aurora A IRES and may be an early event during cancer progression. Taken together, these results identify a novel mechanism contributing to Aurora A kinase over-expression and possibly to immortalization leading to carcinogenesis. PMID:23661421

  3. Role of cyclin D1 amplification and expression in vulvar carcinomas.

    PubMed

    Choschzick, Matthias; Hess, Stephan; Tennstedt, Pierre; Holst, Frederik; Bohlken, Hiltila; Gieseking, Frederike; Mahner, Sven; Woelber, Linn; Simon, Ronald; Sauter, Guido

    2012-09-01

    Cyclin D1 (CCND1) belongs to the family of D-type cyclins involved in cell cycle progression, transcriptional regulation, and cell migration. CCND1 was found to be amplified and overexpressed in a variety of cancers, including some vulvar carcinoma cell lines. To determine the relationship of CCND1 copy number changes and CCND1 protein expression with clinicopathologic features and prognosis, 183 vulvar carcinomas were analyzed on a tissue microarray. Amplification was observed in 32 (22.4%) vulvar cancer specimens and was statistically related to the presence of regional lymph node metastases (P < .001). Detectable CCND1 expression was found in 139 (83.2%) of vulvar carcinomas, and 76 (45.5%) exhibited a moderate or strong expression. Increased levels of CCND1 expression were significantly related to higher patient age (P = .013), positive pN category (P = .004), and negative human papillomavirus status (P < .001). Basaloid as well as verrucous, warty-type, and mixed vulvar carcinomas showed lower CCND1 expression levels than keratinizing or nonkeratinizing tumors (P < .001 and P = .032, respectively). Elevated CCND1 expression levels and amplification of the CCND1 gene were closely connected in the present analysis (P < .001). Patient prognosis was independent from CCND1 amplification status and expression level (P = .57 each). In conclusion, CCND1 is amplified and overexpressed in a substantial proportion of vulvar carcinomas and associated with the occurrence of locoregional lymph node metastases, especially in human papillomavirus-negative tumors.

  4. Over-expression of C/EBP-{alpha} induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Wang Xueqing; Huang Guangcun; Mei Shuang; Qian Jin; Ji Juling; Zhang Jinsheng

    2009-03-06

    Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-{alpha} (C/EBP-{alpha}) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-{alpha} gene (Ad-C/EBP-{alpha}) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-{alpha} resulted in the up-regulation of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) and P53, while P53 expression was regulated by PPAR-{gamma}. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-{gamma} and p53 in the process of apoptosis triggered by C/EBP-{alpha} in HSCs.

  5. Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants.

    PubMed

    Mishra, Ratnesh Chandra; Richa; Grover, Anil

    2016-09-01

    Caseinolytic proteases (Clps) perform the important role of removing protein aggregates from cells, which can otherwise prove to be highly toxic. ClpD system is a two-component protease complex composed of a regulatory ATPase module ClpD and a proteolytic component ClpP. Under desiccation stress condition, rice ClpD1 (OsClpD1) gene encoding for the regulatory subunit, was represented by four variant transcripts differing mainly in the expanse of their N-terminal amino acids. These transcripts were expressed in a differential manner in response to salt, mannitol and polyethylene glycol stresses in rice. Purified OsClpD1.3 protein exhibited intrinsic chaperone activity, shown using citrate synthase as substrate. Arabidopsis (Col-0) plants over-expressing OsClpD1.3 open reading frame downstream to CaMV35S promoter (ClpD1.3 plants) showed higher tolerance to salt and desiccation stresses as compared to wild type plants. ClpD1.3 seedlings also showed enhanced growth during the early stages of seed germination under unstressed, control conditions. The free proline levels and starch breakdown activities were higher in the ClpD1.3 seedlings as compared to the wild type Arabidopsis seedlings. It thus emerges that increasing the potential of ClpD1 chaperoning activity may be of advantage in protection against abiotic stresses. PMID:27457985

  6. Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans.

    PubMed

    Simental-Mendía, M; Lara-Arias, J; Álvarez-Lozano, E; Said-Fernández, S; Soto-Domínguez, A; Padilla-Rivas, G R; Martínez-Rodríguez, H G

    2015-12-01

    Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.

  7. Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro

    SciTech Connect

    Zhang, Huiying; Newman, Donna R.; Bonner, James C.; Sannes, Philip L.

    2012-11-15

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. -- Highlights: ► Primary human lung alveolar type 2 (hAT2) cells and H292 and A549 cells were used. ► Cadmium induced apoptosis in hAT2 cells but not in H292 or A549 cells. ► HSulf-1exacerbates apoptosis induced by cadmium in H292 and A549 but not hAT2 cells.

  8. Redirection of doublecortin-positive cell migration by over-expression of the chemokines MCP-1, MIP-1α and GRO-α in the adult rat brain.

    PubMed

    Tang, S K; Knobloch, R A; Maucksch, C; Connor, B

    2014-02-28

    Inflammation-induced chemoattraction plays a major role in adult subventricular zone (SVZ)-derived precursor cell migration following neural cell loss, in particular through the release of chemokines by activated microglia and macrophages. We previously demonstrated that monocyte chemotactic protein-1 (MCP-1) (chemokine (c-c motif) ligand (CCL)2), macrophage inflammatory protein-1α (MIP-1α) (CCL3) and growth regulatory protein-α (GRO-α) (chemokine (c-x-c motif) ligand (CXCL)1) are up-regulated following neural cell loss in the adult striatum and act as potent chemoattractants for SVZ-derived precursor cells in vitro. Based on these observations, the current study aimed to examine the individual effect of MCP-1, MIP-1α and GRO-α on the migration of adult SVZ-derived neural precursor cells in vivo. To address this without the confounding effects of injury-induced chemotactic cues, adeno-associated viral (AAV)2-mediated in vivo gene transfer was used to ectopically express either MCP-1, MIP-1α or GRO-α, or the control red fluorescent protein (RFP) in the normal adult rat striatum. The extent of doublecortin (Dcx)-positive cell recruitment from the SVZ into the striatal parenchyma was then determined at 4 and 8weeks following AAV2 injection. Ectopic expression either of MCP-1 or MIP-1α in the normal adult rat brain significantly increased the number of Dcx-positive cells and the extent of their migration into the striatum at both 4 and 8weeks after vector injection but did not promote either precursor cell proliferation or neural differentiation. In contrast, while over-expression of GRO-α 4weeks after vector injection induced a significant increase in Dcx-positive cell migration compared to control, this effect was reduced to control levels by 8weeks post injection. Further, direct comparison between MCP-1, MIP-1α and GRO-α at both 4 and 8weeks post vector injection indicated that GRO-α may have a reduced effect in inducing Dcx-positive cell migration

  9. Small-Scale Screening to Large-Scale Over-Expression of Human Membrane Proteins for Structural Studies.

    PubMed

    Chaudhary, Sarika; Saha, Sukanya; Thamminana, Sobrahani; Stroud, Robert M

    2016-01-01

    Membrane protein structural studies are frequently hampered by poor expression. The low natural abundance of these proteins implies a need for utilizing different heterologous expression systems. E. coli and yeast are commonly used expression systems due to rapid cell growth at high cell density, economical production, and ease of manipulation. Here we report a simplified, systematically developed robust strategy from small-scale screening to large-scale over-expression of human integral membrane proteins in the mammalian expression system for structural studies. This methodology streamlines small-scale screening of several different constructs utilizing fluorescence size-exclusion chromatography (FSEC) towards optimization of buffer, additives, and detergents for achieving stability and homogeneity. This is followed by the generation of stable clonal cell lines expressing desired constructs, and lastly large-scale expression for crystallization. These techniques are designed to rapidly advance the structural studies of eukaryotic integral membrane proteins including that of human membrane proteins. PMID:27485338

  10. Over-expression of corticotropin-releasing factor mRNA in inferior olivary neurons of rolling mouse Nagoya.

    PubMed

    Sawada, Kazuhiko; Kawano, Michihiro; Tsuji, Hiroshi; Sakata-Haga, Hiromi; Hisano, Setsuji; Fukui, Yoshihiro

    2003-10-01

    Expression of corticotropin-releasing factor (CRF) mRNA was examined in the inferior olivary nucleus (ION) of an ataxic mutant, rolling mouse Nagoya (RMN) by semi-quantitative in situ hybridization. The most marked difference in the level of CRF mRNA signals between RMN and non-ataxic littermates (control mice) was observed in the beta-subnucleus and ventrolateral protrusion of the ION. The level of signals in these subnuclei was about twofold higher in RMN than in the controls. Signal levels in the dorsal nucleus, principal nucleus and subnucleus A were slightly but significantly higher in RMN than in the controls. In the other subnuclei, there were no differences in signal level between RMN and controls. These results suggest a region-related over-expression of CRF mRNA in the ION of RMN. This may be responsible for the increased sensitivity of some Purkinje cells to glutamate, resulting in ataxic symptoms of RMN.

  11. Ventilatory response to acute hypoxia in transgenic mice over-expressing erythropoietin: effect of acclimation to 3-week hypobaric hypoxia.

    PubMed

    Villafuerte, Francisco C; Cárdenas-Alayza, Rosa; Macarlupú, José Luis; Monge-C, Carlos; León-Velarde, Fabiola

    2007-09-30

    We used transgenic mice constitutively over-expressing erythropoietin ("tg6" mice) and wild-type (wt) mice to investigate whether the high hematocrit (hct), consequence of Epo over-expression affected: (1) the normoxic ventilation (V (E)) and the acute hypoxic ventilatory response (HVR) and decline (HVD), (2) the increase in ventilation observed after chronic exposure to hypobaric hypoxia (430mmHg for 21 days), (3) the respiratory "blunting", and (4) the erythrocythemic response induced by chronic hypoxia exposure. V (E) was found to be similar in tg6 and wt mice in normoxia (FIO2=0.21). Post-acclimation V (E) was significantly elevated in every time point in wt mice at FIO2=0.10 when compared to pre-acclimation values. In contrast, tg6 mice exhibited a non-significant increase in V (E) throughout acute hypoxia exposure. Changes in V (E) are associated with adjustments in tidal volume (V(T)). HVR and HVD were independent of EE in tg6 and wt mice before chornic hypoxia exposure. HVR was significantly greater in wt than in tg6 mice after chronic hypoxia. After acclimation, HVD decreased in tg6 mice. Chronic hypoxia exposure caused hct to increase significantly in wt mice, while only a marginal increase occurred in the tg6 group. Although pre-existent EE does not appear to have an effect on HVR, the observation of alterations on V(T) suggests that it may contribute to time-dependent changes in ventilation and in the acute HVR during exposure to chronic hypoxia. In addition, our results suggest that EE may lead to an early "blunting" of the ventilatory response.

  12. Over-expression of an Na+-and K+-permeable HKT transporter in barley improves salt tolerance.

    PubMed

    Mian, Afaq; Oomen, Ronald J F J; Isayenkov, Stanislav; Sentenac, Hervé; Maathuis, Frans J M; Véry, Anne-Aliénor

    2011-11-01

    Soil salinity is an increasing menace that affects agriculture across the globe. Plant adaptation to high salt concentrations involves integrated functions, including control of Na+ uptake, translocation and compartmentalization. Na+ transporters belonging to the HKT family have been shown to be involved in tolerance to mild salt stress in glycophytes such as Arabidopsis, wheat and rice by contributing to Na+ exclusion from aerial tissues. Here, we have analysed the role of the HKT transporter HKT2;1, which is permeable to K+ and Na+, in barley, a relatively salt-tolerant crop that displays a salt-including behaviour. In Xenopus oocytes, HvHKT2;1 co-transports Na+ and K+ over a large range of concentrations, displaying low affinity for Na+, variable affinity for K+ depending on external Na+ concentration, and inhibition by K+ (K(i) approximately 5 mm). HvHKT2;1 is predominantly expressed in the root cortex. Transcript levels are up-regulated in both roots and shoots by low K+ growth conditions, and in shoots by high Na+ growth conditions. Over-expression of HvHKT2;1 led to enhanced Na+ uptake, higher Na+ concentrations in the xylem sap, and enhanced translocation of Na+ to leaves when plants were grown in the presence of 50 or 100 mm NaCl. Interestingly, these responses were correlated with increased barley salt tolerance. This suggests that one of the factors that limits barley salt tolerance is the capacity to translocate Na+ to the shoot rather than accumulation or compartmentalization of this cation in leaf tissues. Thus, over-expression of HvHKT2;1 leads to increased salt tolerance by reinforcing the salt-including behaviour of barley. PMID:21749504

  13. Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress.

    PubMed

    Rey, P; Gillet, B; Römer, S; Eymery, F; Massimino, J; Peltier, G; Kuntz, M

    2000-03-01

    Proteins homologous to fibrillin, a pepper plastid lipid-associated protein involved in carotenoid storage in fruit chromoplasts, have been recently identified in leaf chloroplasts from several species and shown to be induced upon environmental stress. To further investigate the role of the protein, transgenic Nicotiana tabacum plants over-expressing fibrillin using a constitutive promoter were generated. Transgenics grown under standard light intensities (300 micromol photons m-2 sec-1) were found to contain substantial amounts of fibrillin in flowers and leaves. In leaves, the protein was immunolocalized within chloroplasts in both stromal and thylakoid subfractions. No change was noticed in thylakoid structures from transgenics, but chloroplasts contained an increased number of plastoglobules organized in clusters. In petals, leucoplasts were also found to contain more agglutinated plastoglobules. The effects of environmental factors on fibrillin gene expression and protein localization were studied in tobacco leaves. Less fibrillin was present in plants grown under low light intensities, which can be explained by the involvement of a light-dependent splicing step in the control of fibrillin gene expression in leaves. Analysis of protein subfractions from plants subjected to drought or high light showed that both stresses resulted in fibrillin association with thylakoids. Whereas no growth difference between wild-type (WT) and transgenic plants was noticed under low light conditions, transgenics exhibit a longer main stem, enhanced development of lateral stems and accelerated floral development under higher light intensities. These data suggest that fibrillin-related proteins fulfil an important function in plant development in relation to environmental constraints. PMID:10758499

  14. Expression of Senescence-Associated microRNAs and Target Genes in Cellular Aging and Modulation by Tocotrienol-Rich Fraction

    PubMed Central

    2014-01-01

    Emerging evidences highlight the implication of microRNAs as a posttranscriptional regulator in aging. Several senescence-associated microRNAs (SA-miRNAs) are found to be differentially expressed during cellular senescence. However, the role of dietary compounds on SA-miRNAs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on SA-miRNAs (miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) and established target genes of miR-34a (CCND1, CDK4, and SIRT1) during replicative senescence of human diploid fibroblasts (HDFs). Primary cultures of HDFs at young and senescent were incubated with TRF at 0.5 mg/mL. Taqman microRNA assay showed significant upregulation of miR-24 and miR-34a and downregulation of miR-20a and miR-449a in senescent HDFs (P < 0.05). TRF reduced miR-34a expression in senescent HDFs and increased miR-20a expression in young HDFs and increased miR-449a expression in both young and senescent HDFs. Our results also demonstrated that ectopic expression of miR-34a reduced the expression of CDK4 significantly (P < 0.05). TRF inhibited miR-34a expression thus relieved its inhibition on CDK4 gene expression. No significant change was observed on the expression of CCND1, SIRT1, and miR-34a upstream transcriptional regulator, TP53. In conclusion tocotrienol-rich fraction prevented cellular senescence of human diploid fibroblasts via modulation of SA-miRNAs and target genes expression. PMID:25132913

  15. Identification of Hub Genes Related to the Recovery Phase of Irradiation Injury by Microarray and Integrated Gene Network Analysis

    PubMed Central

    Zhang, Jing; Yang, Yue; Wang, Yin; Zhang, Jinyuan; Wang, Zejian; Yin, Ming; Shen, Xudong

    2011-01-01

    Background Irradiation commonly causes long-term bone marrow injury charactertized by defective HSC self-renewal and a decrease in HSC reserve. However, the effect of high-dose IR on global gene expression during bone marrow recovery remains unknown. Methodology Microarray analysis was used to identify differentially expressed genes that are likely to be critical for bone marrow recovery. Multiple bioinformatics analyses were conducted to identify key hub genes, pathways and biological processes. Principal Findings 1) We identified 1302 differentially expressed genes in murine bone marrow at 3, 7, 11 and 21 days after irradiation. Eleven of these genes are known to be HSC self-renewal associated genes, including Adipoq, Ccl3, Ccnd1, Ccnd2, Cdkn1a, Cxcl12, Junb, Pten, Tal1, Thy1 and Tnf; 2) These 1302 differentially expressed genes function in multiple biological processes of immunity, including hematopoiesis and response to stimuli, and cellular processes including cell proliferation, differentiation, adhesion and signaling; 3) Dynamic Gene Network analysis identified a subgroup of 25 core genes that participate in immune response, regulation of transcription and nucleosome assembly; 4) A comparison of our data with known irradiation-related genes extracted from literature showed 42 genes that matched the results of our microarray analysis, thus demonstrated consistency between studies; 5) Protein-protein interaction network and pathway analyses indicated several essential protein-protein interactions and signaling pathways, including focal adhesion and several immune-related signaling pathways. Conclusions Comparisons to other gene array datasets indicate that global gene expression profiles of irradiation damaged bone marrow show significant differences between injury and recovery phases. Our data suggest that immune response (including hematopoiesis) can be considered as a critical biological process in bone marrow recovery. Several critical hub genes that are

  16. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    NASA Astrophysics Data System (ADS)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  17. [123I]Iodooctyl fenbufen amide as a SPECT tracer for imaging tumors that over-express COX enzymes.

    PubMed

    Huang, Ho-Lien; Yeh, Chun-Nan; Lee, Wei-Yuan; Huang, Ying-Cheng; Chang, Kang-Wei; Lin, Kun-Ju; Tien, Shu-Fan; Su, Wen-Chin; Yang, Ching-Hsiuan; Chen, Jenn-Tzong; Lin, Wuu-Jyh; Fan, Shio-Shio; Yu, Chung-Shan

    2013-04-01

    This study is concerned with the development of an agent for single photon emission computer tomography (SPECT) for imaging inflammation and tumor progression. [(123)I]Iodooctyl fenbufen amide ([(123)I]IOFA) was prepared from the precursor N-octyl-4-oxo-4-(4'-(trimethylstannyl)biphenyl-4-yl)butanamide with a radiochemical yield of 15%, specific activity of 37 GBq/μmol, and radiochemical purity of 95%. Analysis of the binding of [(123)I]IOFA to COX-1 and COX-2 enzymes by using HPLC and a gel filtration column showed a selectivity ratio of 1:1.3. An assay for the competitive inhibition of substrate transfer showed that IOFA exhibited a comparable IC(50) value compared to fenbufen. In the normal rat liver, a lower level and homogeneous pattern of [(123)I]IOFA radioactivity was observed by SPECT. In contrast, in the rat liver with thioacetamide-induced cholangiocarcinoma, a higher uptake and heterogeneous pattern of [(123)I]IOFA radioactivity was seen as hot spots in tumor lesions by SPECT imaging. Importantly, elevated COX-1 and COX-2 expressions from immunostaining were found in the bile ducts of tumor rats but not of normal rats. Therefore, [(123)I]IOFA was found to exhibit the potential for imaging tumors that over-express COX.

  18. Insulin over expression induces heart abnormalities via reactive oxygen species regulation, might be step towards cardiac hypertrophy.

    PubMed

    Mushtaq, S; Ali, T; Gul, M; Javed, Q; Emanueli, C; Murtaza, I

    2015-01-01

    Insulin is known to regulate blood—glucose level and promote its utilization as an energy source in cardiac tissues under normal physiological conditions as well as stimulates signaling pathways that involved cell growth and proliferation. Although recently insulin generated free radicals via NAD(P)H has been documented but the molecular mechanism is still under investigation. The aim of present study is to elucidate the reactive oxygen species (ROS) dependent possible role of insulin in cardiac abnormalities, including hypertrophy by regulation of antioxidants enzyme (SOD) activity. In the current study, 60 cardiac patients and 50 healthy individuals as well as the rat model with insulin administration were under investigation. Oxidant, anti—oxidant biochemical assays, hypertrophic marker expression via immunobloting and histopathology were performed. We observed statistically significant elevation of the reactive oxygen species level in the serum of patients as well as in the insulin administrated rat model, a mild expression of cardiac marker in experimental models along with abnormal histopathology of hearts. However, super oxide dismutase free radical scavenger activity was down regulated upon insulin treatment compared to control rats. Conclusively, the present study showed that over expression of insulin might stimulate cardiac hypertrophic signal via up regulation of free radicals and down regulation of antioxidants enzymes including SOD activity.

  19. Rapid purification of the over-expressed membrane 3 β-hydroxysteroid dehydrogenase in the presence of detergents

    NASA Astrophysics Data System (ADS)

    Zhou, Ming; Breton, Rock; Azzi, Arezki; Lin, Sheng-Xiang

    1996-10-01

    Three-beta hydroxysteroid dehydrogenase / Δ 5-Δ 4 isomerase catalyses a key step in the transformation of all 5-prognen-3β-ol and 5-androsten-3β-ol steroids into the corresponding Δ 4-3-keto-steroids. Human type I 3β-HSD can be found in the subcellular fractions of mitochondria and microsome. A 1.5 kbp cDNA encoding human type I 3β-HSD was inserted into the transfer vector pBlueBac to form plasmid pBB / 3β-HSD. The recombinant baculovirus was obtained by co-transfection of wild type AcNPV genomic DNA and PBB / 3β-HSD in Sf9 cells, then used to infect Sf9 cells to over-express human 3β-HSD protein. The 3β-HSD sample was purified to homogeneity by a rapid procedure, consisting of an anion-exchange and an adsorbance chromatographies, based on FPLC and some detergents application. The whole process was successful with a purification rate of 90 fold and a high recovery (70%). The kinetic study showed a Vmax of 500 nmol/min · mg and a Km of 2.8 μM, being much more active than those reported.

  20. AHR Over-Expression in Papillary Thyroid Carcinoma: Clinical and Molecular Assessments in a Series of Italian Acromegalic Patients with a Long-Term Follow-Up

    PubMed Central

    Mian, Caterina; Ceccato, Filippo; Barollo, Susi; Watutantrige-Fernando, Sara; Albiger, Nora; Regazzo, Daniela; de Lazzari, Paola; Pennelli, Gianmaria; Rotondi, Sandra; Nacamulli, Davide; Pelizzo, Maria Rosa; Jaffrain-Rea, Marie-Lise; Grimaldi, Franco; Occhi, Gianluca; Scaroni, Carla

    2014-01-01

    Aim Acromegaly reportedly carries an increased risk of malignant and benign thyroid tumors, with a prevalence of thyroid cancer of around 3–7%. Germline mutations in the aryl-hydrocarbon receptor (AHR) interacting protein (AIP) have been identified in familial forms of acromegaly. The molecular and endocrine relationships between follicular thyroid growth and GH-secreting pituitary adenoma have yet to be fully established. Our aim was to study the prevalence of differentiated thyroid cancer (DTC) in acromegaly, focusing on the role of genetic events responsible for the onset of thyroid cancer. Methods Germline mutations in the AIP gene were assessed in all patients; BRAF and H-N-K RAS status was analyzed by direct sequencing in thyroid specimens, while immunohistochemistry was used to analyze the protein expression of AIP and AHR. A set of PTCs unrelated to acromegaly was also studied. Results 12 DTCs (10 papillary and 2 follicular carcinomas) were identified in a cohort of 113 acromegalic patients. No differences in GH/IGF-1 levels or disease activity emerged between patients with and without DTC, but the former were older and more often female. BRAF V600E was found in 70% of the papillary thyroid cancers; there were no RAS mutations. AIP protein expression was similar in neoplastic and normal cells, while AHR protein was expressed more in PTCs carrying BRAF mutations than in normal tissue, irrespective of acromegaly status. Conclusions The prevalence of DTC in acromegaly is around 11% and endocrinologists should bear this in mind, especially when examining elderly female patients with uninodular goiter. The DTC risk does not seem to correlate with GH/IGF-1 levels, while it may be associated with BRAF mutations and AHR over-expression. Genetic or epigenetic events probably play a part in promoting thyroid carcinoma. PMID:25019383

  1. Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR).

    PubMed Central

    Diraison, Frédérique; Parton, Laura; Ferré, Pascal; Foufelle, Fabienne; Briscoe, Celia P; Leclerc, Isabelle; Rutter, Guy A

    2004-01-01

    Accumulation of intracellular lipid by pancreatic islet beta-cells has been proposed to inhibit normal glucose-regulated insulin secretion ('glucolipotoxicity'). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for beta-cells at the islet periphery. Real-time PCR (TaqMan) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-gamma (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of beta-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes. PMID:14690455

  2. Over-expression of MAGED4B increases cell migration and growth in oral squamous cell carcinoma and is associated with poor disease outcome.

    PubMed

    Chong, Chan Eng; Lim, Kue Peng; Gan, Chai Phei; Marsh, Christina A; Zain, Rosnah Binti; Abraham, Mannil Thomas; Prime, Stephen S; Teo, Soo-Hwang; Silvio Gutkind, J; Patel, Vyomesh; Cheong, Sok Ching

    2012-08-01

    MAGE proteins have been shown to be good targets for cancer immunotherapy. We demonstrate that MAGED4B is over-expressed in more than 50% of Oral Squamous Cell Carcinoma (OSCC) tissues and the expression of MAGED4B is associated with lymph node metastasis and poor disease specific survival. OSCC cell lines that over-express MAGED4B promote migration in vitro, exhibit an increase in cell growth both in vitro and in vivo, and are more resistant to apoptosis compared to control cells. Our data suggest that MAGED4B over-expression is a driver in oral carcinogenesis and argues strongly that this protein may represent a potential therapeutic target in OSCC. PMID:22459352

  3. miR-155 Over-expression Promotes Genomic Instability by Reducing High-fidelity Polymerase Delta Expression and Activating Error-prone DSB Repair

    PubMed Central

    Czochor, Jennifer R.; Sulkowski, Parker; Glazer, Peter M.

    2016-01-01

    miR-155 is an oncogenic microRNA (miR) that is often over-expressed in cancer and is associated with poor prognosis. miR-155 can target several DNA repair factors including RAD51, MLH1, and MSH6, and its over-expression results in an increased mutation frequency in vitro, although the mechanism has yet to be fully understood. Here, we demonstrate that over-expression of miR-155 drives an increased mutation frequency both in vitro and in vivo, promoting genomic instability by affecting multiple DNA repair pathways. miR-155 over-expression causes a decrease in homologous recombination, but yields a concurrent increase in the error-prone non-homologous end-joining (NHEJ) pathway. Despite repressing established targets MLH1 and MSH6, the identified mutation pattern upon miR-155 over-expression does not resemble that of a mismatch repair-deficient background. Further investigation revealed that all four subunits of polymerase delta, a high-fidelity DNA replication and repair polymerase, are down-regulated at the mRNA level in the context of miR-155 over-expression. FOXO3a, a transcription factor and known target of miR-155, has one or more putative binding site(s) in the promoter of all four polymerase delta subunits. Finally, suppression of FOXO3a by miR-155 or by siRNA knockdown is sufficient to repress the expression of the catalytic subunit of polymerase delta, POLD1, at the protein level, indicating that FOXO3a contributes to the regulation of polymerase delta levels. PMID:26850462

  4. Hypoxia-inducible factor-2 alpha promotes the proliferation of human placenta-derived mesenchymal stem cells through the MAPK/ERK signaling pathway

    PubMed Central

    Zhu, Chengxing; Yu, Jiong; Pan, Qiaoling; Yang, Jinfeng; Hao, Guangshu; Wang, Yingjie; Li, Lanjuan; Cao, Hongcui

    2016-01-01

    Human placenta-derived mesenchymal stem cells (hPMSCs) reside in a physiologically low-oxygen microenvironment. Hypoxia influences a variety of stem cell cellular activities, frequently involving hypoxia-inducible factor-2 alpha (HIF-2α). This research showed that hPMSCs cultured in hypoxic conditions (5% O2) exhibited a more naïve morphology and had a higher proliferative capability and higher HIF-2α expression than hPMSCs cultured in normoxic conditions (21% O2). Similar to the hypoxic cultures, hPMSCs over-expressing HIF-2α showed higher proliferative potential and higher expression of CCND1 (CyclinD1), MYC (c-Myc), POU5F1 (Oct4) and the components of the MAPK/ERK pathway. In contrast, these genes were down-regulated in the HIF-2α-silenced hPMSCs. After adding the MAPK/ERK inhibitor PD0325901, cell growth and the expression of CCND1 and MYC were inhibited. Furthermore, the chromatin immunoprecipitation (ChIP) assay and electrophoretic mobility shift assay (EMSA) showed that HIF-2α bound to the MAPK3 (ERK1) promoter, indicative of its direct regulation of MAPK/ERK components at the transcriptional level during hPMSC expansion. Taken together, our results suggest that HIF-2α facilitated the preservation of hPMSC stemness and promoted their proliferation by regulating CCND1 and MYC through the MAPK/ERK signaling pathway. PMID:27765951

  5. Gene expression profiles in liver of mouse after chronic exposure to drinking water.

    PubMed

    Wu, Bing; Zhang, Yan; Zhao, Dayong; Zhang, Xuxiang; Kong, Zhiming; Cheng, Shupei

    2009-10-01

    cDNA micorarray approach was applied to hepatic transcriptional profile analysis in male mouse (Mus musculus, ICR) to assess the potential health effects of drinking water in Nanjing, China. Mice were treated with continuous exposure to drinking water for 90 days. Hepatic gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 arrays, and pathway analysis was carried out by Molecule Annotation System 2.0 and KEGG pathway database. A total of 836 genes were found to be significantly altered (1.5-fold, P < or = 0.05), including 294 up-regulated genes and 542 down-regulated genes. According to biological pathway analysis, drinking water exposure resulted in aberration of gene expression and biological pathways linked to xenobiotic metabolism, signal transduction, cell cycle and oxidative stress response. Further, deregulation of several genes associated with carcinogenesis or tumor progression including Ccnd1, Egfr, Map2k3, Mcm2, Orc2l and Smad2 was observed. Although transcription changes in identified genes are unlikely to be used as a sole indicator of adverse health effects, the results of this study could enhance our understanding of early toxic effects of drinking water exposure and support future studies on drinking water safety.

  6. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses.

    PubMed

    Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

    2015-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance. PMID:26858694

  7. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses

    PubMed Central

    Goltermann, Lise; Sarusie, Menachem V.; Bentin, Thomas

    2016-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance. PMID:26858694

  8. Chaperonin GroEL/GroES Over-Expression Promotes Aminoglycoside Resistance and Reduces Drug Susceptibilities in Escherichia coli Following Exposure to Sublethal Aminoglycoside Doses.

    PubMed

    Goltermann, Lise; Sarusie, Menachem V; Bentin, Thomas

    2015-01-01

    Antibiotic resistance is an increasing challenge to modern healthcare. Aminoglycoside antibiotics cause translation corruption and protein misfolding and aggregation in Escherichia coli. We previously showed that chaperonin GroEL/GroES depletion and over-expression sensitize and promote short-term tolerance, respectively, to this drug class. Here, we show that chaperonin GroEL/GroES over-expression accelerates acquisition of streptomycin resistance and reduces susceptibility to several other antibiotics following sub-lethal streptomycin antibiotic exposure. Chaperonin buffering could provide a novel mechanism for emergence of antibiotic resistance.

  9. Differential roles of cyclin D1 and D3 in pancreatic ductal adenocarcinoma

    PubMed Central

    2010-01-01

    Background The cyclin D1 (CCND1) and cyclin D3 (CCND3) are frequently co-overexpressed in pancreatic ductal adenocarcinoma (PDAC). Here we examine their differential roles in PDAC. Results CCND1 and CCND3 expression were selectively suppressed by shRNA in PDAC cell lines with expression levels of equal CCND1 and CCND3 (BxPC3), enhanced CCND1 (HPAC) or enhanced CCND3 (PANC1). Suppression of cell proliferation was greater with CCND3 than CCND1 downregulation. CCND3 suppression led to a reduced level of phosphorylated retinoblastoma protein (Ser795p-Rb/p110) and resulted in decreased levels of cyclin A mRNA and protein. A global gene expression analysis identified deregulated genes in D1- or D3-cyclin siRNA-treated PANC1 cells. The downregulated gene targets in CCND3 suppressed cells were significantly enriched in cell cycle associated processes (p < 0.005). In contrast, focal adhesion/actin cytoskeleton, MAPK and NF B signaling appeared to characterize the target genes and their interacting proteins in CCND1 suppressed PANC1 cells. Conclusions Our results suggest that CCND3 is the primary driver of the cell cycle, in cooperation with CCND1 that integrates extracellular mitogenic signaling. We also present evidence that CCND1 plays a role in tumor cell migration. The results provide novel insights for common and differential targets of CCND1 and CCND3 overexpression during pancreatic duct cell carcinogenesis. PMID:20113529

  10. Improved immunogenicity of a H44/76 group B outer membrane vesicle vaccine with over-expressed genome-derived Neisserial antigen 1870.

    PubMed

    Koeberling, Oliver; Welsch, Jo Anne; Granoff, Dan M

    2007-02-26

    A broadly protective vaccine against meningococcal group B disease is not available. We previously reported that an outer membrane vesicle (OMV) vaccine containing over-expressed genome-derived antigen (GNA) 1870 elicited broader protective antibody responses than recombinant GNA1870 or conventional OMV vaccines prepared from a strain that naturally expresses low amounts of GNA1870. Certain wildtype strains such as H44/76 naturally express larger amounts of GNA1870 and, potentially, could be used to prepare an improved OMV vaccine without genetic over-expression of the antigen. We transformed H44/76 with a shuttle vector to over-express variant 1 (v.1) GNA1870 and compared the immunogenicity in mice of OMV vaccines prepared from wildtype H44/76 (v.1), the mutant, and a recombinant v.1 GNA1870 vaccine. Mice immunized with OMV with over-expressed GNA1870 developed broader serum bactericidal and/or greater C3 deposition activity on the surface of encapsulated strains of N. meningitidis than control mice immunized with the OMV vaccine prepared from the wildtype strain, or the rGNA1870 vaccine. When a panel of group B strains from patients in California was tested, sera from mice immunized with the OMV vaccine containing over-expressed GNA1870 were bactericidal against 100% of the v.1 strains. In contrast, only 20% of isolates that expressed subvariants of the v.1 GNA1870 protein were susceptible to bactericidal activity of antibodies elicited by the rGNA1870 or conventional OMV vaccines. Thus, even a modest increase in GNA1870 expression in a strain that naturally is a high producer of GNA1870 results in an OMV vaccine that elicits broader protection against meningococcal disease.

  11. Expression profiling of cell cycle genes in human pancreatic islets with and without type 2 diabetes.

    PubMed

    Taneera, Jalal; Fadista, Joao; Ahlqvist, Emma; Zhang, Mengze; Wierup, Nils; Renström, Erik; Groop, Leif

    2013-08-15

    Microarray gene expression data were used to analyze the expression pattern of cyclin, cyclin-dependent kinase (CDKs) and cyclin-dependent kinase inhibitor (CDKIs) genes from human pancreatic islets with and without type 2 diabetes (T2D). Of the cyclin genes, CCNI was the most expressed. Data obtained from microarray and qRT-PCR showed higher expression of CCND1 in diabetic islets. Among the CDKs, CDK4, CDK8 and CDK9 were highly expressed, while CDK1 was expressed at low level. High expression of CDK18 was observed in diabetic islets. Of the CDKIs, CDKN1A expression was higher in diabetic islets in both microarray and qRT-PCR. Expression of CDKN1A, CDKN2A, CCNI2, CDK3 and CDK16 was correlated with age. Finally, eight SNPs in these genes were associated with T2D in the DIAGRAM database. Our data provide a comprehensive expression pattern of cell cycle genes in human islets. More human studies are required to confirm and reproduce animal studies. PMID:23707792

  12. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

    PubMed

    Yang, Yi-Lin; Serrano, Myrna G; Sheoran, Abhineet S; Manque, Patricio A; Buck, Gregory A; Widmer, Giovanni

    2009-11-01

    The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

  13. Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells

    PubMed Central

    Yang, Yi-Lin; Serrano, Myrna G.; Sheoran, Abhineet S.; Manque, Patricio A.; Buck, Gregory A.; Widmer, Giovanni

    2009-01-01

    The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously. PMID:19631240

  14. Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

    PubMed

    George, Suja; Parida, Ajay

    2011-03-01

    Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

  15. PAI-1 over-expression decreases experimental post-thrombotic vein wall fibrosis by a non-vitronectin dependent mechanism

    PubMed Central

    Obi, Andrea T.; Diaz, Jose A.; Ballard-Lipka, Nicole L.; Roelofs, Karen J.; Farris, Diana M.; Lawrence, Daniel A.; Wakefield, Thomas W.; Henke, Peter K.

    2014-01-01

    SUMMARY Background Factors associated with post-thrombotic syndrome are known clinically, but the underlying cellular processes at the vein wall are not well-delineated. Prior work suggests that vein wall damage does not correlate with thrombus resolution, but rather with plasminogen activator-1 (PAI-1) and matrix metalloproteinase (MMP) activity. Objective We hypothesized that PAI-1 would confer post venous thrombosis (VT) vein wall protection via a Vitronectin (Vn) dependent mechanism. Methods A stasis model of VT was used with harvest over 2 weeks, in wild type (WT), Vn−/−, and PAI-1 overexpressing mice (PAI-1 Tg). Results PAI-1 Tg mice had larger VT at 6 and 14 days, compared to controls, but Vn−/−mice had no alteration of VT resolution. Gene deletion of Vn resulted in increased, rather than expected decrease in circulating PAI-1 activity. While both Vn−/− and PAI-1 Tg had attenuated intimal fibrosis, PAI-1 Tg had significantly less vein wall collagen and a compensatory increase in collagen III gene expression. Both Vn−/− and PAI-1 Tg vein wall had less monocyte chemotactic factor-1, and fewer macrophages (F4/80), with significantly less MMP-2 activity and decreased TIMP-1 antigen. Ex vivo assessment of TGFβ mediated fibrotic response showed that PAI-1 Tg vein walls had increased profibrotic gene expression (collagen I, III, MMP-2 and α-SMA) as compared with controls, opposite of the in vivo response. Conclusions The absence of Vn increases circulating PAI-1, which positively modulates vein wall fibrosis in a dose-dependent manner. Translationally, PAI-1 elevation may decrease vein wall damage after DVT, perhaps by decreasing macrophage-mediated activities. PMID:24943740

  16. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants.

    PubMed

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell-cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  17. c-Myc over-expression in Ramos Burkitt's lymphoma cell line predisposes to iron homeostasis disruption in vitro

    SciTech Connect

    Habel, Marie-Eve; Jung, Daniel . E-mail: djung@hema-quebec.qc.ca

    2006-03-24

    Burkitt's lymphoma is an aggressive B-cell neoplasm resulting from deregulated c-myc expression. We have previously shown that proliferation of Burkitt's lymphoma cell lines such as Ramos is markedly reduced by iron treatment. It has been shown that iron induces expression of c-myc which, owing to its transcriptional regulatory functions, regulates genes involved in iron metabolism. Transient enhancement of c-myc expression by iron could increase the expression of genes involved in iron incorporation, which could lead to an accumulation of intracellular free iron. Here, we have investigated whether cells with a high basal level of c-Myc were more likely to accumulate free iron. Our results suggest that the basal level of c-Myc in Ramos cells is twofold higher than what is seen in HL-60 cells. Moreover, in Ramos cells, where c-Myc is expressed at a high level, H-ferritin expression is down-regulated, transferrin receptor (CD71) expression is increased, and ferritin translation is inhibited. These modifications in iron metabolism, resulting from the strong basal expression of c-Myc, and amplified by iron addition, could lead to a disruption in homeostasis and consequently to growth arrest.

  18. Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells

    PubMed Central

    2010-01-01

    Background Survivin is a dual functioning protein. It inhibits the apoptosis of cancer cells by inhibiting caspases, and also promotes cancer cell growth by stabilizing microtubules during mitosis. Since the molecular chaperone Hsp90 binds and stabilizes survivin, it is widely believed that down-regulation of survivin is one of the important therapeutic functions of Hsp90 inhibitors such as the phase III clinically trialed compound 17-AAG. However, Hsp90 interferes with a number of molecules that up-regulate the intracellular level of survivin, raising the question that clinical use of Hsp90 inhibitors may indirectly induce survivin expression and subsequently enhance cancer anti-drug responses. The purpose of this study is to determine whether targeting Hsp90 can alter survivin expression differently in different cancer cell lines and to explore possible mechanisms that cause the alteration in survivin expression. Results Here, we demonstrated that Hsp90 inhibitors, geldanamycin and 17-AAG, induced the over-expression of survivin in three different human cancer cell lines as shown by Western blotting. Increased survivin mRNA transcripts were observed in 17-AAG and geldanamycin-treated HT-29 and HONE-1 cancer cells. Interestingly, real-time PCR and translation inhibition studies revealed that survivin was over-expressed partially through the up-regulation of protein translation instead of gene transcription in A549 cancer cells. In addition, 17-AAG-treated A549, HONE-1 and HT-29 cells showed reduced proteasomal activity while inhibition of 26S proteasome activity further increased the amount of survivin protein in cells. At the functional level, down-regulation of survivin by siRNA further increased the drug sensitivity to 17-AAG in the tested cancer cell lines. Conclusions We showed for the first time that down-regulation of survivin is not a definite therapeutic function of Hsp90 inhibitors. Instead, targeting Hsp90 with small molecule inhibitors will induce the

  19. Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3

    SciTech Connect

    Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena; Indiveri, Cesare

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer mOCTN3 transport protein has been cloned in pET-21a(+) and over-expressed in Escherichia coli. Black-Right-Pointing-Pointer The expressed mOCTN3 has been purified to homogeneity by Ni-chelating chromatography. Black-Right-Pointing-Pointer The protein solubilised in Triton X-100 has been reconstituted in liposomes. Black-Right-Pointing-Pointer Recombinant mOCTN3 catalyses transport of carnitine by a uniport mode. -- Abstract: pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni{sup 2+}-chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [{sup 3}H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K{sub m} for carnitine was 36 {mu}M. [{sup 3}H]carnitine transport was inhibited by carnitine analogues and some Cys and NH{sub 2} reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.

  20. Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

    PubMed Central

    Michan, Shaday; Juan, Aimee M.; Hurst, Christian G.; Cui, Zhenghao; Evans, Lucy P.; Hatton, Colman J.; Pei, Dorothy T.; Ju, Meihua; Sinclair, David A.; Smith, Lois E. H.; Chen, Jing

    2014-01-01

    Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy. PMID:24416337

  1. Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2

    PubMed Central

    Bhatia, Varnika; Maisnam, Jaya; Jain, Ajay; Sharma, Krishan Kumar; Bhattacharya, Ramcharan

    2015-01-01

    Background and Aims Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae. Methods Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay. Key Results Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure. Conclusions Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of

  2. Cell cycle arrest mediated by a pyridopyrimidine is not abrogated by over-expression of Bcl-2 and cyclin D1.

    PubMed

    Soni, R; Chaudhuri, B

    2001-05-01

    Inhibition of cyclin dependent kinases (Cdks) is of pivotal importance in tumor cell biology as these kinases are the drivers of cell proliferation. This inhibition can be achieved either by naturally occurring biological proteins or by small molecule compounds. They cause cell cycle arrest and/or apoptosis depending upon the specificity and efficacy of the inhibitor in question. We have reported earlier that specific pyridopyrimidines (novel Cdk inhibitors) cause cell cycle arrest in mink lung epithelial cells and the arrest is abrogated by over-expression of Cdk4. In contrast, we show here that one of these inhibitors effectively maintains cell cycle arrest in a leukemic or a breast cancer cell line even after the respective cells over-express an oncogene, either Bcl-2 or cyclin D1. However, in the leukemic cells, Bcl-2 over-expression suppresses apoptosis induced by the pyridopyrimidine. Thus, novel Cdk inhibitors can prove to be useful chemical genetics tools for understanding the underlying mechanisms of growth arrest and/or apoptosis in normal versus tumor cells. This could also lead to the development of improved inhibitors of cell proliferation.

  3. Transcriptional profiling of Vero E6 cells over-expressing SARS-CoV S2 subunit: Insights on viral regulation of apoptosis and proliferation

    SciTech Connect

    Yeung, Y.-S. Yip, C.-W. Hon, C.-C. Chow, Ken Y.C. Ma, Iris C.M. Zeng Fanya Leung, Frederick C.C.

    2008-02-05

    We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by the diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.

  4. Gene expression array analyses predict increased proto-oncogene expression in MMTV induced mammary tumors.

    PubMed

    Popken-Harris, Pamela; Kirchhof, Nicole; Harrison, Ben; Harris, Lester F

    2006-08-01

    Exogenous infection by milk-borne mouse mammary tumor viruses (MMTV) typically induce mouse mammary tumors in genetically susceptible mice at a rate of 90-95% by 1 year of age. In contrast to other transforming retroviruses, MMTV acts as an insertional mutagen and under the influence of steroid hormones induces oncogenic transformation after insertion into the host genome. As these events correspond with increases in adjacent proto-oncogene transcription, we used expression array profiling to determine which commonly associated MMTV insertion site proto-oncogenes were transcriptionally active in MMTV induced mouse mammary tumors. To verify our gene expression array results we developed real-time quantitative RT-PCR assays for the common MMTV insertion site genes found in RIII/Sa mice (int-1/wnt-1, int-2/fgf-3, int-3/Notch 4, and fgf8/AIGF) as well as two genes that were consistently up regulated (CCND1, and MAT-8) and two genes that were consistently down regulated (FN1 and MAT-8) in the MMTV induced tumors as compared to normal mammary gland. Finally, each tumor was also examined histopathologically. Our expression array findings support a model whereby just one or a few common MMTV insertions into the host genome sets up a dominant cascade of events that leave a characteristic molecular signature.

  5. Next generation sequencing in synovial sarcoma reveals novel gene mutations

    PubMed Central

    Vlenterie, Myrella; Hillebrandt-Roeffen, Melissa H.S.; Flucke, Uta E.; Groenen, Patricia J.T.A.; Tops, Bastiaan B.J.; Kamping, Eveline J.; Pfundt, Rolph; de Bruijn, Diederik R.H.; van Kessel, Ad H.M. Geurts; van Krieken, Han J.H.J.M.; van der Graaf, Winette T.A.; Versleijen-Jonkers, Yvonne M.H.

    2015-01-01

    Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults. PMID:26415226

  6. Over-expression of a proline specific aminopeptidase from Aspergillus oryzae JN-412 and its application in collagen degradation.

    PubMed

    Ding, Guo-Wei; Zhou, Nan-Di; Tian, Ya-Ping

    2014-08-01

    A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg(-1) at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7% from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn2+ and Cu2+. DTT, β-mercaptoethanol, EDTA, and ions like Co2+, Mg2+, Mn2+, and Ca2+ had no influence on enzyme activity, whereas Ni2+ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.

  7. Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit.

    PubMed

    Badawi, Ghazi Hamid; Kawano, Naoyoshi; Yamauchi, Yasuo; Shimada, Emi; Sasaki, Ryozo; Kubo, Akihiro; Tanaka, Kiyoshi

    2004-06-01

    The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants. The full length cDNA, coding Arabidopsis thaliana L. APX fused downstream to the chloroplast transit sequence from A. thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin. The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization. Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants. The isolated chloroplasts from this line showed higher APX activity. During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite. The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis. The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H(2)O(2). PMID:15153190

  8. Over-expression of fHbp in Arabdopsis for development of meningococcal serogroup B subunit vaccine.

    PubMed

    Xu, Nuo; Wang, Yunpeng; Ma, Jisheng; Jin, Libo; Xing, Shaochen; Jiang, Chao; Li, Xiaokun

    2016-07-01

    Due to lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitides, the incidence of meningococcal disease remains high. To solve the issue, transgenic plants are used as bioreactors to produce a plant-derived fHbp subunit vaccine. In this study, the fHbp gene was optimized according to the codon usage bias of Arabidopsis thaliana, synthesized artificially, cloned into an expression vector, driven by a seed-specific promoter, and introduced into A. thaliana by Agrobacterium-mediated floral-dip transformation. Transgenic plants were identified by glufosinate selection, quickstix strips for PAT/bar tests and PCR analysis. The five plants showing higher expression of recombinant fHbp were screened through indirect ELISA. Southern blot analysis showed that the transgenic line rHF-22 had a single-copy integration and the highest expression of fHbp. Recombinant fHbp was purified from seeds of rHF-22 by nitrilotriacetic acid-mediated affinity chromatography, and the purity was 82.5%. BALB/c mice were tested for fHbp vaccine protection from lethal MenB infection, and the relative percent survival was found to be 80%. This study indicates that the recombinant fHbp produced from seeds of rHF-22 is a potential candidate for commercial MenB vaccine. It also provides a reference for safe, cheap and large-scale production of other plant-made vaccines. PMID:27119621

  9. The over-expression of survivin enhances the chemotherapeutic efficacy of YM155 in human hepatocellular carcinoma.

    PubMed

    Xia, Hongping; Chen, Jianxiang; Shi, Ming; Deivasigamani, Amudha; Ooi, London Lucien P J; Hui, Kam M

    2015-03-20

    Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. The inability of chemotherapeutic drugs to selectively target HCC tumor cells because of their predominant resistant phenotype to most conventional anticancer agents bestows a major obstacle for the clinical management of HCC. In this report, we have examined and demonstrated the remarkable heterogeneity of expression of survivin and its phosphorylated active form (p-survivin) in HCC patients' tissues and cell lines. Furthermore, the expression of survivin and p-survivin in HCC cell lines was found to be associated with response to the small-molecule survivin suppressant YM155. Therefore, in the HCC cell lines that express elevated level of survivin and p-survivin, YM155 efficiently inhibited their proliferation, induced cell cycle arrest and apoptosis resulting in DNA damage through the dysregulation of cell-cycle checkpoint-related regulatory genes. Importantly, YM155 yielded significantly better therapeutic effect than sorafenib when tested in an orthotopic mouse model using patient-derived HCC xenografts with elevated survivin and p-survivin expression. Our results clearly demonstrated that the level of survivin and p-survivin expression could serve as molecular predictive biomarkers to select potential YM155-responsive patients, in a move towards delivering precision medicine for HCC patients.

  10. Over-expression of ZnT7 increases insulin synthesis and secretion in pancreatic beta-cells by promoting insulin gene transcription

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanism by which zinc regulates insulin synthesis and secretion in pancreatic beta-cells is still unclear. Cellular zinc homeostasis is largely maintained by zinc transporters and intracellular zinc binding proteins. In this study, we demonstrated that zinc transporter 7 (ZnT7, Slc30a7) was co...

  11. Histone deacetylase inhibitor abexinostat affects chromatin organization and gene transcription in normal B cells and in mantle cell lymphoma.

    PubMed

    Markozashvili, Diana; Pichugin, Andrei; Barat, Ana; Camara-Clayette, Valerie; Vasilyeva, Natalia V; Lelièvre, Hélène; Kraus-Berthier, Laurence; Depil, Stéphane; Ribrag, Vincent; Vassetzky, Yegor

    2016-04-15

    Mantle cell lymphoma (MCL) is a rare lymphoma caused by the t(11:14) juxtaposing the cyclin D1 (CCND1) locus on chromosome 11 and the immunoglobulin heavy chain (IgH) locus on chromosome 14. Several new treatments are proposed for MCL, including histone deacetylase inhibitors (HDACi). We have studied gene expression and chromatin organization in the translocated 11q13 locus in MCL cells as compared to lymphoblastoid cell lines as well as the effect of HDACi abexinostat on chromatin organization and gene expression in the 11q13 locus. We have identified a cluster of genes overexpressed in the translocation region on chromosome 11 in MCL cells. Abexinostat provokes a genome-wide disaggregation of heterochromatin. The genes upregulated after the t(11;14) translocation react to the HDACi treatment by increasing their expression, but their gene promoters do not show significant alterations in H3K9Ac and H3K9me2 levels in abexinostat-treated cells.

  12. Assessment of Tumor Heterogeneity, as Evidenced by Gene Expression Profiles, Pathway Activation, and Gene Copy Number, in Patients with Multifocal Invasive Lobular Breast Tumors

    PubMed Central

    Norton, Nadine; Advani, Pooja P.; Serie, Daniel J.; Geiger, Xochiquetzal J.; Necela, Brian M.; Axenfeld, Bianca C.; Kachergus, Jennifer M.; Feathers, Ryan W.; Carr, Jennifer M.; Crook, Julia E.; Moreno-Aspitia, Alvaro; Anastasiadis, Panos Z.; Perez, Edith A.; Thompson, E. Aubrey

    2016-01-01

    Background Invasive lobular carcinoma (ILC) comprises approximately ~10–20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC. Methods We characterized gene expression and copy number in 2 or more foci from 11 patients with MF/MC ILC (all ER+, HER2-) and adjacent normal tissue. RNA and DNA were extracted from 3x1.5mm cores from all foci. Gene expression (730 genes) and copy number (80 genes) were measured using Nanostring PanCancer and Cancer CNV panels. Linear mixed models were employed to compare expression in tumor versus normal samples from the same patient, and to assess heterogeneity (variability) in expression among multiple ILC within an individual. Results 35 and 34 genes were upregulated (FC>2) and down-regulated (FC<0.5) respectively in ILC tumor relative to adjacent normal tissue, q<0.05. 9/34 down-regulated genes (FIGF, RELN, PROM1, SFRP1, MMP7, NTRK2, LAMB3, SPRY2, KIT) had changes larger than CDH1, a hallmark of ILC. Copy number changes in these patients were relatively few but consistent across foci within each patient. Amplification of three genes (CCND1, FADD, ORAOV1) at 11q13.3 was present in 2/11 patients in both foci. We observed significant evidence of within-patient between-foci variability (heterogeneity) in gene expression for 466 genes (p<0.05 with FDR 8%), including CDH1, FIGF, RELN, SFRP1, MMP7, NTRK2, LAMB3, SPRY2 and KIT. Conclusions There was substantial variation in gene expression between ILC foci within patients, including known markers of ILC, suggesting an additional level of complexity that should be addressed. PMID:27078887

  13. New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

    PubMed Central

    Saraiva-Pava, Kathy; Navabi, Nazanin; Skoog, Emma C; Lindén, Sara K; Oleastro, Mónica; Roxo-Rosa, Mónica

    2015-01-01

    AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones’ adherence properties, expression of cell-cell junctions’ markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Lex), Ley and Lea and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce

  14. Cardiac-Specific Over-Expression of Epidermal Growth Factor Receptor 2 (ErbB2) Induces Pro-Survival Pathways and Hypertrophic Cardiomyopathy in Mice

    PubMed Central

    Guo, Xin; Belmonte, Frances; Kang, Byunghak; Bedja, Djahida; Pin, Scott; Tsuchiya, Noriko; Gabrielson, Kathleen

    2012-01-01

    Background Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored. Methodology/Principal Findings We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2–3 times larger than those of control littermates, express increased atrial natriuretic peptide and β-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib. Conclusions/Significance These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a

  15. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

    SciTech Connect

    Jin, Yulan; Purohit, Sharad; Chen, Xueqin; Yi, Bing; She, Jin-Xiong

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

  16. Reversible regulation of cell cycle-related genes by epigallocatechin gallate for hibernation of neonatal human tarsal fibroblasts.

    PubMed

    Bae, Jung Yoon; Kanamune, Jun; Han, Dong-Wook; Matsumura, Kazuaki; Hyon, Suong-Hyu

    2009-01-01

    We investigated the hibernation effect of epigallocatechin-3-O-gallate (EGCG) on neonatal human tarsal fibroblasts (nHTFs) by analyzing the expression of cell cycle-related genes. EGCG application to culture media moderately inhibited the growth of nHTFs, and the removal of EGCG from culture media led to complete recovery of cell growth. EGCG resulted in a slight decrease in the cell population of the S and G(2)/M phases of cell cycle with concomitant increase in that of the G(0)/G(1) phase, but this cell cycle profile was restored to the initial level after EGCG removal. The expression of cyclin D1 (CCND1), CCNE2, CCN-dependent kinase 6 (CDK6), and CDK2 was restored, whereas that of CCNA, CCNB1, and CDK1 was irreversibly attenuated. The expression of a substantial number of genes analyzed by cDNA microarray was affected by EGCG application, and these affected expression levels were restored to the normal levels after EGCG removal. We also found the incorporation of FITC-EGCG into the cytosol of nHTFs and its further nuclear translocation, which might lead to the regulation of the exogenous signals directed to genes for cellular responses including proliferation and cell cycle progression. These results suggest that EGCG temporarily affects not only genes related to the cell cycle but also various other cellular functions.

  17. Comparative gene expression analysis of ovarian carcinoma and normal ovarian epithelium by serial analysis of gene expression.

    PubMed

    Peters, David G; Kudla, Donna M; Deloia, Julie A; Chu, Tian Jiao; Fairfull, Liane; Edwards, Robert P; Ferrell, Robert E

    2005-07-01

    Despite the poor prognosis of ovarian cancer and the importance of early diagnosis, there are no reliable noninvasive biomarkers for detection in the early stages of disease. Therefore, to identify novel ovarian cancer markers with potential utility in early-stage screening protocols, we have undertaken an unbiased and comprehensive analysis of gene expression in primary ovarian tumors and normal human ovarian surface epithelium (HOSE) using Serial Analysis of Gene Expression (SAGE). Specifically, we have generated SAGE libraries from three serous adenocarcinomas of the ovary and, using novel statistical tools, have compared these to SAGE data derived from two pools of normal HOSE. Significantly, in contrast to previous SAGE-based studies, our normal SAGE libraries are not derived from cultured cell lines. We have also compared our data with publicly available SAGE data obtained from primary tumors and "normal" HOSE-derived cell lines. We have thus identified several known and novel genes whose expressions are elevated in ovarian cancer. These include but are not limited to CLDN3, WFDC2, FOLR1, COL18A1, CCND1, and FLJ12988. Furthermore, we found marked differences in gene expression patterns in primary HOSE tissue compared with cultured HOSE. The use of HOSE tissue as a control for these experiments, along with hierarchical clustering analysis, identified several potentially novel biomarkers of ovarian cancer, including TACC3, CD9, GNAI2, AHCY, CCT3, and HMGA1. In summary, these data identify several genes whose elevated expressions have not been observed previously in ovarian cancer, confirm the validity of several existing markers, and provide a foundation for future studies in the understanding and management of this disease. PMID:16030107

  18. Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF.

    PubMed

    Edinger, Nufar; Lebendiker, Mario; Klein, Shoshana; Zigler, Maya; Langut, Yael; Levitzki, Alexander

    2016-01-01

    Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC. PMID:27598772

  19. Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

    PubMed Central

    Edinger, Nufar; Lebendiker, Mario; Klein, Shoshana; Zigler, Maya; Langut, Yael; Levitzki, Alexander

    2016-01-01

    Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC. PMID:27598772

  20. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    PubMed Central

    Maringer, Katherine; Saheb, Entsar; Bush, John

    2014-01-01

    Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA), and dominant negative (DN) forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses. PMID:25157910

  1. Over-expression of DNA-PKcs in renal cell carcinoma regulates mTORC2 activation, HIF-2α expression and cell proliferation

    PubMed Central

    Zheng, Bing; Mao, Jia-Hui; Li, Xiao-Qing; Qian, Lin; Zhu, Hua; Gu, Dong-hua; Pan, Xiao-dong

    2016-01-01

    Here, we demonstrated that DNA-PKcs is over-expressed in multiple human renal cell carcinoma (RCC) tissues and in primary/established human RCCs. Pharmacological or genetic inhibition of DNA-PKcs suppressed proliferation of RCC cells. DNA-PKcs was in the complex of mTOR and SIN1, mediating mTORC2 activation and HIF-2α expression in RCC cells. Inhibiting or silencing DNA-PKcs suppressed AKT Ser-473 phosphorylation and HIF-2α expression. In vivo, DNA-PKcs knockdown or oral administration of the DNA-PKcs inhibitor NU-7441 inhibited AKT Ser-473 phosphorylation, HIF-2α expression and 786-0 RCC xenograft growth in nude mice. We showed that miRNA-101 level was decreased in RCC tissues/cells, which could be responsible for DNA-PKcs overexpression and DNA-PKcs mediated oncogenic actions in RCC cells. We show that DNA-PKcs over-expression regulates mTORC2-AKT activation, HIF-2α expression and RCC cell proliferation. PMID:27412013

  2. Carbon nanoparticle induced cytotoxicity in human mesenchymal stem cells through upregulation of TNF3, NFKBIA and BCL2L1 genes.

    PubMed

    Periasamy, Vaiyapuri S; Athinarayanan, Jegan; Alfawaz, Mohammed A; Alshatwi, Ali A

    2016-02-01

    Carbon based nanomaterials, including carbon nanotubes, graphene, nanodiamond and carbon nanoparticles, have emerged as potential candidates for a wide variety of applications because of their unusual electrical, mechanical, thermal and optical properties. However, our understanding of how increased usage of carbon based nanomaterials could lead to harmful effects in humans and other biological systems is inadequate. Our present investigation is focused on the cellular toxicity of carbon nanoparticles (CNPs) on human mesenchymal stem cells (hMSCs). Following exposure to CNPs, cell viability, nuclear morphological changes, apoptosis and cell cycle progression were monitored. Furthermore, the expression of genes involved in both cell death (e.g., P53, TNF3, CDKN1A, TNFRSF1A, TNFSF10, NFKBIA, BCL2L1) and cell cycle regulation (e.g., PCNA, EGR1, E2F1, CCNG1, CCND1, CCNC, CYCD3) were assessed using qPCR. Our results indicated that CNPs reduce cell viability and cause chromatin condensation and DNA fragmentation. Cell cycle analysis indicated that CNPs affect the cell cycle progression. However, the gene expression measurements confirmed that CNPs significantly upregulated the P53, TNF3, CDKNIA, and NFKBIA genes and downregulated the EGR1 gene in hMSCs. Our findings suggest that CNPs reduce cell viability by disrupting the expression of cell death genes in human mesenchymal stem cell (hMSC). The results of this investigation revealed that CNPs exhibited moderate toxicity on hMSCs.

  3. Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells.

    PubMed

    Chiu, Sou-Tyau; Chang, King-Jen; Ting, Chen-Hung; Shen, Hsi-Che; Li, Hung; Hsieh, Fon-Jou

    2009-09-01

    Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc(Min/+) mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4-ephrin-A and EphB4-ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3-ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes' stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca(2+)-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear beta-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL-Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3-ephrin-B interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect

  4. 5-HT(1A)-receptor over-expressing mice: genotype and sex dependent responses to antidepressants in the forced swim-test.

    PubMed

    Günther, Lydia; Rothe, Julia; Rex, André; Voigt, Jörg-Peter; Millan, Mark J; Fink, Heidrun; Bert, Bettina

    2011-09-01

    Deficiencies in serotonergic neurotransmission are involved in the pathophysiology of depression. Due to its modulatory effect on serotonin (5-HT) release, the 5-HT(1A)-receptor is thought to play a decisive role in the therapy of this mood disorder. However, it is not fully understood how antidepressant effects are mediated by pre- and postsynaptic receptor sites. In this study we examined the impact of postsynaptic 5-HT(1A)-receptor over-expression in corticolimbic areas of male and female mice on the performance in the forced swim-test (FST). Furthermore, we investigated their response to the serotonin selective reuptake inhibitor (SSRI) citalopram in comparison to the selective noradrenaline reuptake inhibitor reboxetine, as well as the partial 5-HT(1A)-receptor agonists, buspirone and S 15535. Additionally, these drugs were evaluated in the open field-test in order to observe effects on motor activity. The density of 5-HT(1A)-receptors in discrete corticolimbic regions was determined in detail by quantitative autoradiography with [(3)H]8-OH-DPAT to investigate genotype as well as sex dependent differences in the expression pattern. [(3)H]8-OH-DPAT binding differed depending on sex with female mice of both genotypes displaying higher receptor binding in distinct brain areas. In the FST untreated male but not female over-expressing (OE) mice showed an antidepressant-like behaviour compared to wild-type (WT) mice. Citalopram yielded an antidepressant effect without influencing locomotor activity in OE mice but not in WT mice. Reboxetine had no antidepressant-like effect in OE mice, but sex-dependently in WT mice. The two partial agonists, buspirone and S 15535 produced no antidepressant-like activity in both genotypes and sexes, but aberrant motor effects. The antidepressant-like phenotype of male transgenic mice accounts for an involvement of postsynaptic 5-HT(1A)-receptors in the FST behaviour. In addition, the selective over-expression of postsynaptic 5-HT(1A

  5. [Genes associated to cancer].

    PubMed

    Peralta-Rodríguez, Raúl; Valdivia, Alejandra; Mendoza, Mónica; Rodríguez, Jade; Marrero, Daniel; Paniagua, Lucero; Romero, Pablo; Taniguchi, Keiko; Salcedo, Mauricio

    2015-01-01

    In 2010, in a cancer genes census, 291 genes were enumerated. These represent near to the 1 % of the total genes, for which there is enough biological evidence that they belong to a new genes classification, known as the cancer genes. These have been defined as the causal genes for sporadic or familiar cancer, when they mutate. The mutation types for these genes includes amplifications, point mutations, deletions, genomic rearranges, amongst others, which lead to a protein over-expression, muting, production of chimeric proteins or a de novo expression. In conjunction these genomic alterations or those of the genetic expression, when they affect specific genes which contribute to the development of cancer, are denominated as cancer genes. It is possible that the list of these alterations will grow longer due to new strategies being developed, for example, the genomic analysis.

  6. Transgenic over-expression of slit2 enhances disruption of blood-brain barrier and increases cell death after traumatic brain injury in mice.

    PubMed

    Li, Shuai; Li, Hang; He, Xiao-Fei; Li, Ge; Zhang, Qun; Liang, Feng-Ying; Jia, Huan-Huan; Li, Jiang-Chao; Huang, Ren; Pei, Zhong; Wang, Li-Jing; Zhang, Yu

    2016-09-19

    Traumatic brain injury (TBI) is the leading cause of mortality and disability among male adolescents and young adults; and mild traumatic brain injury is the most common type of traumatic brain injury. The disruption of blood-brain barrier (BBB) plays an important role in brain trauma. Previously, we have found that slit2, a member of slit protein family, increases permeability of BBB. In the present study, we examined the role of slit2 in the pathogenesis of mild TBI in a mouse model of micro TBI. Rhodamine BandPI (PropidiumIodide) staining were used to detect the permeability of BBB and cell death, respectively. The leakage of Rhodamine B and cell death were significantly increased in Slit2-Tg mice than in C57 control mice after micro TBI. The present results suggest that over expression of slit2 plays a detrimental role in the pathophysiology of mild TBI.

  7. Leaf-disc assay based on transient over-expression in Nicotiana benthamiana to allow functional screening of candidate effectors from aphids.

    PubMed

    Rodriguez, Patricia A; Hogenhout, Saskia A; Bos, Jorunn I B

    2014-01-01

    Aphids, like plant pathogens, are known to form close associations with their host. While probing and feeding, these insects deliver effectors inside the host, which are thought to be involved in suppression of host defenses and/or the release of nutrients. With increasing availability of aphid genome and transcriptome sequencing data, effectors can now be identified using bioinformatics- and proteomics-based approaches. The next step is then to apply functional assays relevant to plant-aphid interactions to identify effector activities. This chapter describes an effective and medium-throughput screen for the identification of effectors that affect aphid fecundity upon in planta over-expression. This assay will allow the identification of aphid effectors with a role in aphid virulence and can be adapted to other plant species amenable to agroinfiltration as well as to other assays based on transient expression, such as RNAi.

  8. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

    PubMed

    Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2014-08-01

    The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage.

  9. Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system.

    PubMed

    Barroso-Chinea, Pedro; Cruz-Muros, Ignacio; Afonso-Oramas, Domingo; Castro-Hernández, Javier; Salas-Hernández, Josmar; Chtarto, Abdelwahed; Luis-Ravelo, Diego; Humbert-Claude, Marie; Tenenbaum, Liliane; González-Hernández, Tomás

    2016-04-01

    The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT-α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein-protein interactions without interfering with DA synthesis. PMID:26777664

  10. Over-expression of HSPA12B protects mice against myocardium ischemic/reperfusion injury through a PPARγ-dependent PI3K/Akt/eNOS pathway

    PubMed Central

    Sun, Yanjun; Ye, Lincai; Jiang, Chuan; Jiang, Jun; Hong, Haifa; Qiu, Lisheng

    2015-01-01

    Acute myocardial ischemia/reperfusion (MIR) injury leads to severe arrhythmias and a high lethality. We aim to determine the effect of heat shock protein A12B (HSPA12B), a newly discovered member of the Hsp70 family, on heart injury parameters following MIR surgery. We used HSPA12B transgenic mice to determine its effects on heart function parameters, infarct size and cellular apoptosis following MIR surgery. Proinflammatory cytokines, oxidative products and anti-oxidative enzymes in the myocardium were measured to evaluate the anti-inflammatory and anti-oxidative effects of HSPA12B over-expression. The role of PPARs/eNOS/PI3k/Akt pathway was investigated using their inhibitors. The alteration of hemodynamic parameters, histopathological, apoptotic and infarct size caused by MIR was greatly attenuated in HSPA12B over-expressed mice. HSPA12B also greatly mitigated the inflammatory response, demonstrated by the decrease in the levels of IL-1β, IL-6, TNF-a and MPO. Anti-oxidative enzymes (SOD, Catalase and GPx) were restored by HSPA12B; oxidative products (8-OHdG, MDA and protein carbonyl) were decreased. HSPA12B activated the PPARγ-dependent eNOS/PI3k/Akt pathway, and the influence of HSPA12B on cardiac function was reversed by the inhibitors of eNOS, PPARγ, Akt and PI3K. Our results present a novel signaling mechanism that HSPA12B protects MIR injury through a PPARγ-dependent PI3K/Akt/eNOS pathway. PMID:26885270

  11. Insertion (8;11) in a renal oncocytoma with multifocal transformation to chromophobe renal cell carcinoma.

    PubMed

    Salido, Marta; Lloreta, Josep; Melero, Carme; García, Mar; Placer, José; Espinet, Blanca; Villa, Olaya; Bielsa, Oscar; Gelabert-Mas, Antoni; Serrano, Sergi; Solé, Francesc

    2005-12-01

    We report the case of a 43-year-old male with multiple tumor foci showing microscopic features of chromophobe renal carcinoma (ChRCC) arising in an oncocytoma. Conventional cytogenetics of fresh tumor cells and fluorescence in situ hybridization (FISH) revealed the following abnormal karyotype: 46,XY,der(8)ins(8;11)(p?;q13),der(11)ins(8;11)inv(11)(q12?p15) with CCND1 (11q13) rearrangement. To our knowledge, chromosome 8 has not been reported as a partner involved in structural rearrangements of 11q13 in oncocytomas. FISH in paraffin tissue sections revealed a rearrangement of CCND1 (11q13) in the oncocytoma cells. The multiple foci of chromophobe carcinoma presented multiple copies of CCND1, suggesting that they represented a transformation from oncocytoma into ChRCC. There was immunohistochemical overexpression of CCND1 in both oncocytoma and chromophobe carcinoma cells. In this case, the correlation of the microscopic findings with changes in CCND1 gene associated to CCND1 overexpression in both components suggest that the ChRCC would have originated from the preexisting oncocytoma. It is not possible to detect, by cytogenetic techniques alone, if the ChRCC component have also the CCND1 rearrangement in addition to the detected polysomy. FISH techniques on paraffin tissue sections may help to identify genetic aberrations such as CCND1 rearrangement in order to establish a diagnosis of oncocytoma.

  12. Radiation-induced, cell cycle-related gene expression in aging hematopoietic stem cells: enigma of their recovery.

    PubMed

    Hirabayashi, Yoko

    2014-03-01

    This paper reviews quantitative and qualitative studies conducted to identify changes in the characteristics of hematopoietic stem/progenitor cells (HSCs/HPCs) with or without radiation exposure. The numerical recovery of HSCs/HPCs after radiation exposure is lower than for other types of cells, an effect that may depend on hierarchical ordering of generation age during blood cell differentiation, from primitive HSCs to various differentiated HPCs. Studies are in progress to evaluate gene expression in bone marrow cells and cells in the lineage-negative, c-Kit(+), stem cell antigen(+) (LKS) fraction from 21-month-old mice, with or without radiation exposure. Preliminary data suggest that cell cycle-related genes, that is, cyclin D1 (Ccnd1), phosphatidylinositol 3 kinase regulatory subunit polypeptide 1 (PiK3r1), and Fyn, are upregulated solely in the LKS fraction from 21-month-old mice irradiated at 6 weeks of age, compared with the LKS fraction from age-matched nonirradiated control mice. Additional studies may provide evidence that the aging phenotype is exaggerated following exposure to ionizing radiation, specifically in the LKS fraction.

  13. Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells.

    PubMed

    Zhan, Fenghuang; Hardin, Johanna; Kordsmeier, Bob; Bumm, Klaus; Zheng, Mingzhong; Tian, Erming; Sanderson, Ralph; Yang, Yang; Wilson, Carla; Zangari, Maurizio; Anaissie, Elias; Morris, Christopher; Muwalla, Firas; van Rhee, Frits; Fassas, Athanasios; Crowley, John; Tricot, Guido; Barlogie, Bart; Shaughnessy, John

    2002-03-01

    Bone marrow plasma cells (PCs) from 74 patients with newly diagnosed multiple myeloma (MM), 5 with monoclonal gammopathy of undetermined significance (MGUS), and 31 healthy volunteers (normal PCs) were purified by CD138(+) selection. Gene expression of purified PCs and 7 MM cell lines were profiled using high-density oligonucleotide microarrays interrogating about 6800 genes. On hierarchical clustering analysis, normal and MM PCs were differentiated and 4 distinct subgroups of MM (MM1, MM2, MM3, and MM4) were identified. The expression pattern of MM1 was similar to normal PCs and MGUS, whereas MM4 was similar to MM cell lines. Clinical parameters linked to poor prognosis, abnormal karyotype (P =.002) and high serum beta(2)-microglobulin levels (P =.0005), were most prevalent in MM4. Also, genes involved in DNA metabolism and cell cycle control were overexpressed in a comparison of MM1 and MM4. In addition, using chi(2) and Wilcoxon rank sum tests, 120 novel candidate disease genes were identified that discriminate normal and malignant PCs (P <.0001); many are involved in adhesion, apoptosis, cell cycle, drug resistance, growth arrest, oncogenesis, signaling, and transcription. A total of 156 genes, including FGFR3 and CCND1, exhibited highly elevated ("spiked") expression in at least 4 of the 74 MM cases (range, 4-25 spikes). Elevated expression of these 2 genes was caused by the translocation t(4;14)(p16;q32) or t(11;14)(q13;q32). Thus, novel candidate MM disease genes have been identified using gene expression profiling and this profiling has led to the development of a gene-based classification system for MM.

  14. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance.

    PubMed

    Kim, Jinhee; Kang, Won-Hee; Hwang, Jeena; Yang, Hee-Bum; Dosun, Kim; Oh, Chang-Sik; Kang, Byoung-Cheorl

    2014-08-01

    The protein-protein interaction between VPg (viral protein genome-linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad-spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge-based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap-binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap-binding pockets, and mutated. Yeast two-hybrid assay and co-immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E-knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild-type were over-expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T1 and T2 transformants demonstrated that the over-expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge-based approaches for the engineering of broad-spectrum resistance in Chinese cabbage. PMID:24417952

  15. Over-expression of astrocytic ET-1 attenuates neuropathic pain by inhibition of ERK1/2 and Akt(s) via activation of ETA receptor.

    PubMed

    Hung, Victor K L; Tai, Lydia W; Qiu, Qiu; Luo, Xin; Wong, K L; Chung, Sookja K; Cheung, C W

    2014-05-01

    A differential role of endothelin-1 (ET-1) in pain processing has recently been suggested. However, the function of central ET-1 in neuropathic pain (NP) has not been fully elucidated to date. We report here the action of endogenous central ET-1 in sciatic nerve ligation-induced NP (SNL-NP) in a transgenic animal model that over-expresses ET-1 in the astrocytes (GET-1 mice). We hypothesized that the over-expression of astrocytic ET-1 would exert anti-allodynic and anti-hyperalgesic effects in NP, as demonstrated by mechanical threshold and plantar withdrawal latency using the von Frey filament and heat stimuli. In our animal model, GET-1 mice showed an increase in the withdrawal threshold and latency in response to the mechanical and thermal stimuli, respectively, in pain behavior tests after SNL. ET-1 and endothelin type A receptor (ETA-R) levels were increased significantly in L4-L6 segments of the spinal cord (ipsilateral to SNL) of GET-1 mice at 7 and 21days after surgery. Moreover, intrathecal administration of a specific ETA-R antagonist, BQ-123, attenuated the anti-allodynic and anti-hyperalgesic phenotype in GET-1 mice. The effects of BQ-123 on the mRNA expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase B/serine protein kinase (Akt(s)) were assessed in the ipsilateral L4-L6 segments harvested 30min after BQ-123 administration on day 7 after surgery. Phosphorylation of ERK1/2 and Akt(s) in the ipsilateral spinal cord of GET-1 mice was reduced following SNL, whereas no reduction was observed after intrathecal injection of BQ-123. In conclusion, our results showed that the xover-expression of astrocytic ET-1 reduced SNL-induced allodynia and hyperalgesia by inhibiting the activation of ERK1/2 and Akt(s) via the ETA-R-mediated pathway.

  16. Oncogene mRNA Imaging with Radionuclide-PNA-Peptides

    SciTech Connect

    Wickstrom, Eric

    2008-03-19

    New cancer gene hybridization probes to carry radionuclides were made. Noninvasive technetium-99m gamma imaging of CCND1 cancer gene activity in human breast cancer tumors in mice was demonstrated, followed by noninvasive technetium-99m imaging of MYC cancer gene activity. Noninvasive imaging of CCND1 cancer gene activity in human breast cancer tumors in mice was demonstrated with a positron-emitting copper-64 probe, followed by noninvasive positron imaging of IRS1 cancer gene activity.

  17. c-CBL E3 Ubiquitin Ligase is Over-Expressed in Cutaneous T-Cell Lymphoma: Its Inhibition Promotes Activation Induced Cell Death

    PubMed Central

    Wu, Jianqiang; Salva, Katrin A.; Wood, Gary S.

    2014-01-01

    Mycosis fungoides (MF) and Sezary syndrome (SS) are two major forms of cutaneous T-cell lymphoma (CTCL) characterized by resistance to apoptosis. A central pathway for T-cell apoptosis is activation-induced cell death (AICD) which is triggered through the T-cell receptor (TCR). This results in upregulation of FAS-ligand (FASL) and subsequent apoptosis through the FAS death receptor pathway. It has been known for more than a decade that TCR signaling is defective in CTCL; however, the underlying mechanism has not been apparent. In this report, we show that the E3 ubiquitin ligase, c-CBL, is over-expressed in CTCL and that its knockdown overcomes defective TCR signaling resulting in phosphorylation of PLCg1, calcium influx, ROS generation, up-regulation of FASL and extrinsic pathway apoptosis in CTCL cells expressing adequate FAS. In CTCL cells with suboptimal FAS expression, FAS can be upregulated epigenetically by derepression of the FAS promoter using methotrexate (MTX) which we showed previously has activity as a DNA methylation inhibitor. Using these combined strategies, FAS-low as well as FAS-high CTCL cells can be killed effectively. PMID:25140833

  18. Interferon-alpha and bortezomib overcome Bcl-2 and Mcl-1 over-expression in melanoma cells by stimulating the extrinsic pathway of apoptosis

    PubMed Central

    Lesinski, Gregory B.; Raig, Ene T.; Guenterberg, Kristan; Brown, Lloyd; Go, Michael R.; Shah, Nisha N.; Lewis, Adrian; Quimper, Megan; Hade, Erinn; Young, Gregory; Chaudhury, Abhik Ray; Ladner, Katherine J.; Guttridge, Denis C.; Bouchard, Page

    2008-01-01

    We hypothesized that interferon-alpha (IFN-α) would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-α induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspases-3, -7, -8, -9, and with cleavage of Bid and PARP. Bortezomib plus IFN-α was effective at inducing apoptosis in melanoma cells that over-expressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The pro-apoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD prior to the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or siRNA targeting Fas. These data suggest that bortezomib and IFN-α act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-α displayed statistically significant anti-tumor activity as compared to either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-α for malignant melanoma. PMID:18922907

  19. [Physiological differences between HPS/PHI over-expressing transgenic and wild-type geraniums under formaldehyde stress revealed by FTIR analysis].

    PubMed

    Tang, Li-juan; Zhang, Ya-nan; Song, Zhong-bang; Zhang, Wei; Huang, Shu-shi; Li, Kun-zhi; Chen, Li-mei

    2012-05-01

    In the present study, FTIR was used to analyze changes in chemical component contents and spectra characters of 3-hexulose-6-phosphate synthase/6-phosphate-3-hexuloisomerase (HPS/PHI) over-expressing transgenic and wild-type (WT) geraniums under formaldehyde (HCHO) stress to examine if FTIR could be a new method for identification of phenotypic differences between the transgenic plants with a photosynthetic HCHO-assimilation pathway and the WT plants. The WT and transgenic geranium plants were treated with 4 mmol x L(-1) HCHO for 0, 1, 2, 3 and 4 days, respectively. The comparison of FTIR spectral characteristics at different time points between the transgenic and WT plants indicated that the contents of carbohydrate, proteins and aliphatic compounds were significantly higher than those in the WT plants after 4 days of HCHO-treatment. This may be due to installation of the photosynthetic HCHO-assimilation pathway in the transgenic geranium, which enhanced its ability to metabolize and assimilate HCHO, thus allowed more HCHO to be fixed to 6-phosphate fructose, and then entered assimilation pathways for synthesis of a variety of intracellular components. The results suggest that FTIR can be a new method to identify the phenotypic differences between transgenic plants with a photosynthetic HCHO-assimilation pathway and WT plants.

  20. Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells.

    PubMed

    Crabtree, Donna; Dodson, Matthew; Ouyang, Xiaosen; Boyer-Guittaut, Michaël; Liang, Qiuli; Ballestas, Mary E; Fineberg, Naomi; Zhang, Jianhua

    2014-03-01

    Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

  1. miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from sporadic amyotrophic lateral sclerosis patients.

    PubMed

    De Felice, Bruna; Annunziata, Anna; Fiorentino, Giuseppe; Borra, Marco; Biffali, Elio; Coppola, Cinzia; Cotrufo, Roberto; Brettschneider, Johannes; Giordana, Maria Luisa; Dalmay, Tamas; Wheeler, Guy; D'Alessandro, Raffaella

    2014-10-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and seriously disabling adult-onset neurological disease. Ninety percent of ALS patients are sporadic cases (sALS) with no clear genetic linkage. Accumulating evidence indicates that various microRNAs (miRNAs), expressed in a spatially and temporally controlled manner in the brain, play a key role in neuronal development. In addition, microRNA dysregulation contributes to some mental disorders and neurodegeneration diseases. In our research, the expression of one selected miRNA, miR-338-3p, which previously we have found over-expressed in blood leukocytes, was studied in several different tissues from sALS patients. For the first time, we detected a specific microRNA disease-related upregulation, miR-338-3p, in blood leukocytes as well in cerebrospinal fluid, serum, and spinal cord from sALS patients. Besides, staining of in situ hybridization showed that the signals of miR-338-3p were localized in the grey matter of spinal cord tissues from sALS autopsied patients. We propose that miRNA profiles found in tissue samples from sALS patients can be relevant to understand sALS pathogenesis and lead to set up effective biomarkers for sALS early diagnosis.

  2. Over-expression of CKS1B activates both MEK/ERK and JAK/STAT3 signaling pathways and promotes myeloma cell drug-resistance

    PubMed Central

    Zangari, Maurizio; Xu, Hongwei; Cao, Thai M.; Xu, Chunjiao; Wu, Yong; Xiao, Fang; Liu, Yinghong; Yang, Ye; Salama, Mohamed; Li, Guiyuan; Tricot, Guido; Zhan, Fenghuang

    2010-01-01

    Here we demonstrate the crucial role of CKS1B in multiple myeloma (MM) progression and define CKS1B-mediated SKP2/p27Kip1-independent down-stream signaling pathways. Forced-expression of CKS1B in MM cells increased cell multidrug-resistance. CKS1B activates STAT3 and MEK/ERK pathways. In contrast, SKP2 knockdown or p27Kip1 over-expression resulted in activation of the STAT3 and MEK/ERK pathways. Further investigations showed that BCL2 is a downstream target of MEK/ERK signaling. Stimulation of STAT3 and MEK/ERK signaling pathways partially abrogated CKS1B knockdown induced MM cell death and growth inhibition. Targeting STAT3 and MEK/ ERK signaling pathways by specific inhibitors induced significant MM cell death and growth inhibition in CKS1B-overexpressing MM cells and their combinations resulted in synergy. Thus, our findings provide a rationale for targeting STAT3 and MEK/ERK/ BCL2 signaling in aggressive CKS1B-overexpressing MM. PMID:20930946

  3. D-dopachrome tautomerase is over-expressed in pancreatic ductal adenocarcinoma and acts cooperatively with macrophage migration inhibitory factor to promote cancer growth.

    PubMed

    Guo, Dawei; Guo, Jinshuai; Yao, Junchao; Jiang, Kun; Hu, Jianhua; Wang, Bo; Liu, Haiyang; Lin, Lin; Sun, Wenyu; Jiang, Xiaofeng

    2016-11-01

    Previous studies have established the important role of MIF in the development of pancreatic ductal adenocarcinoma (PDAC) for both therapeutic and diagnostic perspectives, but little is known about the expression and function of D-dopachrome tautomerase (DDT), a functional homolog of MIF, in PDAC. In the present study, we demonstrated that DDT was over-expressed in PDAC tissues in a pattern correlated with MIF. In the pancreatic cancer cell lines, PANC-1, BXPC-3 and ASPC-1, both DDT and MIF were expressed and co-localized with each other in the endosomal compartments and plasma membrane. Knockdown of DDT and MIF in PANC-1 cells cooperatively inhibited ERK1/2 and AKT phosphorylation, increased p53 expression, and reduced cell proliferation, invasion and tumor formation. These effects were rescued by the re-expression of MIF or DDT, but not by the forced expression of the tautomerase-deficient mutants of DDT and MIF, P1G-DDT and P1G-MIF. Finally, we observed that 4-iodo-6-phenylpyrimidine (4-IPP), a covalent tautomerase inhibitor of both DDT and MIF, attenuated PANC-1 cell proliferation and colony formation in vitro and tumor growth in vivo. Thus, targeting the tautomerase sites of both MIF and DDT may offer more efficient therapeutic benefits to PDAC patients. PMID:27434219

  4. Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

    PubMed

    Pollard, P J; Brière, J J; Alam, N A; Barwell, J; Barclay, E; Wortham, N C; Hunt, T; Mitchell, M; Olpin, S; Moat, S J; Hargreaves, I P; Heales, S J; Chung, Y L; Griffiths, J R; Dalgleish, A; McGrath, J A; Gleeson, M J; Hodgson, S V; Poulsom, R; Rustin, P; Tomlinson, I P M

    2005-08-01

    The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

  5. Over-Expression of Monoacylglycerol Lipase (MGL) in Small Intestine Alters Endocannabinoid Levels and Whole Body Energy Balance, Resulting in Obesity

    PubMed Central

    Chon, Su-Hyoun; Douglass, John D.; Zhou, Yin Xiu; Malik, Nashmia; Dixon, Joseph L.; Brinker, Anita; Quadro, Loredana; Storch, Judith

    2012-01-01

    The function of small intestinal monoacylglycerol lipase (MGL) is unknown. Its expression in this tissue is surprising because one of the primary functions of the small intestine is to convert diet-derived MGs to triacylglycerol (TG), and not to degrade them. To elucidate the function of intestinal MGL, we generated transgenic mice that over-express MGL specifically in small intestine (iMGL mice). After only 3 weeks of high fat feeding, iMGL mice showed an obese phenotype; body weight gain and body fat mass were markedly higher in iMGL mice, along with increased hepatic and plasma TG levels compared to wild type littermates. The iMGL mice were hyperphagic and displayed reduced energy expenditure despite unchanged lean body mass, suggesting that the increased adiposity was due to both increased caloric intake and systemic effects resulting in a hypometabolic rate. The presence of the transgene resulted in lower levels of most MG species in intestinal mucosa, including the endocannabinoid 2-arachidonoyl glycerol (2-AG). The results therefore suggest a role for intestinal MGL, and intestinal 2-AG and perhaps other MG species, in whole body energy balance via regulation of food intake as well as metabolic rate. PMID:22937137

  6. Carbon encapsulated iron oxide nanoparticles surface engineered with polyethylene glycol-folic acid to induce selective hyperthermia in folate over expressed cancer cells.

    PubMed

    Sadhasivam, S; Savitha, S; Wu, Chun-Jen; Lin, Feng-Huei; Stobiński, Leszek

    2015-03-01

    Carbon encapsulated iron oxide nanoparticles (CEIO-NPs) prepared by carbon arc method were successfully applied for in vitro magnetic hyperthermia. The CEIO-NPs were chemically oxidized and surface modified with PEG-FA for selective tumor localization in cancer cells that over expresses the folate receptors (FR(+)). The size, morphology, heating efficiency, biocompatibility and in vitro cell uptake of CEIO-PEG-FA NPs are extensively characterized. The as-prepared nanoparticles have generated quick heating (43-45°C) upon exposure to an alternating magnetic field (AMF) with the saturation magnetization of 25emu/g. The LDH cytotoxic assay demonstrated that the nanoparticle did not affect the viability of normal human fibroblast. The quantitative and cellular uptake studies by TEM confirmed the selective and increased uptake of CEIO-PEG-FA NPs when compared to the CEIO-nanoparticles. In conclusion, CEIO-PEG-FA NPs have the potential to induce magnetic hyperthermia in FR(+) cells via the receptor mediated endocytosis uptake mechanism.

  7. Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense.

    PubMed

    Huergo, Luciano F; Souza, Emanuel M; Steffens, Maria B R; Yates, M Geoffrey; Pedrosa, Fábio O; Chubatsu, Leda S

    2005-03-01

    Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.

  8. Gene Body Methylation can alter Gene Expression and is a Therapeutic Target in Cancer

    PubMed Central

    Yang, Xiaojing; Han, Han; De Carvalho, Daniel D.; Lay, Fides D.; Jones, Peter A.; Liang, Gangning

    2014-01-01

    SUMMARY DNA methylation in promoters is well known to silence genes and is the presumed therapeutic target of methylation inhibitors. Gene body methylation is positively correlated with expression yet its function is unknown. We show that 5-aza-2'-deoxycytidine treatment not only reactivates genes but decreases the over-expression of genes, many of which are involved in metabolic processes regulated by c-MYC. Down-regulation is caused by DNA demethylation of the gene bodies and restoration of high levels of expression requires remethylation by DNMT3B. Gene body methylation may therefore be an unexpected therapeutic target for DNA methylation inhibitors, resulting in the normalization of gene over-expression induced during carcinogenesis. Our results provide direct evidence for a causal relationship between gene body methylation and transcription. PMID:25263941

  9. Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

    SciTech Connect

    Lanza, Daniel C.F.; Trindade, Daniel M.; Assmann, Eliana M.; Kobarg, Joerg

    2008-06-10

    FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, {alpha}- and especially with {gamma}-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.

  10. Wound healing response and xylem differentiation in tobacco plants over-expressing a fungal endopolygalacturonase is mediated by copper amine oxidase activity.

    PubMed

    Cona, Alessandra; Tisi, Alessandra; Ghuge, Sandip Annasaheb; Franchi, Stefano; De Lorenzo, Giulia; Angelini, Riccardo

    2014-09-01

    In this work, we have investigated the involvement of copper amine oxidase (CuAO; EC 1.4.3.21) in wound healing and xylem differentiation of Nicotiana tabacum plants over-expressing a fungal endopolygalacturonase (PG plants), which show constitutively activated defence responses. In petioles and stems of PG plants, we found higher CuAO activity and lower polyamine (PA) levels, particularly putrescine (Put), with respect to wild-type (WT) plants. Upon wounding, a more intense autofluorescence of cell wall phenolics was observed in correspondence of wound surface, extending to epidermis and cortical parenchima only in PG plants. This response was mostly dependent on CuAO activity, as suggested by the reversion of autofluorescence upon supply of 2-bromoethylamine (2-BrEt), a CuAO specific inhibitor. Moreover, in unwounded plants, histochemical analysis revealed a tissue-specific expression of the enzyme in the vascular cambium and neighboring derivative cells of both petioles and stems of PG plants, whereas the corresponding WT tissues appeared unstained or faintly stained. A higher histochemical CuAO activity was also observed in xylem cells of PG plants as compared to WT xylem tissues suggesting a peculiar role of CuAO activity in xylem differentiation in PG plants. Indeed, roots of PG plants exhibited early xylem differentiation, a phenotype consistent with both the higher CuAO and the lower Put levels observed and supported by the 2-BrEt-mediated reversion of early root xylem differentiation and H2O2 accumulation. These results strongly support the relevance of PA-catabolism derived H2O2 in defence responses, such as those signaled by a compromised status of cell wall pectin integrity.

  11. Involvement of over-expressed BMP4 in pentylenetetrazol kindling-induced cell proliferation in the dentate gyrus of adult rats

    SciTech Connect

    Yin Jinbo; Ma Yuxin; Yin Qing; Xu Haiwei . E-mail: haiweixu2001@yahoo.com.cn; An Ning; Liu Shiyong; Fan Xiaotang; Yang Hui . E-mail: huiyang64@yahoo.com

    2007-03-30

    The dentate gyrus (DG) of the hippocampus is one of a few regions in the adult mammalian brain characterized by ongoing neurogenesis. Proliferation of neural precursors in the granule cell layer of the DG has been identified in pentylenetetrazol (PTZ) kindling epilepsy model, however, little is known about the molecular mechanism. We previously reported that the expression pattern of bone morphogenetic proteins-4 (BMP4) mRNA in the hippocampus was developmentally regulated and mainly localized in the DG of the adult. To explore the role of BMP4 in epileptic activity, we detected BMP4 expression in the DG during PTZ kindling process and explore its correlation with cell proliferation combined with bromodeoxyuridine (BrdU) labeling technique. We found that dynamic changes in BMP4 level and BrdU labeled cells dependent on the kindling stage of PTZ induced seizure-prone state. The number of BMP4 mRNA-positive cells and BrdU labeled cells reached the top level 1 day after PTZ kindled, then declined to base level 2 months later. Furthermore, there was a significant correlation between increased BMP4 mRNA expression and increased number of BrdU labeled cells. After effectively blocked expression of BMP4 with antisense oligodeoxynucleotides(ASODN), the BrdU labeled cells in the dentate gyrus subgranular zone(DG-SGZ) and hilus were significantly decreased 16d after First PTZ injection and 1, 3, 7, 14d after kindled respectively. These findings suggest that increased proliferation in the DG of the hippocampus resulted from kindling epilepsy elicited by PTZ maybe be modulated by BMP4 over-expression.

  12. Analyses of GA20ox- and GID1-over-expressing aspen suggest that gibberellins play two distinct roles in wood formation.

    PubMed

    Mauriat, Mélanie; Moritz, Thomas

    2009-06-01

    Gibberellins (GAs) are involved in many aspects of plant development, including shoot growth, flowering and wood formation. Increased levels of bioactive GAs are known to induce xylogenesis and xylem fiber elongation in aspen. However, there is currently little information on the response pathway(s) that mediate GA effects on wood formation. Here we characterize an important element of the GA pathway in hybrid aspen: the GA receptor, GID1. Four orthologs of GID1 were identified in Populus tremula x P. tremuloides (PttGID1.1-1.4). These were functional when expressed in Arabidopsis thaliana, and appear to present a degree of sub-functionalization in hybrid aspen. PttGID1.1 and PttGID1.3 were over-expressed in independent lines of hybrid aspen using either the 35S promoter or a xylem-specific promoter (LMX5). The 35S:PttGID1 over-expressors shared several phenotypic traits previously described in 35S:AtGA20ox1 over-expressors, including rapid growth, increased elongation, and increased xylogenesis. However, their xylem fibers were not elongated, unlike those of 35S:AtGA20ox1 plants. Similar differences in the xylem fiber phenotype were observed when PttGID1.1, PttGID1.3 or AtGA20ox1 were expressed under the control of the LMX5 promoter, suggesting either that PttGID1.1 and PttGID1.3 play no role in fiber elongation or that GA homeostasis is strongly controlled when GA signaling is altered. Our data suggest that GAs are required in two distinct wood-formation processes that have tissue-specific signaling pathways: xylogenesis, as mediated by GA signaling in the cambium, and fiber elongation in the developing xylem.

  13. Ectopic over-expression of the maize beta-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco.

    PubMed

    Kiran, Nagavalli S; Polanská, Lenka; Fohlerová, Radka; Mazura, Pavel; Válková, Martina; Smeral, Miloslav; Zouhar, Jan; Malbeck, Jirí; Dobrev, Petre I; Machácková, Ivana; Brzobohaty, Bretislav

    2006-01-01

    The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

  14. High prevalence of immunoglobulin light chain gene aberrations as revealed by FISH in multiple myeloma and MGUS.

    PubMed

    Türkmen, Seval; Binder, Anastasia; Gerlach, Antje; Niehage, Sylke; Theodora Melissari, Maria; Inandiklioglu, Nihal; Dörken, Bernd; Burmeister, Thomas

    2014-08-01

    Multiple myeloma (MM) is a malignant B-cell neoplasm characterized by an uncontrolled proliferation of aberrant plasma cells in the bone marrow. Chromosome aberrations in MM are complex and represent a hallmark of the disease, involving many chromosomes that are altered both numerically and structurally. Nearly half of the cases are nonhyperdiploid and show IGH translocations with the following partner genes: CCND1, FGFR3 and MMSET, MAF, MAFB, and CCND3. The remaining 50% are grouped into a hyperdiploid group that is characterized by multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. In this study, we analyzed the immunoglobulin light chain kappa (IGK, 2p12) and lambda (IGL, 22q11) loci in 150 cases, mostly with MM but in a few cases monoclonal gammopathy of undetermined significance (MGUS), without IGH translocations. We identified aberrations in 27% (= 40 patients) including rearrangements (12%), gains (12%), and deletions (4.6%). In 6 of 18 patients with IGK or/and IGL rearrangements, we detected a MYC rearrangement which suggests that MYC is the translocation partner in the majority of these cases. PMID:24729354

  15. Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver.

    PubMed

    Santosa, David; Castoldi, Mirco; Paluschinski, Martha; Sommerfeld, Annika; Häussinger, Dieter

    2015-01-01

    microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype. PMID:26195352

  16. Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver.

    PubMed

    Santosa, David; Castoldi, Mirco; Paluschinski, Martha; Sommerfeld, Annika; Häussinger, Dieter

    2015-07-21

    microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.

  17. Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver

    PubMed Central

    Santosa, David; Castoldi, Mirco; Paluschinski, Martha; Sommerfeld, Annika; Häussinger, Dieter

    2015-01-01

    microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype. PMID:26195352

  18. Identification of FGF19 as a prognostic marker and potential driver gene of lung squamous cell carcinomas in Chinese smoking patients

    PubMed Central

    Xia, Weiliang; Li, Ziming; Niu, Xiaomin; Ji, Wenxiang; Yuan, Hong; Xu, Qiang; Luo, Qingquan; Zhang, Jie; Lu, Shun

    2016-01-01

    Comprehensive genomic characterizations of lung squamous cell carcinoma (LSCC) have been performed, but the differences between smokers (S-LSCC) and never smokers (NS-LSCC) are not clear, as NS-LSCC could be considered as a different disease from S-LSCC. In this study we delineated genomic alterations in a cohort of 21 NS-LSCC and 16 S-LSCC patients, and identified common gene mutations and amplifications as previously reported. Inclusion of more NS-LSCC patients enabled us to identify unreported S-LSCC- or NS-LSCC-specific alterations. Importantly, an amplification region containing FGF19, FGF3, FGF4 and CCND1 was found five-times more frequent in S-LSCC than in NS-LSCC. Amplification of FGF19 was validated in independent LSCC samples. Furthermore, FGF19 stimulated LSCC cell growth in vitro. These data implicate FGF19 as a potential driver gene in LSCC with clinic characteristics as smoking. PMID:26943773

  19. Modifying metabolically sensitive histone marks by inhibiting glutamine metabolism affects gene expression and alters cancer cell phenotype.

    PubMed

    Simpson, Natalie E; Tryndyak, Volodymyr P; Pogribna, Marta; Beland, Frederick A; Pogribny, Igor P

    2012-12-01

    The interplay of metabolism and epigenetic regulatory mechanisms has become a focal point for a better understanding of cancer development and progression. In this study, we have acquired data supporting previous observations that demonstrate glutamine metabolism affects histone modifications in human breast cancer cell lines. Treatment of non-invasive epithelial (T-47D and MDA-MB-361) and invasive mesenchymal (MDA-MB-231 and Hs-578T) breast cancer cell lines with the glutaminase inhibitor, Compound 968, resulted in cytotoxicity in all cell lines, with the greatest effect being observed in MDA-MB-231 breast cancer cells. Compound 968-treatment induced significant downregulation of 20 critical cancer-related genes, the majority of which are anti-apoptotic and/or promote metastasis, including AKT, BCL2, BCL2L1, CCND1, CDKN3, ERBB2, ETS1, E2F1, JUN, KITLG, MYB, and MYC. Histone H3K4me3, a mark of transcriptional activation, was reduced at the promoters of all but one of these critical cancer genes. The decrease in histone H3K4me3 at global and gene-specific levels correlated with reduced expression of SETD1 and ASH2L, genes encoding the histone H3K4 methyltransferase complex. Further, the expression of other epigenetic regulatory genes, known to be downregulated during apoptosis (e.g., DNMT1, DNMT3B, SETD1 and SIRT1), was also downregulated by Compound 968. These changes in gene expression and histone modifications were accompanied by the activation of apoptosis, and decreased invasiveness and resistance of MDA-MB-231 cells to chemotherapeutic drug doxorubicin. The results of this study provide evidence to a link between cytotoxicity caused by inhibiting glutamine metabolism with alterations of the epigenome of breast cancer cells and suggest that modification of intracellular metabolism may enhance the efficiency of epigenetic therapy. PMID:23117580

  20. Human VRK1 Is an Early Response Gene and Its Loss Causes a Block in Cell Cycle Progression

    PubMed Central

    Valbuena, Alberto; López-Sánchez, Inmaculada; Lazo, Pedro A.

    2008-01-01

    Background In mammalian cells regulatory proteins controlling the cell cycle are necessary due to the requirements of living in a heterogeneous environment of cell-interactions and growth factors. VRK1 is a novel serine-threonine kinase that phosphorylates several transcription factors and is associated with proliferation phenotypes. Methodology/Principal Findings In this report VRK1 has been identified as regulated in the cell cycle. VRK1 gene expression is activated by the addition of serum to starved cells, indicating it is required for the exit of G0 phase and entry in G1; a response that parallels the re-expression of MYC, FOS and CCND1 (cyclin D1) genes, suggesting that VRK1 is an early-response gene. VRK1 gene expression is also shutdown by serum withdrawal. The human VRK1 gene promoter cloned in a luciferase reporter responds similarly to serum. In response to serum, the level of VRK1 protein expression has a positive correlation with cell proliferation markers such as phosphorylated-Rb or PCNA, and is inversely correlated with cell cycle inhibitors such as p27. The elimination of VRK1 by siRNA results in a G1 block in cell division, and in loss of phosphorylated-Rb, cyclin D1, and other proliferation markers. Elimination of VRK1 by siRNA induces a reduction of cell proliferation. VRK1 colocalizes with p63 in proliferating areas of squamous epithelium, and identifies a subpopulation in the basal layer. Conclusions/Significance VRK1 is an immediate early response gene required for entry in G1, and due to its implication in normal cell proliferation and division, might be a new target for development of inhibitors of cellular proliferation. PMID:18286197

  1. Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)2 to Detect Gamma Glutamyl Transferase Over Expressing Tumors

    PubMed Central

    Khurana, Harleen; Meena, Virendra Kumar; Prakash, Surbhi; Chuttani, Krishna; Chadha, Nidhi; Jaswal, Ambika; Dhawan, Devinder Kumar; Mishra, Anil Kumar; Hazari, Puja Panwar

    2015-01-01

    Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor

  2. Somatostatin receptors over-expression in castration resistant prostate cancer detected by PET/CT: preliminary report of in six patients

    PubMed Central

    Muni, Alfredo; Falchi, Roberta; Zaniboni, Alberto; Barbieri, Roberto; Valmadre, Giuseppe; Minari, Chiara; Casi, Camilla; Rossini, Pierluigi

    2015-01-01

    Prostate cancer (PC) is usually characterized by an excellent prognosis, largely due to little biological aggressiveness and the power of hormonal deprivation therapy. In spite of these favorable characteristics, however, a significant quota of patients does not respond to androgen deprivation therapy (ADT) and develop a progressive disease. Castration-resistant prostate cancer (CRPC) is defined by disease progression in spite of ADT. This progression may show any combination of a rise in serum prostate-specific antigen (PSA), clinical and radiological progression of pre-existing disease, and appearance of new metastases. This event is a striking change in the clinical scenario, since the power of treatment for CRPC patients with distant metastases is very limited. Somatostatin is a hormone produced by neuroendocrine cells. Its distant effects are mediated by the binding to five specific receptors, which are the most striking parameter for neuroendocrine. Various synthetic somatostatin agonists able to bind to the receptors have been synthesized during the past two decades for diagnostic and therapeutic purposes. Octreotide, the most popular of these, is widely used to treat patients affected by neuroendocrine tumors. A number of researches carried out in the past evaluated the possible neuroendocrine differentiation (NED) of PC cells in the castration resistant phase. If proved, the presence of a specific class of receptor on cell’s surfaces should give a potentially biological target to be used for therapy. However, these studies led to contradictory results. Aim of our phase III diagnostic trial was to study “in vivo” the over-expression of somatostatin receptors (SSTRs) in CRPC patients by PET/CT after the administration of the somatostatin analog [68Ga-DOTANOC,1-Nal(3)]-octreotide labeled with 68Ga. Every area of increased uptake corresponding to a metastasis detected with other methods was considered as SSTRs expressing. False positivity to SSTRs

  3. A study on the structural features of SELK, an over-expressed protein in hepatocellular carcinoma, by molecular dynamics simulations in a lipid-water system.

    PubMed

    Polo, Andrea; Guariniello, Stefano; Colonna, Giovanni; Ciliberto, Gennaro; Costantini, Susan

    2016-10-20

    Human SELK is a small trans-membrane selenoprotein characterized by a single trans-membrane helix, while the N-terminal region protrudes into the lumen and the long C-terminal domain into the cytoplasm. SELK is over-expressed in some cancers, like hepatocellular carcinoma; however its precise role in cancer development is presently unknown. SELK is involved in promoting the calcium flux, catalyzing palmitoylation reactions and protein degradation in the endoplasmic reticulum (ER). Therefore, this protein should bind many different proteins like p97/VCP in the supramolecular complex involved in the ER degradation pathway. To study the structural features of SELK in the membrane, we have modeled the protein and then subjected it to molecular dynamics simulations in a lipid-water system. The model shows a N-terminal domain with three β-strands and a short helix, a well-defined trans-membrane helix and a C-terminal domain that lacks a persistent secondary structure and contains long disordered regions. The trajectory analysis during the simulation evidences that: (i) the N-terminal region explores a limited conformational space and is stabilized by intra-peptide H-bonds as well with membrane lipids and water, (ii) the trans-membrane helix was found to be quite stable and (iii) the disordered C-terminal region is stabilized by H-bonds with clustered water molecules as well as by rapidly interchanging intra-peptidic H-bonds, with a structural tendency to compact around the four HUB residues found for this domain. Moreover, N-terminal and C-terminal clusters are distributed differently in the conformational space suggesting that their dynamics are coupled complicatedly through the membrane. Further analyses have shown that the N-terminal has a tendency to pivot around the insertion with the TM-helix through the fluctuations of the three β-strands, which, in turn, show features similar to WW-domains. These results will be useful to study the SELK, SELS and VCP complex

  4. Integrated Genotypic Analysis of Hedgehog-Related Genes Identifies Subgroups of Keratocystic Odontogenic Tumor with Distinct Clinicopathological Features

    PubMed Central

    Shimada, Yasuyuki; Katsube, Ken-ichi; Kabasawa, Yuji; Morita, Kei-ichi; Omura, Ken; Yamaguchi, Akira; Sakamoto, Kei

    2013-01-01

    Keratocystic odontogenic tumor (KCOT) arises as part of Gorlin syndrome (GS) or as a sporadic lesion. Gene mutations and loss of heterozygosity (LOH) of the hedgehog receptor PTCH1 plays an essential role in the pathogenesis of KCOT. However, some KCOT cases lack evidence for gene alteration of PTCH1, suggesting that other genes in the hedgehog pathway may be affected. PTCH2 and SUFU participate in the occurrence of GS-associated tumors, but their roles in KCOT development are unknown. To elucidate the roles of these genes, we enrolled 36 KCOT patients in a study to sequence their entire coding regions of PTCH1, PTCH2 and SUFU. LOH and immunohistochemical expression of these genes, as well as the downstream targets of hedgehog signaling, were examined using surgically-excised KCOT tissues. PTCH1 mutations, including four novel ones, were found in 9 hereditary KCOT patients, but not in sporadic KCOT patients. A pathogenic mutation of PTCH2 or SUFU was not found in any patients. LOH at PTCH1 and SUFU loci correlated with the presence of epithelial budding. KCOT harboring a germline mutation (Type 1) showed nuclear localization of GLI2 and frequent histological findings such as budding and epithelial islands, as well as the highest recurrence rate. KCOT with LOH but without a germline mutation (Type 2) less frequently showed these histological features, and the recurrence rate was lower. KCOT with neither germline mutation nor LOH (Type 3) consisted of two subgroups, Type 3A and 3B, which were characterized by nuclear and cytoplasmic GLI2 localization, respectively. Type 3B rarely exhibited budding and recurrence, behaving as the most amicable entity. The expression patterns of CCND1 and BCL2 tended to correlate with these subgroups. Our data indicates a significant role of PTCH1 and SUFU in the pathogenesis of KCOT, and the genotype-oriented subgroups constitute entities with different potential aggressiveness. PMID:23951062

  5. Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants

    PubMed Central

    Jiang, Shu-Ye; Ma, Ali; Xie, Lifen; Ramachandran, Srinivasan

    2016-01-01

    Rice grains are rich in starch but low in protein with very low level of both lysine and threonine. Thus, it is important to further improve protein quality and quantity, especially to increase lysine and threonine content in rice grains. We artificially synthesized two new genes by fusing endogenous rice genes with lysine (K)/threonine (T) motif (TKTKK) coding sequences. They were designated as TKTKK1 and TKTKK2 and their encoded proteins consist of 73.1% and 83.5% of lysine/threonine, respectively. These two genes were under the control of 35S promoter and were independently introduced into the rice genome to generate transgenic plants. Our data showed that overexpression of TKTKK1 generated stable proteins with expected molecular weight and the transgenic rice seeds significantly increased lysine, threonine, total amino acids and crude protein content by 33.87%, 21.21%, 19.43% and 20.45%, respectively when compared with wild type control; significant improvement was also observed in transgenic rice seeds overexpressing TKTKK2. However, limited improvement in protein quality and quantity was observed in transgenic seeds carrying tandom array of these two new genes. Our data provide the basis and alternative strategy on further improving protein quality and quantity in other crops or vegetable plants by synthetic biology. PMID:27677708

  6. Contextualizing the Genes Altered in Bladder Neoplasms in Pediatric andTeen Patients Allows Identifying Two Main Classes of Biological ProcessesInvolved and New Potential Therapeutic Targets

    PubMed Central

    Porrello, A.; Piergentili, R. b

    2016-01-01

    Research on bladder neoplasms in pediatric and teen patients (BNPTP) has described 21 genes, which are variously involved in this disease and are mostly responsible for deregulated cell proliferation. However, due to the limited number of publications on this subject, it is still unclear what type of relationships there are among these genes and which are the chances that, while having different molecular functions, they i) act as downstream effector genes of well-known pro- or anti- proliferative stimuli and/or interplay with biochemical pathways having oncological relevance or ii) are specific and, possibly, early biomarkers of these pathologies. A Gene Ontology (GO)-based analysis showed that these 21 genes are involved in biological processes, which can be split into two main classes: cell regulation-based and differentiation/development-based. In order to understand the involvement/overlapping with main cancer-related pathways, we performed a meta-analysis dependent on the 189 oncogenic signatures of the Molecular Signatures Database (OSMSD) curated by the Broad Institute. We generated a binary matrix with 53 gene signatures having at least one hit; this analysis i) suggests that some genes of the original list show inconsistencies and might need to be experimentally re- assessed or evaluated as biomarkers (in particular, ACTA2) and ii) allows hypothesizing that important (proto)oncogenes (E2F3, ERBB2/HER2, CCND1, WNT1, and YAP1) and (putative) tumor suppressors (BRCA1, RBBP8/CTIP, and RB1-RBL2/p130) may participate in the onset of this disease or worsen the observed phenotype, thus expanding the list of possible molecular targets for the treatment of BNPTP. PMID:27013923

  7. Proinflammatory cytokines decrease the expression of genes critical for RPE function

    PubMed Central

    Samuel, William; Boyce, Kaifa; Cherukuri, Aswini; Duncan, Todd; Jaworski, Cynthia; Nagineni, Chandrasekharam N.; Redmond, T. Michael

    2016-01-01

    Purpose Proinflammatory cytokines interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1β) secreted by infiltrating lymphocytes or macrophages may play a role in triggering RPE dysfunction associated with age-related macular degeneration (AMD). Binding of these proinflammatory cytokines to their specific receptors residing on the RPE cell surface can activate signaling pathways that, in turn, may dysregulate cellular gene expression. The purpose of the present study was to investigate whether IFN-γ, TNF-α, and IL-1β have an adverse effect on the expression of genes essential for RPE function, employing the RPE cell line ARPE-19 as a model system. Methods ARPE-19 cells were cultured for 3–4 months until they exhibited epithelial morphology and expressed mRNAs for visual cycle genes. The differentiated cells were treated with IFN-γ, TNF-α, and/or IL-1β, and gene expression was analyzed with real-time PCR analysis. Western immunoblotting was employed for the detection of proteins. Results Proinflammatory cytokines (IFN-γ + TNF-α + IL-1β) greatly increased the expression of chemokines and cytokines in cultured ARPE-19 cells that exhibited RPE characteristics. However, this response was accompanied by markedly decreased expression of genes important for RPE function, such as CDH1, RPE65, RDH5, RDH10, TYR, and MERTK. This was associated with decreased expression of the genes MITF, TRPM1, and TRPM3, as well as microRNAs miR-204 and miR-211, which are known to regulate RPE-specific gene expression. The decreased expression of the epithelial marker gene CDH1 was associated with increased expression of mesenchymal marker genes (CDH2, VIM, and CCND1) and epithelial–mesenchymal transition (EMT) promoting transcription factor genes (ZEB1 and SNAI1). Conclusions RPE cells exposed to proinflammatory cytokines IFN-γ, TNF-α, and IL-1β showed decreased expression of key genes involved in the visual cycle, epithelial morphology

  8. Gene expression profiling reveals underlying molecular mechanisms of the early stages of tamoxifen-induced rat hepatocarcinogenesis

    SciTech Connect

    Pogribny, Igor P. Bagnyukova, Tetyana V.; Tryndyak, Volodymyr P.; Muskhelishvili, Levan; Rodriguez-Juarez, Rocio; Kovalchuk, Olga; Han Tao; Fuscoe, James C.; Ross, Sharon A.; Beland, Frederick A.

    2007-11-15

    Tamoxifen is a widely used anti-estrogenic drug for chemotherapy and, more recently, for the chemoprevention of breast cancer. Despite the indisputable benefits of tamoxifen in preventing the occurrence and re-occurrence of breast cancer, the use of tamoxifen has been shown to induce non-alcoholic steatohepatitis, which is a life-threatening fatty liver disease with a risk of progression to cirrhosis and hepatocellular carcinoma. In recent years, the high-throughput microarray technology for large-scale analysis of gene expression has become a powerful tool for increasing the understanding of the molecular mechanisms of carcinogenesis and for identifying new biomarkers with diagnostic and predictive values. In the present study, we used the high-throughput microarray technology to determine the gene expression profiles in the liver during early stages of tamoxifen-induced rat hepatocarcinogenesis. Female Fisher 344 rats were fed a 420 ppm tamoxifen containing diet for 12 or 24 weeks, and gene expression profiles were determined in liver of control and tamoxifen-exposed rats. The results indicate that early stages of tamoxifen-induced liver carcinogenesis are characterized by alterations in several major cellular pathways, specifically those involved in the tamoxifen metabolism, lipid metabolism, cell cycle signaling, and apoptosis/cell proliferation control. One of the most prominent changes during early stages of tamoxifen-induced hepatocarcinogenesis is dysregulation of signaling pathways in cell cycle progression from the G{sub 1} to S phase, evidenced by the progressive and sustained increase in expression of the Pdgfc, Calb3, Ets1, and Ccnd1 genes accompanied by the elevated level of the PI3K, p-PI3K, Akt1/2, Akt3, and cyclin B, D1, and D3 proteins. The early appearance of these alterations suggests their importance in the mechanism of neoplastic cell transformation induced by tamoxifen.

  9. Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana

    PubMed Central

    Wang, Tao; Sun, Ming-Yang; Wang, Xue-Song; Li, Wen-Bin; Li, Yong-Guang

    2016-01-01

    Flowering is a pivotal event in the life cycle of plants. miR172 has been widely confirmed to play critical roles in flowering time control by regulating its target gene expression in Arabidopsis. However, the role of its counterpart in soybean remains largely unclear. In the present study, we found that the gma-miR172a was regulated by a GIGANTEA ortholog, GmGIa, in soybean through miRNA metabolism. The expression analysis revealed that gma-miR172a has a pattern of diurnal rhythm expression and its abundance increased rapidly as plants grew until the initiation of flowering phase in soybean. One target gene of gma-miR172a, Glyma03g33470, was predicted and verified using a modified RLM 5′-RACE (RNA ligase-mediated rapid amplification of 5′ cDNA ends) assay. Overexpression of gma-miR172a exhibited an early flowering phenotype and the expression of FT, AP1 and LFY were simultaneously increased in gma-miR172a-transgenic Arabidopsis plants, suggesting that the early flowering phenotype was associated with up-regulation of these genes. The overexpression of the gma-miR172a-resistant version of Glyma03g33470 weakened early flowering phenotype in the toe1 mutant of Arabidopsis. Taken together, our results suggested that gma-miR172a played an important role in GmGIa-mediated flowering by repressing Glyma03g33470, which in turn increased the expression of FT, AP1 and LFY to promote flowering in soybean. PMID:27136537

  10. Transgenic tobacco plants over expressing cold regulated protein CbCOR15b from Capsella bursa-pastoris exhibit enhanced cold tolerance.

    PubMed

    Wu, Lihua; Zhou, Mingqi; Shen, Chen; Liang, Jing; Lin, Juan

    2012-09-15

    Low temperature is among the most significant abiotic stresses, restricting the habitats of sessile plants and reducing crop productivity. Cold regulated (COR) genes are low temperature-responsive genes expressing under regulation of a specific signal transduction pathway, which is designated C-repeat-binding-factor (CBF) signaling pathway. In the present article, cold bioassay showed that the transcript level of cold regulated gene CbCOR15b from shepherd's purse (Capsella bursa-pastoris) was obviously elevated under cold treatments. Reverse transcription-PCR (RT-PCR) and GUS report system revealed that unlike AtCOR15b, CbCOR15b expressed not only in leaves but also in stems and maturation zone of roots. When transgenic tobacco plants ectopically expressing CbCOR15b were exposed to chilling and freezing temperatures, they displayed more cold tolerance compared to control plants. According to the electrolyte leakage, the relative water content, the glucose content and the phenotype observation, CbCOR15b transformants suffered less damage under cold stress. Further investigation of the subcellular localization of CbCOR15b by transient expression of fusion protein CbCOR15b-GFP revealed that it was localized exclusively in the chloroplasts of tobacco mesophyll cells and in the cytoplasm of onion epidermal cells. It can be concluded that CbCOR15b which located in the chloroplasts and in the cytoplasm of cells without chloroplasts was involved in cold response of C. bursa-pastoris and conferred enhanced cold tolerance in transgenic tobacco plants.

  11. Over-expression of the β-carboxysomal CcmM protein in Synechococcus PCC7942 reveals a tight co-regulation of carboxysomal carbonic anhydrase (CcaA) and M58 content.

    PubMed

    Long, Benedict M; Rae, Benjamin D; Badger, Murray R; Price, G Dean

    2011-09-01

    Carboxysomes, containing the cell's complement of RuBisCO surrounded by a specialized protein shell, are a central component of the cyanobacterial CO(2)-concentrating mechanism. The ratio of two forms of the β-carboxysomal protein CcmM (M58 and M35) may affect the carboxysomal carbonic anhydrase (CcaA) content. We have over-expressed both M35 and M58 in the β-cyanobacterium Synechococcus PCC7942. Over-expression of M58 resulted in a marked increase in the amount of this protein in carboxysomes at the expense of M35, with a concomitant increase in the observed CcaA content of carboxysomes. Conversely, M35 over-expression diminished M58 content of carboxysomes and led to a decrease in CcaA content. Carboxysomes of air-grown wild-type cells contained slightly elevated CcaA and M58 content and slightly lower M35 content compared to their 2% CO(2)-grown counterparts. Over a range of CcmM expression levels, there was a strong correlation between M58 and CcaA content, indicating a constant carboxysomal M58:CcaA stoichiometry. These results also confirm a role for M58 in the recruitment of CcaA into the carboxysome and suggest a tight regulation of M35 and M58 translation is required to produce carboxysomes with an appropriate CA content. Analysis of carboxysomal protein ratios, resulting from the afore-mentioned over-expression studies, revealed that β-carboxysomal protein stoichiometries are relatively flexible. Determination of absolute protein quantities supports the hypothesis that M35 is distributed throughout the β-carboxysome. A modified β-carboxysome packing model is presented.

  12. CDC2 mediates progestin initiated endometrial stromal cell proliferation: a PR signaling to gene expression independently of its binding to chromatin.

    PubMed

    Vallejo, Griselda; La Greca, Alejandro D; Tarifa-Reischle, Inti C; Mestre-Citrinovitz, Ana C; Ballaré, Cecilia; Beato, Miguel; Saragüeta, Patricia

    2014-01-01

    Although non-genomic steroid receptor pathways have been studied over the past decade, little is known about the direct gene expression changes that take place as a consequence of their activation. Progesterone controls proliferation of rat endometrial stromal cells during the peri-implantation phase of pregnancy. We showed that picomolar concentration of progestin R5020 mimics this control in UIII endometrial stromal cells via ERK1-2 and AKT activation mediated by interaction of Progesterone Receptor (PR) with Estrogen Receptor beta (ERb) and without transcriptional activity of endogenous PR and ER. Here we identify early downstream targets of cytoplasmic PR signaling and their possible role in endometrial stromal cell proliferation. Microarray analysis of global gene expression changes in UIII cells treated for 45 min with progestin identified 97 up- and 341 down-regulated genes. The most over-represented molecular functions were transcription factors and regulatory factors associated with cell proliferation and cell cycle, a large fraction of which were repressors down-regulated by hormone. Further analysis verified that progestins regulate Ccnd1, JunD, Usf1, Gfi1, Cyr61, and Cdkn1b through PR-mediated activation of ligand-free ER, ERK1-2 or AKT, in the absence of genomic PR binding. ChIP experiments show that progestin promoted the interaction of USF1 with the proximal promoter of the Cdc2 gene. Usf1 knockdown abolished Cdc2 progestin-dependent transcriptional regulation and cell proliferation, which also blocked Cdc2 knockdown. We conclude that progestin-induced proliferation of endometrial stromal cells is mediated by ERK1-2 and AKT dependent early regulation of USF1, which directly induces Cdc2. To our knowledge, this is the first description of early target genes of progestin-activated classical PR via crosstalk with protein kinases and independently of hormone receptor binding to the genomic targets. PMID:24859236

  13. Biological mechanisms underlying the ultraviolet radiation-induced formation of skin wrinkling and sagging II: over-expression of neprilysin plays an essential role.

    PubMed

    Imokawa, Genji; Nakajima, Hiroaki; Ishida, Koichi

    2015-04-08

    Our previous studies strongly indicated that the up-regulated activity of skin fibroblast-derived elastase plays a pivotal role in wrinkling and/or sagging of the skin via the impairment of elastic fiber configuration and the subsequent loss of skin elasticity. Fortunately, we succeeded in identifying human skin fibroblast-derived elastase as a previously known enzyme, neprilysin or neutral endopeptidase (NEP). We have also characterized epithelial-mesenchymal paracrine cytokine interactions between UVB-exposed-keratinocytes and dermal fibroblasts and found that interleukin-1α and granulocyte macrophage colony stimulatory factor (GM-CSF) are intrinsic cytokines secreted by UVB-exposed keratinocytes that stimulate the expression of neprilysin by fibroblasts. On the other hand, direct UVA exposure of human fibroblasts significantly stimulates the secretion of IL-6 and also elicits a significant increase in the gene expression of matrix metallo-protease(MMP)-1 as well as neprilysin (to a lesser extent), which is followed by distinct increases in their protein and enzymatic activity levels. Direct UVA exposure of human keratinocytes also stimulates the secretion of IL-6, IL-8 and GM-CSF but not of IL-1 and endothelin-1. These findings suggest that GM-CSF secreted by UVA-exposed keratinocytes as well as IL-6 secreted by UVA-exposed dermal fibroblasts play important and additional roles in UVA-induced sagging and wrinkling by up-regulation of neprilysin and MMP-1, respectively, in dermal fibroblasts.

  14. The CCND1 870G>A polymorphism is a risk factor for t(11;14)(q13;q32) multiple myeloma

    PubMed Central

    Chen, Bowang; Försti, Asta; Hosking, Fay J; Broderick, Peter; Ma, Yussanne P; Dobbins, Sara E; Hose, Dirk; Walker, Brian A; Davies, Faith E; Kaiser, Martin F; Li, Ni L; Gregory, Walter A; Jackson, Graham H; Witzens-Harig, Mathias; Neben, Kai; Hoffmann, Per; Nöthen, Markus M; Mühleisen, Thomas W; Eisele, Lewin; Ross, Fiona M; Jauch, Anna; Goldschmidt, Hartmut; Houlston, Richard S; Morgan, Gareth J; Hemminki, Kari

    2016-01-01

    A number of specific chromosomal abnormalities define the subgroups of multiple myeloma. In a meta-analysis of two genomewide association studies of multiple myeloma totaling 1,661 patients we investigated risk for developing a specific tumor karyotype. The t(11;14) (q13;q32) translocation in which CCDN1 is placed under the control of the immunoglobin heavy chain enhancer was strongly associated with the CCDN1 870G>A polymorphism (P =7.96 x10-11). These results provide for a model in which a constitutional genetic factor is associated with risk of a specific chromosomal translocation. PMID:23502783

  15. Over-expression of JcDGAT1 from Jatropha curcas increases seed oil levels and alters oil quality in transgenic Arabidopsis thaliana.

    PubMed

    Misra, Aparna; Khan, Kasim; Niranjan, Abhishek; Nath, Pravendra; Sane, Vidhu A

    2013-12-01

    The increasing consumption of fossil fuels and petroleum products is leading to their rapid depletion and is a matter of concern around the globe. Substitutes of fossil fuels are required to sustain the pace of economic development. In this context, oil from the non food crops (biofuel) has shown potential to substitute fossil fuels. Jatropha curcas is an excellent shrub spread and naturalized across the globe. Its oil contains a high percentage of unsaturated fatty acids (about 78-84% of total fatty acid content) making the oil suitable for biodiesel production. Despite its high oil content, it has been poorly studied in terms of important enzymes/genes responsible for oil biosynthesis. Here, we describe the isolation of the full length cDNA clone of JcDGAT1, a key enzyme involved in oil biosynthesis, from J. curcas seeds and manipulation of oil content and composition in transgenic Arabidopsis plants by its expression. Transcript analysis of JcDGAT1 reveals a gradual increase from early seed development to its maturation. Homozygous transgenic Arabidopsis lines expressing JcDGAT1 both under CaMV35S promoter and a seed specific promoter show an enhanced level of total oil content (up by 30-41%) in seeds but do not show any phenotypic differences. In addition, our studies also show alterations in the oil composition through JcDGAT1 expression. While the levels of saturated FAs such as palmitate and stearate in the oil do not change, there is significant reproducible decrease in the levels of oleic acid and a concomitant increase in levels of linolenic acid both under the CaMV35S promoter as well as the seed specific promoter. Our studies thus confirm that DGAT is involved in flux control in oil biosynthesis and show that JcDGAT1 could be used specifically to manipulate and improve oil content and composition in plants. PMID:24125179

  16. Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase delta.

    PubMed

    Abe, Hiroko; Fujita, Yasuko; Takaoka, Yuki; Kurita, Eri; Yano, Shuntaro; Tanaka, Naotaka; Nakayama, Ken-ichi

    2009-09-01

    Ethanol damages the cell membrane and functional proteins, gradually reducing cell viability, and leading to cell death during fermentation which impairs effective bioethanol production by budding yeast Saccharomyces cerevisiae. To obtain more suitable strains for bioethanol production and to gain a better understanding of ethanol tolerance, ethanol-tolerant mutants were isolated using the novel mutagenesis technique based on the disparity theory of evolution. According to this theory evolution can be accelerated by affecting the lagging-strand synthesis in which DNA polymerase delta is involved. Expression of the pol3-01 gene, a proofreading-deficient of DNA polymerase delta, in S. cerevisiae W303-1A grown under conditions of increasing ethanol concentration resulted in three ethanol-tolerant mutants (YFY1, YFY2 and YFY3), which could grow in medium containing 13% ethanol. Ethanol productivity also increased in YFY strains compared to the wild-type strain in medium containing 25% glucose. Cell morphology of YFY strain cells was normal even in the presence of 8% ethanol, whereas W303-1A cells were expanded by a big vacuole. Furthermore, two of these mutants were also resistant to high-temperature, Calcofluor white and NaCl. Expression levels of TPS1 and TSL1, which are responsible for trehalose biosynthesis, were higher in YFY strains relative to W303-1A, resulting in high levels of intracellular trehalose in YFY strains. This contributed to the multiple-stress tolerance that makes YFY strains suitable for the production of bioethanol.

  17. Cloning, over-expression and characterization of an alkali-tolerant endo-β-1,4-mannanase from Penicillium freii F63.

    PubMed

    Wang, Yawei; Shi, Pengjun; Luo, Huiying; Bai, Yingguo; Huang, Huoqing; Yang, Peilong; Xiong, Hairong; Yao, Bin

    2012-06-01

    A glycosyl hydrolase family 5 endo-β-mannanase gene (man5F63) was cloned from Penicillium freii F63 and overexpressed in Pichia pastoris. man5F63 contained an open reading frame of 1260 bp that encoded a polypeptide of 419 amino acids including a putative 18-residue signal peptide. The recombinant enzyme (rMan5F63) was secreted into the culture supernatant to near electrophoretic homogeneity with a high yield (1.1 gl(-1) in flask). Its apparent molecular weight was approximately 72.0 kDa, 29.0 kDa higher than the theoretical molecular mass. rMan5F63 was optimal at pH 4.5 and 60 °C and exhibited good stability over a broad pH range from acidic to alkaline (>85.0% activity at pH 4.0-9.0, >70.0% activity at pH 10.0 and 43.7% even at pH 12.0). The activity of rMan5F63 was significantly enhanced in the presence of Co(2+), Cu(2+), Mn(2+) and β-mercaptoethanol and was strongly inhibited by Hg(2+) and SDS. The specific activity, K(m) and V(max) values were 47.5 U mg(-1), 7.8 mg ml(-1) and 70.4 μmol min(-1)mg(-1), respectively, for locust bean gum, and 40.3 U mg(-1), 2.3 mg ml(-1) and 61.7 μmol min(-1)mg(-1), respectively, for konjac flour. All these favorable enzymatic properties make it cost-effective to commercialization and valuable in various industries.

  18. Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors.

    PubMed

    Saarelainen, Kati S; Ranna, Martin; Rabe, Holger; Sinkkonen, Saku T; Möykkynen, Tommi; Uusi-Oukari, Mikko; Linden, Anni-Maija; Lüddens, Hartmut; Korpi, Esa R

    2008-04-01

    The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.

  19. CD90/THY1 is over-expressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

    SciTech Connect

    True, Lawrence D.; Zhang, Hui; Ye, Mingliang; Huang, Chung-Ying; Nelson, Peter S.; Von Haller, Priska D.; Tjoelker, Larry W.; Kim, Jong Seo; Qian, Weijun; Smith, Richard D.; Ellis, William J.; Liebeskind, Emily S.; Liu, Alvin Y.

    2010-10-01

    A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of Nglycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared to non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, approximately 5-to-10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma plays a role in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ~90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, Western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

  20. Genetic variation in the immunosuppression pathway genes and breast cancer susceptibility: a pooled analysis of 42,510 cases and 40,577 controls from the Breast Cancer Association Consortium.

    PubMed

    Lei, Jieping; Rudolph, Anja; Moysich, Kirsten B; Behrens, Sabine; Goode, Ellen L; Bolla, Manjeet K; Dennis, Joe; Dunning, Alison M; Easton, Douglas F; Wang, Qin; Benitez, Javier; Hopper, John L; Southey, Melissa C; Schmidt, Marjanka K; Broeks, Annegien; Fasching, Peter A; Haeberle, Lothar; Peto, Julian; Dos-Santos-Silva, Isabel; Sawyer, Elinor J; Tomlinson, Ian; Burwinkel, Barbara; Marmé, Frederik; Guénel, Pascal; Truong, Thérèse; Bojesen, Stig E; Flyger, Henrik; Nielsen, Sune F; Nordestgaard, Børge G; González-Neira, Anna; Menéndez, Primitiva; Anton-Culver, Hoda; Neuhausen, Susan L; Brenner, Hermann; Arndt, Volker; Meindl, Alfons; Schmutzler, Rita K; Brauch, Hiltrud; Hamann, Ute; Nevanlinna, Heli; Fagerholm, Rainer; Dörk, Thilo; Bogdanova, Natalia V; Mannermaa, Arto; Hartikainen, Jaana M; Van Dijck, Laurien; Smeets, Ann; Flesch-Janys, Dieter; Eilber, Ursula; Radice, Paolo; Peterlongo, Paolo; Couch, Fergus J; Hallberg, Emily; Giles, Graham G; Milne, Roger L; Haiman, Christopher A; Schumacher, Fredrick; Simard, Jacques; Goldberg, Mark S; Kristensen, Vessela; Borresen-Dale, Anne-Lise; Zheng, Wei; Beeghly-Fadiel, Alicia; Winqvist, Robert; Grip, Mervi; Andrulis, Irene L; Glendon, Gord; García-Closas, Montserrat; Figueroa, Jonine; Czene, Kamila; Brand, Judith S; Darabi, Hatef; Eriksson, Mikael; Hall, Per; Li, Jingmei; Cox, Angela; Cross, Simon S; Pharoah, Paul D P; Shah, Mitul; Kabisch, Maria; Torres, Diana; Jakubowska, Anna; Lubinski, Jan; Ademuyiwa, Foluso; Ambrosone, Christine B; Swerdlow, Anthony; Jones, Michael; Chang-Claude, Jenny

    2016-01-01

    Immunosuppression plays a pivotal role in assisting tumors to evade immune destruction and promoting tumor development. We hypothesized that genetic variation in the immunosuppression pathway genes may be implicated in breast cancer tumorigenesis. We included 42,510 female breast cancer cases and 40,577 controls of European ancestry from 37 studies in the Breast Cancer Association Consortium (2015) with available genotype data for 3595 single nucleotide polymorphisms (SNPs) in 133 candidate genes. Associations between genotyped SNPs and overall breast cancer risk, and secondarily according to estrogen receptor (ER) status, were assessed using multiple logistic regression models. Gene-level associations were assessed based on principal component analysis. Gene expression analyses were conducted using RNA sequencing level 3 data from The Cancer Genome Atlas for 989 breast tumor samples and 113 matched normal tissue samples. SNP rs1905339 (A>G) in the STAT3 region was associated with an increased breast cancer risk (per allele odds ratio 1.05, 95 % confidence interval 1.03-1.08; p value = 1.4 × 10(-6)). The association did not differ significantly by ER status. On the gene level, in addition to TGFBR2 and CCND1, IL5 and GM-CSF showed the strongest associations with overall breast cancer risk (p value = 1.0 × 10(-3) and 7.0 × 10(-3), respectively). Furthermore, STAT3 and IL5 but not GM-CSF were differentially expressed between breast tumor tissue and normal tissue (p value = 2.5 × 10(-3), 4.5 × 10(-4) and 0.63, respectively). Our data provide evidence that the immunosuppression pathway genes STAT3, IL5, and GM-CSF may be novel susceptibility loci for breast cancer in women of European ancestry.

  1. Diversion of carbon flux from gibberellin to steviol biosynthesis by over-expressing SrKA13H induced dwarfism and abnormality in pollen germination and seed set behaviour of transgenic Arabidopsis.

    PubMed

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2015-07-01

    This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 μg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 μg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set.

  2. Diversion of carbon flux from gibberellin to steviol biosynthesis by over-expressing SrKA13H induced dwarfism and abnormality in pollen germination and seed set behaviour of transgenic Arabidopsis.

    PubMed

    Guleria, Praveen; Masand, Shikha; Yadav, Sudesh Kumar

    2015-07-01

    This paper documents the engineering of Arabidopsis thaliana for the ectopic over-expression of SrKA13H (ent-kaurenoic acid-13 hydroxylase) cDNA from Stevia rebaudiana. HPLC analysis revealed the significant accumulation of steviol (1-3 μg g(-1) DW) in two independent transgenic Arabidopsis lines over-expressing SrKA13H compared with the control. Independent of the steviol concentrations detected, both transgenic lines showed similar reductions in endogenous bioactive gibberellins (GA1 and GA4). They possessed phenotypic similarity to gibberellin-deficient mutants. The reduction in endogenous gibberellin content was found to be responsible for dwarfism in the transgenics. The exogenous application of GA3 could rescue the transgenics from dwarfism. The hypocotyl, rosette area, and stem length were all considerably reduced in the transgenics. A noteworthy decrease in pollen viability was noticed and, similarly, a retardation of 60-80% in pollen germination rate was observed. The exogenous application of steviol (0.2, 0.5, and 1.0 μg ml(-1)) did not influence pollen germination efficiency. This has suggested that in planta formation of steviol was not responsible for the observed changes in transgenic Arabidopsis. Further, the seed yield of the transgenics was reduced by 24-48%. Hence, this study reports for the first time that over-expression of SrKA13H cDNA in Arabidopsis has diverted the gibberellin biosynthetic route towards steviol biosynthesis. The Arabidopsis transgenics showed a significant reduction in endogenous gibberellins that might be responsible for the dwarfism, and the abnormal behaviour of pollen germination and seed set. PMID:25954046

  3. Cyclin D type does not influence cell cycle response to DNA damage caused by ionizing radiation in multiple myeloma tumours.

    PubMed

    Smith, Dean; Mann, David; Yong, Kwee

    2016-06-01

    Multiple myeloma (MM) is characterized by over-expression of cyclin D1 (CCND1) or D2 (CCND2), which control G1 phase cell-cycle progression. Proteolytic degradation of CCND1 (but not CCND2), resulting in G1 arrest, is reported in non-MM cells post-DNA damage, affecting DNA repair and survival. We examined the effect of ionizing radiation (IR) on D-cyclin levels and cell-cycle kinetics of MM cells, exploring differences based on D-cyclin expression. We showed that CCND1 is downregulated, whereas CCND2 is not, following IR. This did not lead to hypo-phosphorylation of retinoblastoma protein or G1 arrest. Both CCND1- and CCND2-expressing MM cells arrested in S/G2/M, and did not differ in other cell-cycle proteins or sensitivity to IR. When treated with a CDK4/6 inhibitor, both CCND1 and CCND2 MM cells arrested in G1 and therefore are subject to physiological regulation at this checkpoint. Immunoprecipitation showed that, despite CCND1 degradation following IR, sufficient protein remains bound to CDK4/6 to prevent G1 arrest. Aberrant expression of CCND1 driven from the IGH promoter in t(11;14) MM cells maintains progression through G1 to arrest in S/G2/M. Differential expression of D-cyclin does not appear to affect cell-cycle response to IR, and is unlikely to underlie differential sensitivity to DNA damage. PMID:27146121

  4. Primary over-expression of AβPP in muscle does not lead to the development of inclusion body myositis in a new lineage of the MCK-AβPP transgenic mouse

    PubMed Central

    Luo, Yue-Bei; Johnsen, Russell D; Griffiths, Lisa; Needham, Merrilee; Fabian, Victoria A; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-01-01

    The aim of this study is to determine whether primary over-expression of AβPP in skeletal muscle results in the development of features of inclusion body myositis (IBM) in a new lineage of the MCK-AβPP transgenic mouse. Quantitative histological, immunohistochemical and western blotting studies were performed on muscles from 3 to 18 month old transgenic and wild-type C57BL6/SJL mice. Electron microscopy was also performed on muscle sections from selected animals. Although western blotting confirmed that there was over-expression of full length AβPP in transgenic mouse muscles, deposition of amyloid-β and fibrillar amyloid could not be demonstrated histochemically or with electron microscopy. Additionally, other changes typical of IBM such as rimmed vacuoles, cytochrome C oxidase-deficient fibres, upregulation of MHC antigens, lymphocytic inflammatory infiltration and T cell fibre invasion were absent. The most prominent finding in both transgenic and wild-type animals was the presence of tubular aggregates which was age-related and largely restricted to male animals. Expression of full length AβPP in this MCK-AβPP mouse lineage did not reach the levels required for immunodetection or deposition of amyloid-β as in the original transgenic strains, and was not associated with the development of pathological features of IBM. These negative results emphasise the potential pitfalls of re-deriving transgenic mouse strains in different laboratories. PMID:24205796

  5. Diesel exhaust particles induce the over expression of tumor necrosis factor-alpha (TNF-alpha) gene in alvelor machrophage and failed to induce apoptosis through activation of nuclear factor-kappaB (NF-kappaB)

    EPA Science Inventory

    Exposure to particulate matter (PM2.5-10), including diesel exhaust particles (DEP) has been reported to induce lung injury and exacerbation of asthma and chronic obstructive pulmonary disease. Alveolar macrophages play a major role in the lung's response to inhaled particles and...

  6. The nitrogen responsive transcriptome in potato (Solanum tuberosum L.) reveals significant gene regulatory motifs.

    PubMed

    Gálvez, José Héctor; Tai, Helen H; Lagüe, Martin; Zebarth, Bernie J; Strömvik, Martina V

    2016-01-01

    Nitrogen (N) is the most important nutrient for the growth of potato (Solanum tuberosum L.). Foliar gene expression in potato plants with and without N supplementation at 180 kg N ha(-1) was compared at mid-season. Genes with consistent differences in foliar expression due to N supplementation over three cultivars and two developmental time points were examined. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed with supplemented N. Functional relationships between over-expressed genes were found. The main metabolic pathway represented among differentially expressed genes was amino acid metabolism. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed and nine overrepresented motifs were found using three motif discovery algorithms (Seeder, Weeder and MEME). These results point to coordinated gene regulation at the transcriptional level controlling steady state potato responses to N sufficiency. PMID:27193058

  7. The nitrogen responsive transcriptome in potato (Solanum tuberosum L.) reveals significant gene regulatory motifs.

    PubMed

    Gálvez, José Héctor; Tai, Helen H; Lagüe, Martin; Zebarth, Bernie J; Strömvik, Martina V

    2016-05-19

    Nitrogen (N) is the most important nutrient for the growth of potato (Solanum tuberosum L.). Foliar gene expression in potato plants with and without N supplementation at 180 kg N ha(-1) was compared at mid-season. Genes with consistent differences in foliar expression due to N supplementation over three cultivars and two developmental time points were examined. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed with supplemented N. Functional relationships between over-expressed genes were found. The main metabolic pathway represented among differentially expressed genes was amino acid metabolism. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed and nine overrepresented motifs were found using three motif discovery algorithms (Seeder, Weeder and MEME). These results point to coordinated gene regulation at the transcriptional level controlling steady state potato responses to N sufficiency.

  8. The nitrogen responsive transcriptome in potato (Solanum tuberosum L.) reveals significant gene regulatory motifs

    PubMed Central

    Gálvez, José Héctor; Tai, Helen H.; Lagüe, Martin; Zebarth, Bernie J.; Strömvik, Martina V.

    2016-01-01

    Nitrogen (N) is the most important nutrient for the growth of potato (Solanum tuberosum L.). Foliar gene expression in potato plants with and without N supplementation at 180 kg N ha−1 was compared at mid-season. Genes with consistent differences in foliar expression due to N supplementation over three cultivars and two developmental time points were examined. In total, thirty genes were found to be over-expressed and nine genes were found to be under-expressed with supplemented N. Functional relationships between over-expressed genes were found. The main metabolic pathway represented among differentially expressed genes was amino acid metabolism. The 1000 bp upstream flanking regions of the differentially expressed genes were analysed and nine overrepresented motifs were found using three motif discovery algorithms (Seeder, Weeder and MEME). These results point to coordinated gene regulation at the transcriptional level controlling steady state potato responses to N sufficiency. PMID:27193058

  9. Cyclin D1, a novel molecular marker of minimal residual disease, in metastatic neuroblastoma.

    PubMed

    Cheung, Irene Y; Feng, Yi; Vickers, Andrew; Gerald, William; Cheung, Nai-Kong V

    2007-04-01

    Accurate monitoring of minimal residual disease (MRD) is critical for the management of metastatic neuroblastoma (NB). We evaluated cyclin D1 (CCND1), a cell-cycle control gene, as a novel MRD marker of NB. Using quantitative reverse transcriptase-polymerase chain reaction, we studied CCND1 expression in 133 solid tumors of different histological types, including 39 NB tumors, and examined its potential clinical utility as an early response marker in the bone marrows before and after treatment of 118 stage 4 patients enrolled after induction chemotherapy in an immunotherapy protocol. Based on 40 normal marrow and peripheral blood samples, a CCND1 transcript value greater than the mean + 2 SD was defined as positive. Sensitivity of this assay was one NB cell in 10(6) normal mononuclear cells. CCND1 transcript levels were high in NB, breast cancer, and Ewing family tumors. Among the NB patients evaluated, early (2.5 months from protocol entry) marrow response was strongly associated with both progression-free (P=0.0001) and overall survival (P=0.0006). CCND1 response remained predictive of survival among a subset of 66 patients who had no histological evidence of marrow disease before immunotherapy. We conclude that CCND1 has potential clinical utility as a novel molecular marker of MRD in the bone marrow of patients with metastatic NB.

  10. JMJD2A attenuation affects cell cycle and tumourigenic inflammatory gene regulation in lipopolysaccharide stimulated neuroectodermal stem cells

    SciTech Connect

    Das, Amitabh; Chai, Jin Choul; Jung, Kyoung Hwa; Das, Nando Dulal; Kang, Sung Chul; Lee, Young Seek; Seo, Hyemyung; Chai, Young Gyu

    2014-11-01

    JMJD2A is a lysine trimethyl-specific histone demethylase that is highly expressed in a variety of tumours. The role of JMJD2A in tumour progression remains unclear. The objectives of this study were to identify JMJD2A-regulated genes and understand the function of JMJD2A in p53-null neuroectodermal stem cells (p53{sup −/−} NE-4Cs). We determined the effect of LPS as a model of inflammation in p53{sup −/−} NE-4Cs and investigated whether the epigenetic modifier JMJD2A alter the expression of tumourigenic inflammatory genes. Global gene expression was measured in JMJD2A knockdown (kd) p53{sup −/−} NE-4Cs and in LPS-stimulated JMJD2A-kd p53{sup −/−} NE-4C cells. JMJD2A attenuation significantly down-regulated genes were Cdca2, Ccnd2, Ccnd1, Crebbp, IL6rα, and Stat3 related with cell cycle, proliferation, and inflammatory-disease responses. Importantly, some tumour-suppressor genes including Dapk3, Timp2 and TFPI were significantly up-regulated but were not affected by silencing of the JMJD2B. Furthermore, we confirmed the attenuation of JMJD2A also down-regulated Cdca2, Ccnd2, Crebbp, and Rest in primary NSCs isolated from the forebrains of E15 embryos of C57/BL6J mice with effective p53 inhibitor pifithrin-α (PFT-α). Transcription factor (TF) motif analysis revealed known binding patterns for CDC5, MYC, and CREB, as well as three novel motifs in JMJD2A-regulated genes. IPA established molecular networks. The molecular network signatures and functional gene-expression profiling data from this study warrants further investigation as an effective therapeutic target, and studies to elucidate the molecular mechanism of JMJD2A-kd-dependent effects in neuroectodermal stem cells should be performed. - Highlights: • Significant up-regulation of epigenetic modifier JMJD2A mRNA upon LPS treatment. • Inhibition of JMJD2A attenuated key inflammatory and tumourigenic genes. • Establishing IPA based functional genomics in JMJD2A-attenuated p53{sup

  11. Effect of angiotensin II type 1 receptor blocker on renal function, arterial blood pressure and parathyroid hormone related protein over expression in cadmium induced nephrotoxicity in adult male rats

    PubMed Central

    Ahmed, Marwa A

    2013-01-01

    Objective: To study the possible effect of angiotensin II type 1 Receptor blocker (AT1 blocker) on renal function, arterial blood pressure and parathyroid hormone related protein over expression in cadmium induced nephrotoxicity in adult male rats. Forty five rats were divided randomly into a control (group I), group II, received cadmium chloride at a dose of 5 mg/kg/day, orally, for nine weeks, group III received telmisartan (TEL) treatment (1 mg/kg/day, orally) one week before cadmium administration and continued for ten weeks. Results: Telmisartan significantly reduced blood urea nitrogen (BUN) and serum creatinine levels which were increased significantly by cadmium. Telmisartan significantly suppressed lipid peroxidation, compensated deficits in the antioxidant defenses (super oxide dismutase (SOD) level and catalase activity), decreased the elevations of nitric oxide (NO) and cadmium ion concentrations in renal tissue observed in Cd-treated rats. Group III had a significant decrease of urinary levels of total protein, N-acetyl-β-d-glucosaminidase (NAG), alkaline phosphatase (ALP) and γ-glutamyl-transpeptidase (GGT) and urinary 8-isoprostanes than those of group II. Telmisartan decreased the systolic blood pressure significantly than those of group II. Histopathological examination revealed that cadmium-induced renal tissue damage was ameliorated by telmisartan treatment. Immunohistochemical analysis revealed that telmisartan significantly decreased the cadmium-induced overexpression of parathyroid hormone receptor 1 (PTHR1) in renal tissue. RT-PCR analysis showed that Cd increased renal expression of PTHrP; however telmisartan could decrease the expression of PTHrP in group III. Conclusion: Blocking AT1 receptors significantly decreases PTHrP over expression and ameliorates renal dysfunction in Cd induced nephrotoxicity. These data suggest that Ang II might contribute to pathophysiology and deleterious effects in cadmium nephrotoxicity. PMID:23750309

  12. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  13. miR-630 targets IGF1R to regulate response to HER-targeting drugs and overall cancer cell progression in HER2 over-expressing breast cancer

    PubMed Central

    2014-01-01

    Background While the treatment of HER2 over-expressing breast cancer with recent HER-targeted drugs has been highly effective for some patients, primary (also known as innate) or acquired resistance limits the success of these drugs. microRNAs have potential as diagnostic, prognostic and predictive biomarkers, as well as replacement therapies. Here we investigated the role of microRNA-630 (miR-630) in breast cancer progression and as a predictive biomarker for response to HER-targeting drugs, ultimately yielding potential as a therapeutic approach to add value to these drugs. Methods We investigated the levels of intra- and extracellular miR-630 in cells and conditioned media from breast cancer cell lines with either innate- or acquired- resistance to HER-targeting lapatinib and neratinib, compared to their corresponding drug sensitive cell lines, using qPCR. To support the role of miR-630 in breast cancer, we examined the clinical relevance of this miRNA in breast cancer tumours versus matched peritumours. Transfection of miR-630 mimics and inhibitors was used to manipulate the expression of miR-630 to assess effects on response to HER-targeting drugs (lapatinib, neratinib and afatinib). Other phenotypic changes associated with cellular aggressiveness were evaluated by motility, invasion and anoikis assays. TargetScan prediction software, qPCR, immunoblotting and ELISAs, were used to assess miR-630’s regulation of mRNA, proteins and their phosphorylated forms. Results We established that introducing miR-630 into cells with innate- or acquired- resistance to HER-drugs significantly restored the efficacy of lapatinib, neratinib and afatinib; through a mechanism which we have determined to, at least partly, involve miR-630’s regulation of IGF1R. Conversely, we demonstrated that blocking miR-630 induced resistance/insensitivity to these drugs. Cellular motility, invasion, and anoikis were also observed as significantly altered by miR-630 manipulation, whereby

  14. MicroRNA library screening identifies growth-suppressive microRNAs that regulate genes involved in cell cycle progression and apoptosis.

    PubMed

    Choi, Young-Chul; Yoon, Sena; Byun, Yuree; Lee, Gangtae; Kee, Honghwan; Jeong, Yongsu; Yoon, Jaeseung; Baek, Kwanghee

    2015-12-10

    Micro(mi)RNAs play important and varied roles in tumorigenesis; however, the full repertoire of miRNAs that affect cancer cell growth is not known. In this study, an miRNA library was screened to identify those that affect the growth of A549 tumor cells. Among 300 miRNAs, miR-28-5p, -323-5p, -510-5p, -552-3p, and -608 were the most effective in inhibiting cell growth. More specifically, overexpressing miR-28-5p, -323-5p, and -510-5p induced G1 arrest, as determined by flow cytometry, whereas that of miR-608 induced cell death in a caspase-dependent manner. Moreover, several genes involved in apoptosis and cell cycle progression were downregulated upon overexpression of each of the five miRNAs, with the functional targets of miR-552-3p and miR-608 confirmed by microarray, quantitative real-time PCR, and luciferase reporter assay. In miR-608-transfected cells, B cell lymphoma 2-like 1 (BCL2L1), D-type cyclin 1 (CCND1), CCND3, cytochrome b5 reductase 3 (CYB5R3), phosphoinositide 3-kinase regulatory subunit 2 (PIK3R2), specificity protein 1 (SP1), and phosphorylated Akt were all downregulated, while Bcl-2-interacting killer (BIK) was upregulated. Moreover, miR-608 was determined to have a suppressive function on tumor growth in an NCI-H460 xenograft model. These findings provide insights into the roles of five miRNAs in growth inhibition and their potential function as cancer therapeutics. PMID:26485640

  15. Dichlorodiphenyltrichloroethane technical mixture regulates cell cycle and apoptosis genes through the activation of CAR and ERα in mouse livers

    SciTech Connect

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2013-09-01

    Dichlorodiphenyltrichloroethane (DDT) is a widely used organochlorine pesticide and a xenoestrogen that promotes rodent hepatomegaly and tumours. A recent study has shown significant correlation between DDT serum concentration and liver cancer incidence in humans, but the underlying mechanisms remain elusive. We hypothesised that a mixture of DDT isomers could exert effects on the liver through pathways instead of classical ERs. The acute effects of a DDT mixture containing the two major isomers p,p′-DDT (85%) and o,p′-DDT (15%) on CAR and ERα receptors and their cell cycle and apoptosis target genes were studied in mouse livers. ChIP results demonstrated increased CAR and ERα recruitment to their specific target gene binding sites in response to the DDT mixture. The results of real-time RT-PCR were consistent with the ChIP data and demonstrated that the DDT was able to activate both CAR and ERα in mouse livers, leading to target gene transcriptional increases including Cyp2b10, Gadd45β, cMyc, Mdm2, Ccnd1, cFos and E2f1. Western blot analysis demonstrated increases in cell cycle progression proteins cMyc, Cyclin D1, CDK4 and E2f1 and anti-apoptosis proteins Mdm2 and Gadd45β. In addition, DDT exposure led to Rb phosphorylation. Increases in cell cycle progression and anti-apoptosis proteins were accompanied by a decrease in p53 content and its transcriptional activity. However, the DDT was unable to stimulate the β-catenin signalling pathway, which can play an important role in hepatocyte proliferation. Thus, our results indicate that DDT treatment may result in cell cycle progression and apoptosis inhibition through CAR- and ERα-mediated gene activation in mouse livers. These findings suggest that the proliferative and anti-apoptotic conditions induced by CAR and ERα activation may be important contributors to the early stages of hepatocarcinogenesis as produced by DDT in rodent livers. - Highlights: • DDT activated both CAR and ERα and their cell

  16. Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPP1) and protein phosphatase type 1 (PP1) in Drosophila: consequences of over-expression of NIPP1 in flies and suppression by co-expression of PP1.

    PubMed Central

    Parker, Louise; Gross, Sascha; Beullens, Monique; Bollen, Mathieu; Bennett, Daimark; Alphey, Luke

    2002-01-01

    The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates. We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system. NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro, binding to RNA and PP1c, and localization to nuclear speckles. However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila. NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown. Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages. The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function. Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo, suggesting that the NIPP1Dm-PP1c holoenzyme is not normally limiting in Drosophila. These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1's role as a phosphatase regulator is conserved in Drosophila. PMID:12358598

  17. Elevated glutathione level does not protect against chronic alcohol mediated apoptosis in recombinant human hepatoma cell line VL-17A over-expressing alcohol metabolizing enzymes--alcohol dehydrogenase and Cytochrome P450 2E1.

    PubMed

    Chandrasekaran, Karthikeyan; Swaminathan, Kavitha; Kumar, S Mathan; Chatterjee, Suvro; Clemens, Dahn L; Dey, Aparajita

    2011-06-01

    Chronic consumption of alcohol leads to liver injury. Ethanol-inducible Cytochrome P450 2E1 (CYP2E1) plays a critical role in alcohol mediated oxidative stress due to its ability to metabolize ethanol. In the present study, using the recombinant human hepatoma cell line VL-17A that over-expresses the alcohol metabolizing enzymes-alcohol dehydrogenase (ADH) and CYP2E1; and control HepG2 cells, the mechanism and mode of cell death due to chronic ethanol exposure were studied. Untreated VL-17A cells exhibited apoptosis and oxidative stress when compared with untreated HepG2 cells. Chronic alcohol exposure, i.e., 100 mM ethanol treatment for 72 h caused a significant decrease in viability (47%) in VL-17A cells but not in HepG2 cells. Chronic ethanol mediated cell death in VL-17A cells was predominantly apoptotic, with increased oxidative stress as the underlying mechanism. Chronic ethanol exposure of VL-17A cells resulted in 1.1- to 2.5-fold increased levels of ADH and CYP2E1. Interestingly, the level of the antioxidant GSH was found to be 3-fold upregulated in VL-17A cells treated with ethanol, which may be a metabolic adaptation to the persistent and overwhelming oxidative stress. In conclusion, the increased GSH level may not be sufficient enough to protect VL-17A cells from chronic alcohol mediated oxidative stress and resultant apoptosis. PMID:21414402

  18. Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach.

    PubMed

    Tozlu, S; Girault, I; Vacher, S; Vendrell, J; Andrieu, C; Spyratos, F; Cohen, P; Lidereau, R; Bieche, I

    2006-12-01

    The estrogen receptor alpha (ERalpha) plays a critical role in the pathogenesis and clinical behavior of breast cancer. To obtain further insights into the molecular basis of estrogen-dependent forms of this malignancy, we used real-time quantitative reverse transcription (RT)-PCR to compare the mRNA expression of 560 selected genes in ERalpha-positive and ERalpha-negative breast tumors. Fifty-one (9.1%) of the 560 genes were significantly upregulated in ERalpha-positive breast tumors compared with ERalpha-negative breast tumors. In addition to well-known ERalpha-induced genes (PGR, TFF1/PS2, BCL2, ERBB4, CCND1, etc.) and genes recently identified by cDNA microarray-based approaches (GATA3, TFF3, MYB, STC2, HPN/HEPSIN, FOXA1, XBP1, SLC39A6/LIV-1, etc.), an appreciable number of novel genes were identified, many of, which were weakly expressed. This validates the use of large-scale real-time RT-PCR as a method complementary to cDNA microarrays for molecular tumor profiling. Most of the new genes identified here encoded secreted proteins (SEMA3B and CLU), growth factors (BDNF, FGF2 and EGF), growth factor receptors (IL6ST, PTPRT, RET, VEGFR1 and FGFR2) or metabolic enzymes (CYP2B6, CA12, ACADSB, NAT1, LRBA, SLC7A2 and SULT2B1). Importantly, we also identified a large number of genes encoding proteins with either pro-apoptotic (PUMA, NOXA and TATP73) or anti-apoptotic properties (BCL2, DNTP73 and TRAILR3). Surprisingly, only a small proportion of the 51 genes identified in breast tumor biopsy specimens were confirmed to be ERalpha-regulated and/or E2-regulated in vitro (cultured cell lines). Therefore, this study identified a limited number of genes and signaling pathways, which better delineate the role of ERalpha in breast cancer. Some of the genes identified here could be useful for diagnosis or for predicting endocrine responsiveness, and could form the basis for novel therapeutic strategies.

  19. Interaction between amiodarone and hepatitis-C virus nucleotide inhibitors in human induced pluripotent stem cell-derived cardiomyocytes and HEK-293 Cav1.2 over-expressing cells.

    PubMed

    Lagrutta, Armando; Zeng, Haoyu; Imredy, John; Balasubramanian, Bharathi; Dech, Spencer; Lis, Edward; Wang, Jixin; Zhai, Jin; DeGeorge, Joseph; Sannajust, Frederick

    2016-10-01

    Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels.

  20. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib

    PubMed Central

    Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

    2014-01-01

    Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF “like” ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation. PMID:25249545

  1. Neurotensin (NTS) and its receptor (NTSR1) causes EGFR, HER2 and HER3 over-expression and their autocrine/paracrine activation in lung tumors, confirming responsiveness to erlotinib.

    PubMed

    Younes, Mohamad; Wu, Zherui; Dupouy, Sandra; Lupo, Audrey Mansuet; Mourra, Najat; Takahashi, Takashi; Fléjou, Jean François; Trédaniel, Jean; Régnard, Jean François; Damotte, Diane; Alifano, Marco; Forgez, Patricia

    2014-09-30

    Alterations in the signaling pathways of epidermal growth factor receptors (HERs) are associated with tumor aggressiveness. Neurotensin (NTS) and its high affinity receptor (NTSR1) are up regulated in 60% of lung cancers. In a previous clinical study, NTSR1 overexpression was shown to predict a poor prognosis for 5 year overall survival in a selected population of stage I lung adenocarcinomas treated by surgery alone. In a second study, shown here, the frequent and high expression of NTSR1 was correlated with a pejorative prognosis in 389 patients with stage I to III lung adenocarcinoma, and was an independent prognosis marker. Interactions between NTS and NTSR1 induce pro-oncogenic biological effects associated with neoplastic processes and tumor progression. Here we highlight the cellular mechanisms activated by Neurotensin (NTS) and its high affinity receptor (NTSR1) contributing to lung cancer cell aggressiveness. We show that the NTS autocrine and/or paracrine regulation causes EGFR, HER2, and HER3 over-expression and activation in lung tumor cells. The EGFR and HER3 autocrine activation is mediated by MMP1 activation and EGF "like" ligands (HB-EGF, Neuregulin 1) release. By establishing autocrine and/or paracrine NTS regulation, we show that tumor growth is modulated according to NTS expression, with a low growth rate in those tumors that do not express NTS. Accordingly, xenografted tumors expressing NTS and NTSR1 showed a positive response to erlotinib, whereas tumors void of NTSR1 expression had no detectable response. This is consistent with the presence of a NTS autocrine loop, leading to the sustained activation of EGFR and responsible for cancer aggressiveness. We propose the use of NTS/NTSR1 tumor expression, as a biomarker for the use of EGFR tyrosine kinase inhibitors in patients lacking EGFR mutation.

  2. High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

    PubMed Central

    2012-01-01

    Background To balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled plasma – mass spectrometry (ICP-MS) we quantify Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn in 11890 mutant strains, including 4940 haploid and 1127 diploid deletion strains, and 5798 over expression strains. Results We identified 1065 strains with an altered ionome, including 584 haploid and 35 diploid deletion strains, and 446 over expression strains. Disruption of protein metabolism or trafficking has the highest likelihood of causing large ionomic changes, with gene dosage also being important. Gene over expression produced more extreme ionomic changes, but over expression and loss of function phenotypes are generally not related. Ionomic clustering revealed the existence of only a small number of possible ionomic profiles suggesting fitness tradeoffs that constrain the ionome. Clustering also identified important roles for the mitochondria, vacuole and ESCRT pathway in regulation of the ionome. Network analysis identified hub genes such as PMR1 in Mn homeostasis, novel members of ionomic networks such as SMF3 in vacuolar retrieval of Mn, and cross-talk between the mitochondria and the vacuole. All yeast ionomic data can be searched and downloaded at http://www.ionomicshub.org. Conclusions Here, we demonstrate the power of high-throughput ICP-MS analysis to functionally dissect the ionome on a genome-wide scale. The information this reveals has the potential to benefit both human health and agriculture. PMID:23151179

  3. Elevated autophagy gene expression in adipose tissue of obese humans: A potential non-cell-cycle-dependent function of E2F1

    PubMed Central

    Haim, Yulia; Blüher, Matthias; Slutsky, Noa; Goldstein, Nir; Klöting, Nora; Harman-Boehm, Ilana; Kirshtein, Boris; Ginsberg, Doron; Gericke, Martin; Guiu Jurado, Esther; Kovsan, Julia; Tarnovscki, Tanya; Kachko, Leonid; Bashan, Nava; Gepner, Yiftach; Shai, Iris; Rudich, Assaf

    2015-01-01

    Autophagy genes' expression is upregulated in visceral fat in human obesity, associating with obesity-related cardio-metabolic risk. E2F1 (E2F transcription factor 1) was shown in cancer cells to transcriptionally regulate autophagy. We hypothesize that E2F1 regulates adipocyte autophagy in obesity, associating with endocrine/metabolic dysfunction, thereby, representing non-cell-cycle function of this transcription factor. E2F1 protein (N=69) and mRNA (N=437) were elevated in visceral fat of obese humans, correlating with increased expression of ATG5 (autophagy-related 5), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), but not with proliferation/cell-cycle markers. Elevated E2F1 mainly characterized the adipocyte fraction, whereas MKI67 (marker of proliferation Ki-67) was elevated in the stromal-vascular fraction of adipose tissue. In human visceral fat explants, chromatin-immunoprecipitation revealed body mass index (BMI)-correlated increase in E2F1 binding to the promoter of MAP1LC3B, but not to the classical cell cycle E2F1 target, CCND1 (cyclin D1). Clinically, omental fat E2F1 expression correlated with insulin resistance, circulating free-fatty-acids (FFA), and with decreased circulating ADIPOQ/adiponectin, associations attenuated by adjustment for autophagy genes. Overexpression of E2F1 in HEK293 cells enhanced promoter activity of several autophagy genes and autophagic flux, and sensitized to further activation of autophagy by TNF. Conversely, mouse embryonic fibroblast (MEF)-derived adipocytes from e2f1 knockout mice (e2f1−/−) exhibited lower autophagy gene expression and flux, were more insulin sensitive, and secreted more ADIPOQ. Furthermore, e2f1−/− MEF-derived adipocytes, and autophagy-deficient (by Atg7 siRNA) adipocytes were resistant to cytokines-induced decrease in ADIPOQ secretion. Jointly, upregulated E2F1 sensitizes adipose tissue autophagy to inflammatory stimuli, linking visceral obesity to adipose and systemic

  4. Influence of mitochondria on gene expression in a citrus cybrid.

    PubMed

    Bassene, Jean-Baptiste; Froelicher, Yann; Navarro, Luis; Ollitrault, Patrick; Ancillo, Gema

    2011-06-01

    The production of cybrids, combining nucleus of a species with alien cytoplasmic organelles, is a valuable method used for improvement of various crops. Several citrus cybrids have been created by somatic hybridization. These genotypes are interesting models to analyze the impact of cytoplasmic genome change on nuclear genome expression. Herein, we report genome-wide gene expression analysis in leaves of a citrus cybrid between C. reticulata cv 'Willowleaf mandarin' and C. limon cv 'Eureka lemon' compared with its lemon parent, using a Citrus 20K cDNA microarray. Molecular analysis showed that this cybrid possesses nuclear and chloroplast genomes of Eureka lemon plus mitochondria from Willowleaf mandarin and, therefore, can be considered as a lemon bearing foreign mitochondria. Mandarin mitochondria influenced the expression of a large set of lemon nuclear genes causing an over-expression of 480 of them and repression of 39 genes. Quantitative real-time RT-PCR further confirmed the credibility of microarray data. Genes over-expressed in cybrid leaves are predominantly attributed to the functional category "cellular protein metabolism" whereas in the down-regulated none functional category was enriched. Overall, mitochondria replacement affected different nuclear genes including particularly genes predicted to be involved in mitochondrial retrograde signaling. Mitochondria regulate all cell structures even chloroplast status. These results suggest that nuclear gene expression is modulated with respect to new information received from the foreign organelle, with the final objective to suit specific needs to ensure better cell physiological balance.

  5. Tetracycline-inducible system for regulation of skeletal muscle-specific gene expression in transgenic mice

    NASA Technical Reports Server (NTRS)

    Grill, Mischala A.; Bales, Mark A.; Fought, Amber N.; Rosburg, Kristopher C.; Munger, Stephanie J.; Antin, Parker B.

    2003-01-01

    Tightly regulated control of over-expression is often necessary to study one aspect or time point of gene function and, in transgenesis, may help to avoid lethal effects and complications caused by ubiquitous over-expression. We have utilized the benefits of an optimized tet-on system and a modified muscle creatine kinase (MCK) promoter to generate a skeletal muscle-specific, doxycycline (Dox) controlled over-expression system in transgenic mice. A DNA construct was generated in which the codon optimized reverse tetracycline transactivator (rtTA) was placed under control of a skeletal muscle-specific version of the mouse MCK promoter. Transgenic mice containing this construct expressed rtTA almost exclusively in skeletal muscles. These mice were crossed to a second transgenic line containing a bi-directional promoter centered on a tet responder element driving both a luciferase reporter gene and a tagged gene of interest; in this case the calpain inhibitor calpastatin. Compound hemizygous mice showed high level, Dox dependent muscle-specific luciferase activity often exceeding 10,000-fold over non-muscle tissues of the same mouse. Western and immunocytochemical analysis demonstrated similar Dox dependent muscle-specific induction of the tagged calpastatin protein. These findings demonstrate the effectiveness and flexibility of the tet-on system to provide a tightly regulated over-expression system in adult skeletal muscle. The MCKrtTA transgenic lines can be combined with other transgenic responder lines for skeletal muscle-specific over-expression of any target gene of interest.

  6. Genes and gene regulation

    SciTech Connect

    MacLean, N.

    1988-01-01

    Genetics has long been a central topic for biologists, and recent progress has captured the public imagination as well. This book addresses questions that are at the leading edge of this continually advancing discipline. In tune with the increasing emphasis on molecular biology and genetic engineering, this text emphasizes the molecular aspects of gene expression, and the evolution of gene sequence organization and control. It reviews the genetic material of viruses, bacteria, and of higher organisms. Cells and organisms are compared in terms of gene numbers, their arrangements within a cell, and the control mechanisms which regulate the activity of genes.

  7. Over-expression of the miRNA cluster at chromosome 14q32 in the alcoholic brain correlates with suppression of predicted target mRNA required for oligodendrocyte proliferation.

    PubMed

    Manzardo, A M; Gunewardena, S; Butler, M G

    2013-09-10

    We examined miRNA expression from RNA isolated from the frontal cortex (Broadman area 9) of 9 alcoholics (6 males, 3 females, mean age 48 years) and 9 matched controls using both the Affymetrix GeneChip miRNA 2.0 and Human Exon 1.0 ST Arrays to further characterize genetic influences in alcoholism and the effects of alcohol consumption on predicted target mRNA expression. A total of 12 human miRNAs were significantly up-regulated in alcohol dependent subjects (fold change≥1.5, false discovery rate (FDR)≤0.3; p<0.05) compared with controls including a cluster of 4 miRNAs (e.g., miR-377, miR-379) from the maternally expressed 14q32 chromosome region. The status of the up-regulated miRNAs was supported using the high-throughput method of exon microarrays showing decreased predicted mRNA gene target expression as anticipated from the same RNA aliquot. Predicted mRNA targets were involved in cellular adhesion (e.g., THBS2), tissue differentiation (e.g., CHN2), neuronal migration (e.g., NDE1), myelination (e.g., UGT8, CNP) and oligodendrocyte proliferation (e.g., ENPP2, SEMA4D1). Our data support an association of alcoholism with up-regulation of a cluster of miRNAs located in the genomic imprinted domain on chromosome 14q32 with their predicted gene targets involved with oligodendrocyte growth, differentiation and signaling. PMID:23747354

  8. Heterologous over-expression of ACC SYNTHASE8 (ACS8) in Populus tremula x P. alba clone 717-1B4 results in elevated levels of ethylene and induces stem dwarfism and reduced leaf size through separate genetic pathways.

    PubMed

    Plett, Jonathan M; Williams, Martin; LeClair, Gaetan; Regan, Sharon; Beardmore, Tannis

    2014-01-01

    Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism. PMID:25414707

  9. Heterologous over-expression of ACC SYNTHASE8 (ACS8) in Populus tremula x P. alba clone 717-1B4 results in elevated levels of ethylene and induces stem dwarfism and reduced leaf size through separate genetic pathways

    PubMed Central

    Plett, Jonathan M.; Williams, Martin; LeClair, Gaetan; Regan, Sharon; Beardmore, Tannis

    2014-01-01

    Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism. PMID:25414707

  10. Characterization of Glossy1-homologous genes in rice involved in leaf wax accumulation and drought resistance.

    PubMed

    Islam, Mohammad Asadul; Du, Hao; Ning, Jing; Ye, Haiyan; Xiong, Lizhong

    2009-07-01

    The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. Glossy 1 (GL1) is one of the reported genes controlling wax synthesis. This study analyzed GL1-homologous genes in Oryza sativa and characterized the key members of this family involved in wax synthesis and stress resistance. Sequence analysis revealed 11 homologous genes of GL1 in rice, designated OsGL1-1 to OsGL1-11. OsGL1-1, -2 and -3 are closely related to GL1. OsGL1-4, -5, -6, and -7 are closely related to Arabidopsis CER1 that is involved in cuticular wax biosynthesis. OsGL1-8, -9, -10 and -11 are closely related to SUR2 encoding a putative sterol desaturase also involved in epicuticular wax biosynthesis. These genes showed variable expression levels in different tissues and organs of rice, and most of them were induced by abiotic stresses. Compared to the wild type, the OsGL1-2-over-expression rice exhibited more wax crystallization and a thicker epicuticular layer; while the mutant of this gene showed less wax crystallization and a thinner cuticular layer. Chlorophyll leaching experiment suggested that the cuticular permeability was decreased and increased in the over-expression lines and the mutant, respectively. Quantification analysis of wax composition by GC-MS revealed a significant reduction of total cuticular wax in the mutant and increase of total cuticular wax in the over-expression plants. Compared to the over-expression and wild type plants, the osgl1-2 mutant was more sensitive to drought stress at reproductive stage, suggesting an important role of this gene in drought resistance.

  11. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...

  12. Absence of repellents in Ustilago maydis induces genes encoding small secreted proteins.

    PubMed

    Teertstra, Wieke R; Krijgsheld, Pauline; Wösten, Han A B

    2011-08-01

    The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.

  13. Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC

    PubMed Central

    Forestier, Anne; Douki, Thierry; De Rosa, Viviana; Béal, David; Rachidi, Walid

    2015-01-01

    Repair of oxidative DNA damage, particularly Base Excision Repair (BER), impairment is often associated with Alzheimer’s disease pathology. Here, we aimed at investigating the complete Nucleotide Excision Repair (NER), a DNA repair pathway involved in the removal of bulky DNA adducts, status in an Alzheimer-like cell line. The level of DNA damage was quantified using mass spectrometry, NER gene expression was assessed by qPCR, and the NER protein activity was analysed through a modified version of the COMET assay. Interestingly, we found that in the presence of the Amyloid β peptide (Aβ), NER factors were upregulated at the mRNA level and that NER capacities were also specifically increased following oxidative stress. Surprisingly, NER capacities were not differentially improved following a typical NER-triggering of ultraviolet C (UVC) stress. Oxidative stress generates a differential and specific DNA damage response in the presence of Aβ. We hypothesized that the release of NER components such as DNA damage binding protein 2 (DDB2) and Xeroderma Pigmentosum complementation group C protein (XPC) following oxidative stress might putatively involve their apoptotic role rather than DNA repair function. PMID:26263968

  14. Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy.

    PubMed

    Danda, Ravikanth; Krishnan, Gopinath; Ganapathy, Kalaivani; Krishnan, Uma Maheswari; Vikas, Khetan; Elchuri, Sailaja; Chatterjee, Nivedita; Krishnakumar, Subramanian

    2013-01-01

    In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus-thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3'UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM (+ve)/let-7b(down-regulated)), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM (-ve)/let-7b(up-regulated)), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM (+ve)/let-7b(up-regulated)) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells. PMID:24391761

  15. Regulatory roles of spnT, a novel gene located within transposon TnTIR.

    PubMed

    Wei, Jun-Rong; Soo, Po-Chi; Horng, Yu-Tze; Hsieh, Shang-Chen; Tsai, Yu-Huan; Swift, Simon; Withers, Helen; Williams, Paul; Lai, Hsin-Chih

    2006-09-29

    The transposon TnTIR contains spnIR quorum-sensing system regulating sliding motility and the production of nuclease, biosurfactant, and prodigiosin in Serratia marcescens. Within TnTIR, a gene named spnT is upstream of and co-transcribed with spnI. SpnT is a cytoplasmic protein and its level peaks during early stationary phase. spnT over-expression resulted in inhibition of sliding motility and synthesis of prodigiosin, and biosurfactant similar to spnR. spnT but not spnR over-expression induced cell elongation and aberrant DNA replication in S. marcescens and Escherichia coli strains. In comparison with wild-type E. coli strain, over-expression of spnT in an E. coli priA and dnaC double-mutant strain did not lead to the aberrant cell morphology phenotypes, suggesting SpnT may act through the recombination-dependent DNA replication system. As spnT over-expression inhibited swarming but not swimming motility, SpnT may indirectly function as a negative regulator of surface-dependent migration and secondary metabolite production.

  16. Gene-based and semantic structure of the Gene Ontology as a complex network

    NASA Astrophysics Data System (ADS)

    Coronnello, Claudia; Tumminello, Michele; Miccichè, Salvatore

    2016-09-01

    The last decade has seen the advent and consolidation of ontology based tools for the identification and biological interpretation of classes of genes, such as the Gene Ontology. The Gene Ontology (GO) is constantly evolving over time. The information accumulated time-by-time and included in the GO is encoded in the definition of terms and in the setting up of semantic relations amongst terms. Here we investigate the Gene Ontology from a complex network perspective. We consider the semantic network of terms naturally associated with the semantic relationships provided by the Gene Ontology consortium. Moreover, the GO is a natural example of bipartite network of terms and genes. Here we are interested in studying the properties of the projected network of terms, i.e. a gene-based weighted network of GO terms, in which a link between any two terms is set if at least one gene is annotated in both terms. One aim of the present paper is to compare the structural properties of the semantic and the gene-based network. The relative importance of terms is very similar in the two networks, but the community structure changes. We show that in some cases GO terms that appear to be distinct from a semantic point of view are instead connected, and appear in the same community when considering their gene content. The identification of such gene-based communities of terms might therefore be the basis of a simple protocol aiming at improving the semantic structure of GO. Information about terms that share large gene content might also be important from a biomedical point of view, as it might reveal how genes over-expressed in a certain term also affect other biological processes, molecular functions and cellular components not directly linked according to GO semantics.

  17. Evaluation of efflux pump gene expression among drug susceptible and drug resistant strains of Mycobacterium tuberculosis from Iran.

    PubMed

    Kardan Yamchi, Jalil; Haeili, Mehri; Gizaw Feyisa, Seifu; Kazemian, Hossein; Hashemi Shahraki, Abdolrazagh; Zahednamazi, Fatemeh; Imani Fooladi, Abbas Ali; Feizabadi, Mohammad Mehdi

    2015-12-01

    Absence of mutations within the genes encoding drug targets in some phenotypically drug resistant strains of Mycobacterium tuberculosis suggests possible involvement of alternative mechanisms such as over-expression of efflux pumps. We investigated the expression level of Rv1410c, Rv2459, Rv1218c and Rv1273c efflux pumps gene by real-time quantitative reverse transcription PCR (qRT-PCR) in 31 clinical isolates of M. tuberculosis. Susceptibility to first-line drugs was performed using the proportion method. Twenty one isolates were characterized with drug resistance (DR), and among them 12 showed a significantly elevated level of expression (>4 fold) for at least one of the studied genes encoding for efflux pumps. Point mutations in the katG (codons 315 or 335) and rpoB (codons 456 and 441) genes were found in 42.85% and 66.6% of drug resistant isolates, respectively. Only one isolate showed mutation at position -15 of the inhA promoter region. Among the 7 isolates (33.33%) which had no mutation in the studied regions of drug target genes, 5 isolates showed over-expression for efflux pumps. Our results demonstrated that over-expression of efflux pumps can contribute to drug resistance in M. tuberculosis.

  18. A predictor for predicting Escherichia coli transcriptome and the effects of gene perturbations

    PubMed Central

    2014-01-01

    Background A means to predict the effects of gene over-expression, knockouts, and environmental stimuli in silico is useful for system biologists to develop and test hypotheses. Several studies had predicted the expression of all Escherichia coli genes from sequences and reported a correlation of 0.301 between predicted and actual expression. However, these do not allow biologists to study the effects of gene perturbations on the native transcriptome. Results We developed a predictor to predict transcriptome-scale gene expression from a small number (n = 59) of known gene expressions using gene co-expression network, which can be used to predict the effects of over-expressions and knockdowns on E. coli transcriptome. In terms of transcriptome prediction, our results show that the correlation between predicted and actual expression value is 0.467, which is similar to the microarray intra-array variation (p-value = 0.348), suggesting that intra-array variation accounts for a substantial portion of the transcriptome prediction error. In terms of predicting the effects of gene perturbation(s), our results suggest that the expression of 83% of the genes affected by perturbation can be predicted within 40% of error and the correlation between predicted and actual expression values among the affected genes to be 0.698. With the ability to predict the effects of gene perturbations, we demonstrated that our predictor has the potential to estimate the effects of varying gene expression level on the native transcriptome. Conclusion We present a potential means to predict an entire transcriptome and a tool to estimate the effects of gene perturbations for E. coli, which will aid biologists in hypothesis development. This study forms the baseline for future work in using gene co-expression network for gene expression prediction. PMID:24884349

  19. Dissection of the oncogenic MYCN transcriptional network reveals a large set of clinically relevant cell cycle genes as drivers of neuroblastoma tumorigenesis.

    PubMed

    Murphy, Derek M; Buckley, Patrick G; Bryan, Kenneth; Watters, Karen M; Koster, Jan; van Sluis, Peter; Molenaar, Jan; Versteeg, Rogier; Stallings, Raymond L

    2011-06-01

    Amplification of the oncogenic transcription factor MYCN plays a major role in the pathogenesis of several pediatric cancers, including neuroblastoma, medulloblastoma, and rhabodomyosarcoma. For neuroblastoma, MYCN amplification is the most powerful genetic predictor of poor patient survival, yet the mechanism by which MYCN drives tumorigenesis is only partially understood. To gain an insight into the distribution of MYCN binding and to identify clinically relevant MYCN target genes, we performed an integrated analysis of MYCN ChIP-chip and mRNA expression using the MYCN repressible SHEP-21N neuroblastoma cell line. We hypothesized that genes exclusively MYCN bound in SHEP-21N cells over-expressing MYCN would be enriched for direct targets which contribute to the process of disease progression. Integrated analysis revealed that MYCN drives tumorigenesis predominantly as a positive regulator of target gene transcription. A high proportion of genes (24%) that are MYCN bound and up-regulated in the SHEP-21N model are significantly associated with poor overall patient survival (OS) in a set of 88 tumors. In contrast, the proportion of genes down-regulated when bound by MYCN in the SHEP-21N model and which are significantly associated with poor overall patient survival when under-expressed in primary tumors was significantly lower (5%). Gene ontology analysis determined a highly statistically significant enrichment for cell cycle related genes within the over-expressed MYCN target group which were also associated with poor OS. We conclude that the over-expression of MYCN leads to aberrant binding and over-expression of genes associated with cell cycle regulation which are significantly correlated with poor OS and MYCN amplification.

  20. Inducible gene expression in transgenic Xenopus embryos.

    PubMed

    Wheeler, G N; Hamilton, F S; Hoppler, S

    2000-07-13

    The amphibian Xenopus laevis has been successfully used for many years as a model system for studying vertebrate development. Because of technical limitations, however, molecular investigations have mainly concentrated on early stages. We have developed a straightforward method for stage-specific induction of gene expression in transgenic Xenopus embryos [1] [2]. This method is based on the Xenopus heat shock protein 70 (Xhsp70 [3]) promoter driving the expression of desired gene products. We found that ubiquitous expression of the transgene is induced upon relatively mild heat treatment. Green fluorescent protein (GFP) was used as a marker to monitor successful induction of gene expression in transgenic embryos. We used this method to study the stage specificity of Wnt signalling function. Transient ectopic Wnt-8 expression during early neurulation was sufficient to repress anterior head development and this capacity was restricted to early stages of neurulation. By transient over-expression at different stages of development, we show that frizzled-7 disrupted morphogenesis sequentially from anterior to posterior along the dorsal axis as development proceeds. These results demonstrate that this method for inducible gene expression in transgenic Xenopus embryos will be a very powerful tool for temporal analysis of gene function and for studying molecular mechanisms of vertebrate organogenesis.

  1. Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function

    SciTech Connect

    Yoshihara, Takashi; Collado, Denise; Hamaguchi, Masaaki . E-mail: hamaguchi@fordham.edu

    2007-07-13

    The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.

  2. Hen uterine gene expression profiling during eggshell formation reveals putative proteins involved in the supply of minerals or in the shell mineralization process

    PubMed Central

    2014-01-01

    Background The chicken eggshell is a natural mechanical barrier to protect egg components from physical damage and microbial penetration. Its integrity and strength is critical for the development of the embryo or to ensure for consumers a table egg free of pathogens. This study compared global gene expression in laying hen uterus in the presence or absence of shell calcification in order to characterize gene products involved in the supply of minerals and / or the shell biomineralization process. Results Microarrays were used to identify a repertoire of 302 over-expressed genes during shell calcification. GO terms enrichment was performed to provide a global interpretation of the functions of the over-expressed genes, and revealed that the most over-represented proteins are related to reproductive functions. Our analysis identified 16 gene products encoding proteins involved in mineral supply, and allowed updating of the general model describing uterine ion transporters during eggshell calcification. A list of 57 proteins potentially secreted into the uterine fluid to be active in the mineralization process was also established. They were classified according to their potential functions (biomineralization, proteoglycans, molecular chaperone, antimicrobials and proteases/antiproteases). Conclusions Our study provides detailed descriptions of genes and corresponding proteins over-expressed when the shell is mineralizing. Some of these proteins involved in the supply of minerals and influencing the shell fabric to protect the egg contents are potentially useful biological markers for the genetic improvement of eggshell quality. PMID:24649854

  3. ObRb downregulation increases breast cancer cell sensitivity to tamoxifen.

    PubMed

    Qian, Yingying; Shi, Dongmin; Qiu, Jinrong; Zhu, Fang; Qian, Jing; He, Shaohua; Shu, Yongqian; Yin, Yongmei; Chen, Xiaofeng

    2015-09-01

    Leptin is a potent adipokine that plays an important role in the progression of breast cancer and interferes with the action of tamoxifen. We investigated the molecular mechanism underlying the effect of leptin on tamoxifen resistance in breast cancer cells that express leptin receptor (ObRb), and evaluated the impact of ObRb suppression on tamoxifen treatment in MCF-7 and tamoxifen-resistant (TAM-R) cells. Leptin-induced signaling pathway activation was examined by qRT-PCR and Western blotting. Chromatin immunoprecipitation assays were performed to further examine the binding of estrogen receptor (ER) α on the promoter of cyclin D1 (CCND1) gene. The effects of combined ObRb knockdown and tamoxifen treatment were evaluated in MCF-7 and TAM-R cells. We found that the enhanced proliferation effects induced by leptin were related to extracellular-signal-regulated kinase (ERK) 1/2 and signal transducers and activators of transcription (STAT) 3 signaling pathway activation and CCND1 upregulation. Leptin enhanced CCND1 gene transcription by inducing the binding of ERα to the promoter of CCND1 gene. ObRb knockdown significantly enhanced the inhibitory effects of tamoxifen on TAM-R cell proliferation and survival. This study suggested that long-term endocrine therapy facilitates leptin and ObRb overexpression in breast cancer cells, which attenuates the inhibitory effect of tamoxifen by activating both the ERK1/2 and STAT3 signaling pathways and upregulating CCND1 gene expression. Combination therapy involving ObRb knockdown and tamoxifen treatment may be an alternative therapeutic option for tamoxifen-resistant breast cancer.

  4. Characteristics of carboxylesterase genes and their expression-level between acaricide-susceptible and resistant Tetranychus cinnabarinus (Boisduval).

    PubMed

    Wei, Peng; Shi, Li; Shen, Guangmao; Xu, Zhifeng; Liu, Jialu; Pan, Yu; He, Lin

    2016-07-01

    Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation. PMID:27265830

  5. Evolvability and hierarchy in rewired bacterial gene networks

    PubMed Central

    Isalan, Mark; Lemerle, Caroline; Michalodimitrakis, Konstantinos; Beltrao, Pedro; Horn, Carsten; Raineri, Emanuele; Garriga-Canut, Mireia; Serrano, Luis

    2009-01-01

    Sequencing DNA from several organisms has revealed that duplication and drift of existing genes have primarily molded the contents of a given genome. Though the effect of knocking out or over-expressing a particular gene has been studied in many organisms, no study has systematically explored the effect of adding new links in a biological network. To explore network evolvability, we constructed 598 recombinations of promoters (including regulatory regions) with different transcription or σ-factor genes in Escherichia coli, added over a wild-type genetic background. Here we show that ~95% of new networks are tolerated by the bacteria, that very few alter growth, and that expression level correlates with factor position in the wild-type network hierarchy. Most importantly, we find that certain networks consistently survive over the wild-type under various selection pressures. Therefore new links in the network are rarely a barrier for evolution and can even confer a fitness advantage. PMID:18421347

  6. Immunologic Applications of Conditional Gene Modification Technology in the Mouse

    PubMed Central

    Sharma, Suveena; Zhu, Jinfang

    2014-01-01

    Since the success of homologous recombination in altering mouse genome and the discovery of Cre-loxP system, the combination of these two breakthroughs has created important applications for studying the immune system in the mouse. Here, we briefly summarize the general principles of this technology and its applications in studying immune cell development and responses; such implications include conditional gene knockout and inducible and/or tissue-specific gene over-expression, as well as lineage fate mapping. We then discuss the pros and cons of a few commonly used Cre-expressing mouse lines for studying lymphocyte development and functions. We also raise several general issues, such as efficiency of gene deletion, leaky activity of Cre, and Cre toxicity, all of which may have profound impacts on data interpretation. Finally, we selectively list some useful links to the Web sites as valuable mouse resources. PMID:24700321

  7. Association of cyclin D1 genotype with breast cancer risk and survival.

    PubMed

    Shu, Xiao Ou; Moore, Derek B; Cai, Qiuyin; Cheng, Jiarong; Wen, Wanqing; Pierce, Larry; Cai, Hui; Gao, Yu-Tang; Zheng, Wei

    2005-01-01

    Cyclin D1 (CCND1) is a key cell cycle regulatory protein that governs cell cycle progression from the G(1) to S phase. A common polymorphism (A870G) in exon 4 of the CCND1 gene produces an alternate transcript (transcript-b) that preferentially encodes a protein with enhanced cell transformation activity and possible prolonged half-life. We evaluated the association of CCND1 A870G polymorphism with breast cancer risk and survival in 1,130 breast cancer cases and 1,196 controls who participated in the Shanghai Breast Cancer Study. Approximately 81% of cases and 79% of controls carried the A allele at A870G of the CCND1 gene [odds ratio, 1.1; 95% confidence interval (95% CI), 0.9-1.4]. As lightly stronger but nonsignificant association was found for the A allele among younger women (odds ratio, 1.3; 95% CI, 0.9-1.8). However, this polymorphism seems to modify the effect of hormonal exposures on postmenopausal breast cancer, as the positive associations of postmenopausal breast cancer with body mass index (Pfor interaction = 0.02) and waist-to-hip ratios (P for interaction < 0.03; all Ps are two sided) were only observed among women who carry the A allele at A870G of the CCND1 gene. Following up with this cohort of patients for an average of 4.84 years, we found that the CCND1 A870G polymorphism was inversely associated with overall and disease-free survival, particularly among women with late stage or estrogen/progesterone receptor-negative breast cancer. The adjusted hazard ratios for disease-free survival associated with GA and AA genotypes were 0.94 (95% CI, 0.49-1.82) and 0.41 (95% CI, 0.19-0.91) for tumor-node-metastasis stage III to IV breast cancer, and 0.35 (95% CI, 0.15-0.80) and 0.32 (95% CI, 0.13-0.79) for estrogen/progesterone receptor-negative breast cancer. This study suggests that CCND1 A870G polymorphism may modify the postmenopausal breast cancer risk associated with hormonal exposure and predict survival after breast cancer diagnosis. PMID:15668481

  8. Identification of a cis-regulatory element by transient analysis of co-ordinately regulated genes

    PubMed Central

    Dare, Andrew P; Schaffer, Robert J; Lin-Wang, Kui; Allan, Andrew C; Hellens, Roger P

    2008-01-01

    Background Transcription factors (TFs) co-ordinately regulate target genes that are dispersed throughout the genome. This co-ordinate regulation is achieved, in part, through the interaction of transcription factors with conserved cis-regulatory motifs that are in close proximity to the target genes. While much is known about the families of transcription factors that regulate gene expression in plants, there are few well characterised cis-regulatory motifs. In Arabidopsis, over-expression of the MYB transcription factor PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) leads to transgenic plants with elevated anthocyanin levels due to the co-ordinated up-regulation of genes in the anthocyanin biosynthetic pathway. In addition to the anthocyanin biosynthetic genes, there are a number of un-associated genes that also change in expression level. This may be a direct or indirect consequence of the over-expression of PAP1. Results Oligo array analysis of PAP1 over-expression Arabidopsis plants identified genes co-ordinately up-regulated in response to the elevated expression of this transcription factor. Transient assays on the promoter regions of 33 of these up-regulated genes identified eight promoter fragments that were transactivated by PAP1. Bioinformatic analysis on these promoters revealed a common cis-regulatory motif that we showed is required for PAP1 dependent transactivation. Conclusion Co-ordinated gene regulation by individual transcription factors is a complex collection of both direct and indirect effects. Transient transactivation assays provide a rapid method to identify direct target genes from indirect target genes. Bioinformatic analysis of the promoters of these direct target genes is able to locate motifs that are common to this sub-set of promoters, which is impossible to identify with the larger set of direct and indirect target genes. While this type of analysis does not prove a direct interaction between protein and DNA, it does provide a tool to

  9. Pristinamycin-inducible gene regulation in mycobacteria.

    PubMed

    Forti, Francesca; Crosta, Andrea; Ghisotti, Daniela

    2009-03-25

    In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin I. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer. Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction; both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin I. Thus it showed to be an effective inducible system in mycobacteria. PMID:19428723

  10. NME genes in epithelial morphogenesis

    PubMed Central

    2012-01-01

    The NME family of genes encodes highly conserved multifunctional proteins that have been shown to participate in nucleic acid metabolism, energy homeostasis, cell signaling, and cancer progression. Some family members, particularly isoforms 1 and 2, have attracted extensive interests because of their potential anti-metastasis activity. Unfortunately, there have been few consensus mechanistic explanations for this critical function because of the numerous molecular functions ascribed to these proteins, including nucleoside diphosphate kinase, protein kinase, nuclease, transcription factor, growth factor, among others. In addition, different studies showed contradictory prognostic correlations between NME expression levels and tumor progression in clinical samples. Thus, analyses using pliable in vivo systems have become critical for unraveling at least some aspects of the complex functions of this family of genes. Recent works using the Drosophila genetic system have suggested a role for NME in regulating epithelial cell motility and morphogenesis, which has also been demonstrated in mammalian epithelial cell culture. This function is mediated by promoting internalization of growth factor receptors in motile epithelial cells, and the adherens junction components such as E-cadherin and β-catenin in epithelia that form the tissue linings. Interestingly, NME genes in epithelial cells appear to function in a defined range of expression levels. Either down-regulation or over-expression can perturb epithelial integrity, resulting in different aspects of epithelial abnormality. Such biphasic functions provide a plausible explanation for the documented anti-metastatic activity and the suspected oncogenic function. This review summarizes these recent findings and discusses their implications. PMID:21336542

  11. A prognostic gene expression signature in infratentorial ependymoma

    PubMed Central

    Wani, Khalida; Armstrong, Terri; Vera-Bolanos, Elizabeth; Raghunathan, Aditya; Ellison, David; Gilbertson, Richard; Vaillant, Brian; Goldman, Stewart; Packer, Roger J.; Fouladi, Maryam; Pollack, Ian; Mikkelsen, Tom; Prados, Michael; Omuro, Antonio; Soffietti, Riccardo; Ledoux, Alicia; Wilson, Charmaine; Long, Lihong; Gilbert, Mark; Aldape, Ken

    2013-01-01

    Patients with ependymoma exhibit a wide range of clinical outcomes that is currently unexplained by clinical or histological factors. Little is known regarding molecular biomarkers that could predict clinical behavior. Since recent data suggests that these tumors display biological characteristics according to their location (cerebral vs. infratentorial vs. spinal cord), rather than explore a broad spectrum of ependymoma, we focused on molecular alterations in ependymomas arising in the infratentorial compartment. Unsupervised clustering of available gene expression microarray data revealed two major subgroups of infratentorial ependymoma. Group 1 tumors over expressed genes that were associated with mesenchyme, Group 2 tumors showed no distinct gene ontologies. To assess the prognostic significance of these gene expression subgroups, real-time reverse-transcriptase polymerase chain reaction assays were performed on genes defining the subgroups in a training set. This resulted in a 10-gene prognostic signature. Multivariate analysis showed that the 10-gene signature was an independent predictor of recurrence-free survival after adjusting for clinical factors. Evaluation of an external dataset describing subgroups of infratentorial ependymomas showed concordance of subgroup definition, including validation of the mesenchymal subclass. Importantly, the 10-gene signature was validated as a predictor of recurrence-free survival in this dataset. Taken together, the results indicate a link between clinical outcome and biologically-identified subsets of infratentorial ependymoma and offer the potential for prognostic testing to estimate clinical aggressiveness in these tumors. PMID:22322993

  12. A prognostic gene expression signature in infratentorial ependymoma.

    PubMed

    Wani, Khalida; Armstrong, Terri S; Vera-Bolanos, Elizabeth; Raghunathan, Aditya; Ellison, David; Gilbertson, Richard; Vaillant, Brian; Goldman, Stewart; Packer, Roger J; Fouladi, Maryam; Pollack, Ian; Mikkelsen, Tom; Prados, Michael; Omuro, Antonio; Soffietti, Riccardo; Ledoux, Alicia; Wilson, Charmaine; Long, Lihong; Gilbert, Mark R; Aldape, Ken

    2012-05-01

    Patients with ependymoma exhibit a wide range of clinical outcomes that are currently unexplained by clinical or histological factors. Little is known regarding molecular biomarkers that could predict clinical behavior. Since recent data suggest that these tumors display biological characteristics according to their location (cerebral vs. infratentorial vs. spinal cord), rather than explore a broad spectrum of ependymoma, we focused on molecular alterations in ependymomas arising in the infratentorial compartment. Unsupervised clustering of available gene expression microarray data revealed two major subgroups of infratentorial ependymoma. Group 1 tumors over expressed genes that were associated with mesenchyme, Group 2 tumors showed no distinct gene ontologies. To assess the prognostic significance of these gene expression subgroups, real-time reverse transcriptase polymerase chain reaction assays were performed on genes defining the subgroups in a training set. This resulted in a 10-gene prognostic signature. Multivariate analysis showed that the 10-gene signature was an independent predictor of recurrence-free survival after adjusting for clinical factors. Evaluation of an external dataset describing subgroups of infratentorial ependymomas showed concordance of subgroup definition, including validation of the mesenchymal subclass. Importantly, the 10-gene signature was validated as a predictor of recurrence-free survival in this dataset. Taken together, the results indicate a link between clinical outcome and biologically identified subsets of infratentorial ependymoma and offer the potential for prognostic testing to estimate clinical aggressiveness in these tumors.

  13. Endosymbiotic gene transfer and transcriptional regulation of transferred genes in Paulinella chromatophora.

    PubMed

    Nowack, Eva C M; Vogel, Heiko; Groth, Marco; Grossman, Arthur R; Melkonian, Michael; Glöckner, Gernot

    2011-01-01

    Paulinella chromatophora is a cercozoan amoeba that contains "chromatophores," which are photosynthetic inclusions of cyanobacterial origin. The recent discovery that chromatophores evolved independently of plastids, underwent major genome reduction, and transferred at least two genes to the host nucleus has highlighted P. chromatophora as a model to infer early steps in the evolution of photosynthetic organelles. However, owing to the paucity of nuclear genome sequence data, the extent of endosymbiotic gene transfer (EGT) and host symbiont regulation are currently unknown. A combination of 454 and Illumina next generation sequencing enabled us to generate a comprehensive reference transcriptome data set for P. chromatophora on which we mapped short Illumina cDNA reads generated from cultures from the dark and light phases of a diel cycle. Combined with extensive phylogenetic analyses of the deduced protein sequences, these data revealed that 1) about 0.3-0.8% of the nuclear genes were obtained by EGT compared with 11-14% in the Plantae, 2) transferred genes show a distinct bias in that many encode small proteins involved in photosynthesis and photoacclimation, 3) host cells established control over expression of transferred genes, and 4) not only EGT, but to a minor extent also horizontal gene transfer from organisms that presumably served as food sources, helped to shape the nuclear genome of P. chromatophora. The identification of a significant number of transferred genes involved in photosynthesis and photoacclimation of thylakoid membranes as well as the observed transcriptional regulation of these genes strongly implies import of the encoded gene products into chromatophores, a feature previously thought to be restricted to canonical organelles. Thus, a possible mechanism by which P. chromatophora exerts control over the performance of its newly acquired photosynthetic organelle may involve controlling the expression of nuclear-encoded chromatophore

  14. Gene doping.

    PubMed

    Harridge, Stephen D R; Velloso, Cristiana P

    2008-01-01

    Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities.

  15. Gene Regulatory Networks Elucidating Huanglongbing Disease Mechanisms

    PubMed Central

    Martinelli, Federico; Reagan, Russell L.; Uratsu, Sandra L.; Phu, My L.; Albrecht, Ute; Zhao, Weixiang; Davis, Cristina E.; Bowman, Kim D.; Dandekar, Abhaya M.

    2013-01-01

    Next-generation sequencing was exploited to gain deeper insight into the response to infection by Candidatus liberibacter asiaticus (CaLas), especially the immune disregulation and metabolic dysfunction caused by source-sink disruption. Previous fruit transcriptome data were compared with additional RNA-Seq data in three tissues: immature fruit, and young and mature leaves. Four categories of orchard trees were studied: symptomatic, asymptomatic, apparently healthy, and healthy. Principal component analysis found distinct expression patterns between immature and mature fruits and leaf samples for all four categories of trees. A predicted protein – protein interaction network identified HLB-regulated genes for sugar transporters playing key roles in the overall plant responses. Gene set and pathway enrichment analyses highlight the role of sucrose and starch metabolism in disease symptom development in all tissues. HLB-regulated genes (glucose-phosphate-transporter, invertase, starch-related genes) would likely determine the source-sink relationship disruption. In infected leaves, transcriptomic changes were observed for light reactions genes (downregulation), sucrose metabolism (upregulation), and starch biosynthesis (upregulation). In parallel, symptomatic fruits over-expressed genes involved in photosynthesis, sucrose and raffinose metabolism, and downregulated starch biosynthesis. We visualized gene networks between tissues inducing a source-sink shift. CaLas alters the hormone crosstalk, resulting in weak and ineffective tissue-specific plant immune responses necessary for bacterial clearance. Accordingly, expression of WRKYs (including WRKY70) was higher in fruits than in leaves. Systemic acquired responses were inadequately activated in young leaves, generally considered the sites where most new infections occur. PMID:24086326

  16. Up-regulation of the interferon-related genes in BRCA2 knockout epithelial cells

    PubMed Central

    Xu, Hong; Xian, Jian; Vire, Emmanuelle; McKinney, Steven; Wong, Jason; Wei, Vivien; Tong, Rebecca; Kouzarides, Tony; Caldas, Carlos; Aparicio, Samuel

    2016-01-01

    BRCA2 mutations are significantly associated with early onset breast cancer, and the tumour suppressing function of BRCA2 has been attributed to its involvement in homologous recombination [1]-mediated DNA repair. In order to identify additional functions of BRCA2, we generated BRCA2-knockout HCT116 human colorectal carcinoma cells. Using genome-wide microarray analyses, we have discovered a link between the loss of BRCA2 and the up-regulation of a subset of interferon (IFN)-related genes, including APOBEC3F and APOBEC3G. The over-expression of IFN-related genes was confirmed in different human BRCA2−/− and mouse Brca2−/− tumour cell lines, and was independent of either senescence or apoptosis. In isogenic wild type BRCA2 cells, we observed over-expression of IFN-related genes after treatment with DNA-damaging agents, and following ionizing radiation. Cells with endogenous DNA damage because of defective BRCA1 or RAD51 also exhibited over-expression of IFN-related genes. Transcriptional activity of the IFN-stimulated response element (ISRE) was increased in BRCA2 knockout cells, and the expression of BRCA2 greatly decreased IFN-α stimulated ISRE reporter activity, suggesting that BRCA2 directly represses the expression of IFN-related genes through the ISRE. Finally, the colony forming capacity of BRCA2 knockout cells was significantly reduced in the presence of either IFN-β or IFN-γ, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. PMID:25043256

  17. Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas.

    PubMed

    Wang, Dongxu; Chen, Xianju; Song, Yuning; Lv, Qinyan; Lai, Liangxue; Li, Zhanjun

    2014-09-01

    Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.

  18. Screening of genes involved in cell migration in Dictyostelium.

    PubMed

    Nagasaki, Akira; Uyeda, Taro Q P

    2008-03-10

    A single cell of wild-type Dictyostelium discoideum forms a visible colony on a plastic dish in several days, but due to enhanced cell migration, amiB-null mutant cells scatter over a large area and do not form noticeable colonies. Here, with an aim to identify genes involved in cell migration, we isolated suppresser mutants of amiB-null mutants that restore the ability to form colonies. From REMI (restriction enzyme-mediated integration)-mutagenized pool of double-mutants, we identified 18 responsible genes from them. These genes can be categorized into several biological processes. One cell line, Sab16 (Suppressor of amiB) was chosen for further analysis, which had a disrupted phospholipase D pldB gene. To confirm the role of pldB gene in cell migration, we knocked out the pldB gene and over-expressed gfp-pldB in wild-type cells. GFP-PLDB localized to plasma membrane and on vesicles, and in migrating cells, at the protruding regions of pseudopodia. Migration speed of vegetative pldB-null cells was reduced to 73% of that of the wild-type. These results suggest that PLDB plays an important role in migration in Dictyostelium cells, and that our screening system is useful for the identification of genes involved in cell migration. PMID:18164290

  19. Upregulated, 7q21-22 amplicon candidate gene SHFM1 confers oncogenic advantage by suppressing p53 function in gastric cancer.

    PubMed

    Tamilzhalagan, Sembulingam; Muthuswami, Muthulakshmi; Periasamy, Jayaprakash; Lee, Ming Hui; Rha, Sun Young; Tan, Patrick; Ganesan, Kumaresan

    2015-06-01

    Chromosomal aberrations are hallmarks of cancers and the locus of frequent genomic amplifications often harbors key cancer driver genes. Many genomic amplicons remain larger with hundreds of genes and the key drivers remain to be identified by an amplification-wide systematic analysis. The 7q21.12-q22.3 genomic amplification is frequent in gastric cancers which occur in ~10% of the patients and multiple cell lines. This 7q21.12-q22.3 amplicon has not yet been completely analyzed towards identifying the driver genes and their functional contribution in oncogenesis. The amplitude and prevalence indicate the important role conferred by this amplicon in gastric cancers. Among the 159 genes of this amplicon, 12 genes are found over-expressed in primary gastric tumors and cell lines. Many of the over-expressed genes show negative association with p53 transcriptional activity. RNAi based functional screening of the genes reveal, SHFM1 as key gastric cancer driver gene. SHFM1 confers cell cycle progression and resistance to p53 stabilizing drugs in gastric cancer cells. SHFM1 also activates Src, MAPK/ERK and PI3K/Akt signaling pathways. This is the first integrative genomic investigation of 7q21.12-q22.3 amplicon revealing the potential oncogenic candidacy of 12 genes. The oncogenic contribution of SHFM1, mediated by the p53 suppressive feature has been demonstrated in gastric cancer cells.

  20. Trichoderma genes

    SciTech Connect

    Foreman, Pamela; Goedegebuur, Frits; Van Solingen, Pieter; Ward, Michael

    2012-06-19

    Described herein are novel gene sequences isolated from Trichoderma reesei. Two genes encoding proteins comprising a cellulose binding domain, one encoding an arabionfuranosidase and one encoding an acetylxylanesterase are described. The sequences, CIP1 and CIP2, contain a cellulose binding domain. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

  1. [Gene therapy].

    PubMed

    Rodríguez-Fragoso, L

    1997-01-01

    In the last years there has been much progress in our understanding of molecular mechanisms in the pathogenesis of disease. In this review we provide an overview of gene therapy, its most actualized techniques for gene delivery, and we give specific examples of laboratory and clinical achievements to date. The development of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. As a result, concepts and methods that would have been considered purely science fiction 50 years ago are now used in the treatment of diseases. The widespread application of gene therapy technology to many diseases is already breaking down the traditional boundaries of modern medicine. However, despite its progress, several key technical drawbacks need to be overcome before gene therapy can be used safely and effectively in clinical settings. Technological developments, particularly in the areas of gene delivery and cell transplantation, will be critical for the successful practice of gene therapy.

  2. Gene Therapy

    PubMed Central

    Baum, Bruce J

    2014-01-01

    Applications of gene therapy have been evaluated in virtually every oral tissue, and many of these have proved successful at least in animal models. While gene therapy will not be used routinely in the next decade, practitioners of oral medicine should be aware of the potential of this novel type of treatment that doubtless will benefit many patients with oral diseases. PMID:24372817

  3. Molecular dissection of the roles of the SOD genes in mammalian response to low dose irradiation

    SciTech Connect

    Eric Y. Chuang

    2006-08-31

    It has been long recognized that a significant fraction of the radiation-induced genetic damage to cells are caused by secondary oxidative species. Internal cellular defense systems against oxidative stress play significant roles in countering genetic damage induced by ionizing radiation. The role of the detoxifying enzymes may be even more prominent in the case of low-dose, low-LET irradiation, as the majority of genetic damage may be caused by secondary oxidative species. In this study we have attempted to decipher the roles of the superoxide dismutase (SOD) genes, which are responsible for detoxifying the superoxide anions. We used adenovirus vectors to deliver RNA interference (RNAi or siRNA) technology to down-regulate the expression levels of the SOD genes. We have also over-expressed the SOD genes by use of recombinant adenovirus vectors. Cells infected with the vectors were then subjected to low dose γ-irradiation. Total RNA were extracted from the exposed cells and the expression of 9000 genes were profiled by use of cDNA microarrays. The result showed that low dose radiation had clear effects on gene expression in HCT116 cells. Both over-expression and down-regulation of the SOD1 gene can change the expression profiles of sub-groups of genes. Close to 200 of the 9000 genes examined showed over two-fold difference in expression under various conditions. Genes with changed expression pattern belong to many categories that include: early growth response, DNA-repair, ion transport, apoptosis, and cytokine response.

  4. Adr1 and Cat8 Mediate Coactivator Recruitment and Chromatin Remodeling at Glucose-Regulated Genes

    PubMed Central

    Biddick, Rhiannon K.; Law, G. Lynn; Young, Elton T.

    2008-01-01

    Background Adr1 and Cat8 co-regulate numerous glucose-repressed genes in S. cerevisiae, presenting a unique opportunity to explore their individual roles in coactivator recruitment, chromatin remodeling, and transcription. Methodology/Principal Findings We determined the individual contributions of Cat8 and Adr1 on the expression of a cohort of glucose-repressed genes and found three broad categories: genes that need both activators for full derepression, genes that rely mostly on Cat8 and genes that require only Adr1. Through combined expression and recruitment data, along with analysis of chromatin remodeling at two of these genes, ADH2 and FBP1, we clarified how these activators achieve this wide range of co-regulation. We find that Adr1 and Cat8 are not intrinsically different in their abilities to recruit coactivators but rather, promoter context appears to dictate which activator is responsible for recruitment to specific genes. These promoter-specific contributions are also apparent in the chromatin remodeling that accompanies derepression: ADH2 requires both Adr1 and Cat8, whereas, at FBP1, significant remodeling occurs with Cat8 alone. Although over-expression of Adr1 can compensate for loss of Cat8 at many genes in terms of both activation and chromatin remodeling, this over-expression cannot complement all of the cat8Δ phenotypes. Conclusions/Significance Thus, at many of the glucose-repressed genes, Cat8 and Adr1 appear to have interchangeable roles and promoter architecture may dictate the roles of these activators. PMID:18197247

  5. Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes

    PubMed Central

    Sharili, Amir S.; Kenny, Fiona N.; Vartiainen, Maria K.; Connelly, John T.

    2016-01-01

    The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type β-actin or β-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration. PMID:27650314

  6. Nuclear actin modulates cell motility via transcriptional regulation of adhesive and cytoskeletal genes.

    PubMed

    Sharili, Amir S; Kenny, Fiona N; Vartiainen, Maria K; Connelly, John T

    2016-01-01

    The actin cytoskeleton is a classic biomechanical mediator of cell migration. While it is known that actin also shuttles in and out of the nucleus, its functions within this compartment remain poorly understood. In this study, we investigated how nuclear actin regulates keratinocyte gene expression and cell behavior. Gene expression profiling of normal HaCaT keratinocytes compared to HaCaTs over-expressing wild-type β-actin or β-actin tagged with a nuclear localization sequence (NLS-actin), identified multiple adhesive and cytoskeletal genes, such as MYL9, ITGB1, and VCL, which were significantly down-regulated in keratinocytes with high levels of nuclear actin. In addition, genes associated with transcriptional regulation and apoptosis were up-regulated in cells over expressing NLS-actin. Functionally, accumulation of actin in the nucleus altered cytoskeletal and focal adhesion organization and inhibited cell motility. Exclusion of endogenous actin from the nucleus by knocking down Importin 9 reversed this phenotype and enhanced cell migration. Based on these findings, we conclude that the level of actin in the nucleus is a transcriptional regulator for tuning keratinocyte migration. PMID:27650314

  7. A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat.

    PubMed

    Faheem, Muhammad; Li, Yingbo; Arshad, Muhammad; Jiangyue, Cheng; Jia, Zhao; Wang, Zongkuan; Xiao, Jin; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Yu, Feifei; Zhang, Ruiqi; Xie, Qi; Wang, Xiue

    2016-04-13

    In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a') and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-V(C57A) did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function.

  8. A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat.

    PubMed

    Faheem, Muhammad; Li, Yingbo; Arshad, Muhammad; Jiangyue, Cheng; Jia, Zhao; Wang, Zongkuan; Xiao, Jin; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Yu, Feifei; Zhang, Ruiqi; Xie, Qi; Wang, Xiue

    2016-01-01

    In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a') and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-V(C57A) did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function. PMID:27071705

  9. A disulphide isomerase gene (PDI-V) from Haynaldia villosa contributes to powdery mildew resistance in common wheat

    PubMed Central

    Faheem, Muhammad; Li, Yingbo; Arshad, Muhammad; Jiangyue, Cheng; Jia, Zhao; Wang, Zongkuan; Xiao, Jin; Wang, Haiyan; Cao, Aizhong; Xing, Liping; Yu, Feifei; Zhang, Ruiqi; Xie, Qi; Wang, Xiue

    2016-01-01

    In this study, we report the contribution of a PDI-like gene from wheat wild relative Haynaldia villosa in combating powdery mildew. PDI-V protein contains two conserved thioredoxin (TRX) active domains (a and a′) and an inactive domain (b). PDI-V interacted with E3 ligase CMPG1-V protein, which is a positive regulator of powdery mildew response. PDI-V was mono-ubiquitinated by CMPG1-V without degradation being detected. PDI-V was located on H. villosa chromosome 5V and encoded for a protein located in the endoplasmic reticulum. Bgt infection in leaves of H. villosa induced PDI-V expression. Virus induced gene silencing of PDIs in a T. durum-H. villosa amphiploid compromised the resistance. Single cell transient over-expression of PDI-V or a truncated version containing the active TXR domain a decreased the haustorial index in moderately susceptible wheat cultivar Yangmai 158. Stable transgenic lines over-expressing PDI-V in Yangmai 158 displayed improved powdery mildew resistance at both the seedling and adult stages. By contrast over-expression of point-mutated PDI-VC57A did not increase the level of resistance in Yangmai 158. The above results indicate a pivotal role of PDI-V in powdery mildew resistance and showed that conserved TRX domain a is critical for its function. PMID:27071705

  10. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed Central

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S.

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar ‘Morex’. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar ‘Morex’ or the full resistance reaction requires the presence of several PEI genes. PMID:26937960

  11. Drosophila Myc is required for normal DREF gene expression

    SciTech Connect

    Dang Thi Phuong Thao; Seto, Hirokazu; Yamaguchi, Masamitsu

    2008-01-01

    The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm{sup 4}/Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm{sup 2}/dm{sup 2} homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression.

  12. Vector systems for prenatal gene therapy: principles of adenovirus design and production.

    PubMed

    Alba, Raul; Baker, Andrew H; Nicklin, Stuart A

    2012-01-01

    Adenoviruses have many attributes, which have made them one of the most widely investigated vectors for gene therapy applications. These include ease of genetic manipulation to produce replication-deficient vectors, ability to readily generate high titer stocks, efficiency of gene delivery into many cell types, and ability to encode large genetic inserts. Recent advances in adenoviral vector engineering have included the ability to genetically manipulate the tropism of the vector by engineering of the major capsid proteins, particularly fiber and hexon. Furthermore, simple replication-deficient adenoviral vectors deleted for expression of a single gene have been complemented by the development of systems in which the majority of adenoviral genes are deleted, generating sophisticated Ad vectors which can mediate sustained transgene expression following a single delivery. This chapter outlines methods for developing simple transgene over expressing Ad vectors and detailed strategies to engineer mutations into the major capsid proteins.

  13. Gene family structure, expression and functional analysis of HD-Zip III genes in angiosperm and gymnosperm forest trees

    PubMed Central

    2010-01-01

    Background Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups. Results Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with

  14. Gene networks specific for innate immunity define post-traumatic stress disorder.

    PubMed

    Breen, M S; Maihofer, A X; Glatt, S J; Tylee, D S; Chandler, S D; Tsuang, M T; Risbrough, V B; Baker, D G; O'Connor, D T; Nievergelt, C M; Woelk, C H

    2015-12-01

    The molecular factors involved in the development of Post-Traumatic Stress Disorder (PTSD) remain poorly understood. Previous transcriptomic studies investigating the mechanisms of PTSD apply targeted approaches to identify individual genes under a cross-sectional framework lack a holistic view of the behaviours and properties of these genes at the system-level. Here we sought to apply an unsupervised gene-network based approach to a prospective experimental design using whole-transcriptome RNA-Seq gene expression from peripheral blood leukocytes of U.S. Marines (N=188), obtained both pre- and post-deployment to conflict zones. We identified discrete groups of co-regulated genes (i.e., co-expression modules) and tested them for association to PTSD. We identified one module at both pre- and post-deployment containing putative causal signatures for PTSD development displaying an over-expression of genes enriched for functions of innate-immune response and interferon signalling (Type-I and Type-II). Importantly, these results were replicated in a second non-overlapping independent dataset of U.S. Marines (N=96), further outlining the role of innate immune and interferon signalling genes within co-expression modules to explain at least part of the causal pathophysiology for PTSD development. A second module, consequential of trauma exposure, contained PTSD resiliency signatures and an over-expression of genes involved in hemostasis and wound responsiveness suggesting that chronic levels of stress impair proper wound healing during/after exposure to the battlefield while highlighting the role of the hemostatic system as a clinical indicator of chronic-based stress. These findings provide novel insights for early preventative measures and advanced PTSD detection, which may lead to interventions that delay or perhaps abrogate the development of PTSD.

  15. Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer.

    PubMed

    Castillo, Sandra D; Angulo, Barbara; Suarez-Gauthier, Ana; Melchor, Lorenzo; Medina, Pedro P; Sanchez-Verde, Lydia; Torres-Lanzas, Juan; Pita, Guillermo; Benitez, Javier; Sanchez-Cespedes, Montse

    2010-09-01

    The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

  16. Transgenic tobacco plants overexpressing a grass PpEXP1 gene exhibit enhanced tolerance to heat stress.

    PubMed

    Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

    2014-01-01

    Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars.

  17. Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

    PubMed Central

    Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

    2014-01-01

    Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars. PMID:25003197

  18. Gene expression profiles of hepatocytes treated with La (NO3) 3 of rare earth in rats

    PubMed Central

    Zhao, Hui; Hao, Wei-Dong; Xu, Hou-En; Shang, Lan-Qin; Lu, You-Yong

    2004-01-01

    AIM: To compare the gene expression between La (NO3) 3-exposed and control rats in vivo. METHODS: Rats were fed La (NO3) 3 once daily at a dose of 20 mg/kg for one month by gavage. Gene expression of hepatocytes was detected using mRNA differential display (DD) technique and cDNA microarray and compared between treated and control groups. RESULTS: Six differentially expressed sequence tags were cloned by DD, of which five were up regulated and one was down regulated in treated rats. Two sequences were determined. One band was novel. The other shared 100% sequence homology with AU080263 Sugano mouse brain mncb Mus musculus cDNA clone MNCb-5435 5’. With DNA microarray, 136 differentially expressed genes were identified including 131 over-expressed genes and 5 under-expressed genes. Most of these differentially expressed genes were cell signal and transmission genes, genes associated with metabolism, protein translation and synthesis. CONCLUSION: La (NO3) 3 could change the expression levels of some kinds of genes. Further analysis of the differentially expressed genes would be helpful for understanding the wide biological effect spectrum of rare earth elements. PMID:15162537

  19. How difficult is inference of mammalian causal gene regulatory networks?

    PubMed

    Djordjevic, Djordje; Yang, Andrian; Zadoorian, Armella; Rungrugeecharoen, Kevin; Ho, Joshua W K

    2014-01-01

    Gene regulatory networks (GRNs) play a central role in systems biology, especially in the study of mammalian organ development. One key question remains largely unanswered: Is it possible to infer mammalian causal GRNs using observable gene co-expression patterns alone? We assembled two mouse GRN datasets (embryonic tooth and heart) and matching microarray gene expression profiles to systematically investigate the difficulties of mammalian causal GRN inference. The GRNs were assembled based on > 2,000 pieces of experimental genetic perturbation evidence from manually reading > 150 primary research articles. Each piece of perturbation evidence records the qualitative change of the expression of one gene following knock-down or over-expression of another gene. Our data have thorough annotation of tissue types and embryonic stages, as well as the type of regulation (activation, inhibition and no effect), which uniquely allows us to estimate both sensitivity and specificity of the inference of tissue specific causal GRN edges. Using these unprecedented datasets, we found that gene co-expression does not reliably distinguish true positive from false positive interactions, making inference of GRN in mammalian development very difficult. Nonetheless, if we have expression profiling data from genetic or molecular perturbation experiments, such as gene knock-out or signalling stimulation, it is possible to use the set of differentially expressed genes to recover causal regulatory relationships with good sensitivity and specificity. Our result supports the importance of using perturbation experimental data in causal network reconstruction. Furthermore, we showed that causal gene regulatory relationship can be highly cell type or developmental stage specific, suggesting the importance of employing expression profiles from homogeneous cell populations. This study provides essential datasets and empirical evidence to guide the development of new GRN inference methods for

  20. Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice.

    PubMed

    Hu, Honghong; You, Jun; Fang, Yujie; Zhu, Xiaoyi; Qi, Zhuyun; Xiong, Lizhong

    2008-05-01

    Plants respond to adverse environment by initiating a series of signaling processes including activation of transcription factors that can regulate expression of arrays of genes for stress response and adaptation. NAC (NAM, ATAF, and CUC) is a plant specific transcription factor family with diverse roles in development and stress regulation. In this report, a stress-responsive NAC gene (SNAC2) isolated from upland rice IRA109 (Oryza sativa L. ssp japonica) was characterized for its role in stress tolerance. SNAC2 was proven to have transactivation and DNA-binding activities in yeast and the SNAC2-GFP fusion protein was localized in the rice nuclei. Northern blot and SNAC2 promoter activity analyses suggest that SNAC2 gene was induced by drought, salinity, cold, wounding, and abscisic acid (ABA) treatment. The SNAC2 gene was over-expressed in japonica rice Zhonghua 11 to test the effect on improving stress tolerance. More than 50% of the transgenic plants remained vigorous when all WT plants died after severe cold stress (4-8 degrees C for 5 days). The transgenic plants had higher cell membrane stability than wild type during the cold stress. The transgenic rice had significantly higher germination and growth rate than WT under high salinity conditions. Over-expression of SNAC2 can also improve the tolerance to PEG treatment. In addition, the SNAC2-overexpressing plants showed significantly increased sensitivity to ABA. DNA chip profiling analysis of transgenic plants revealed many up-regulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, heat shock protein, GDSL-like lipase, and phenylalanine ammonia lyase. Interestingly, none of the up-regulated genes in the SNAC2-overexpressing plants matched the genes up-regulated in the transgenic plants over-expressing other stress responsive NAC genes reported previously. These data suggest SNAC2 is a novel stress

  1. Effects of brain IKKβ gene silencing by small interfering RNA on P-glycoprotein expression and brain damage in the rat kainic acid-induced seizure model.

    PubMed

    Yu, Nian; Liu, Hao; Zhang, Yan-Fang; Su, Ling-Ying; Liu, Xin-Hong; Li, Le-Chao; Hao, Jin-Bo; Huang, Xian-Jing; Di, Qing

    2014-01-01

    Multidrug resistance mediated by over-expression of P-glycoprotein (P-gp) in brain is an important mechanism accounting for the drug-therapy failure in epilepsy. Over-expression of P-gp in epilepsy rat brain may be regulated by inflammation and nuclear factor-kappa B (NF-κB) activation. Inhibitory κ B kinase subunit β (IKKβ) is an up-stream molecular controlling NF-κB activation. With the small interfering RNA (siRNA) technique and kainic acid (KA)-induced rat epileptic seizure model, the present study was aimed to further evaluate the role of NF-κB inhibition, via blocking IKKβ gene transcription, in the epileptic brain P-gp over-expression, seizure susceptibility, and post-seizure brain damage. siRNA targeting IKKβ was administered to rats via intracerebroventricular injection before seizure induction by KA microinjection; scrambled siRNA was used as control. Brain mRNA and protein levels of IKKβ and P-gp were detected by RT-PCR and immunohistochemistry. NF-κB activity was measured by electrophoretic mobility shift assay. Latency to grade III or V seizure onset was recorded, brain damage was evaluated by neuronal cell counting and epileptiform activity was monitored by electroencephalography. IKKβ siRNA pre-treatment inhibited NF-κB activation and abolished P-gp over-expression in KA-induced epileptic rat brain, accompanied by decreased seizure susceptibility. These findings suggested that epileptogenic-induced P-gp over-expression could be regulated by IKKβ through the NF-κB pathway. PMID:24040792

  2. Designer Genes.

    ERIC Educational Resources Information Center

    Miller, Judith; Miller, Mark

    1983-01-01

    Genetic technologies may soon help fill some of the most important needs of humanity from food to energy to health care. The research of major designer genes companies and reasons why the initial mad rush for biotechnology has slowed are reviewed. (SR)

  3. Attention Genes

    ERIC Educational Resources Information Center

    Posner, Michael I.; Rothbart, Mary K.; Sheese, Brad E.

    2007-01-01

    A major problem for developmental science is understanding how the cognitive and emotional networks important in carrying out mental processes can be related to individual differences. The last five years have seen major advances in establishing links between alleles of specific genes and the neural networks underlying aspects of attention. These…

  4. Constitutive expression of a grapevine polygalacturonase-inhibiting protein affects gene expression and cell wall properties in uninfected tobacco

    PubMed Central

    2011-01-01

    Background Polygalacturonase-inhibiting proteins (PGIPs) directly limit the effective ingress of fungal pathogens by inhibiting cell wall-degrading endopolygalacturonases (ePGs). Transgenic tobacco plants over-expressing grapevine (Vitis vinifera) Vvpgip1 have previously been shown to be resistant to Botrytis infection. In this study we characterized two of these PGIP over-expressing lines with known resistance phenotypes by gene expression and hormone profiling in the absence of pathogen infection. Results Global gene expression was performed by a cross-species microarray approach using a potato cDNA microarray. The degree of potential cross-hybridization between probes was modeled by a novel computational workflow designed in-house. Probe annotations were updated by predicting probe-to-transcript hybridizations and combining information derived from other plant species. Comparing uninfected Vvpgip1-overexpressing lines to wild-type (WT), 318 probes showed significant change in expression. Functional groups of genes involved in metabolism and associated to the cell wall were identified and consequent cell wall analysis revealed increased lignin-levels in the transgenic lines, but no major differences in cell wall-derived polysaccharides. GO enrichment analysis also identified genes responsive to auxin, which was supported by elevated indole-acetic acid (IAA) levels in the transgenic lines. Finally, a down-regulation of xyloglucan endotransglycosylase/hydrolases (XTHs), which are important in cell wall remodeling, was linked to a decrease in total XTH activity. Conclusions This evaluation of PGIP over-expressing plants performed under pathogen-free conditions to exclude the classical PGIP-ePG inhibition interaction indicates additional roles for PGIPs beyond the inhibition of ePGs. PMID:22078230

  5. Boron induced expression of some stress-related genes in tomato.