Science.gov

Sample records for ccnd1 gene over-expression

  1. Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma.

    PubMed

    Masamha, Chioniso Patience; Albrecht, Todd R; Wagner, Eric J

    2016-03-29

    The t(11;14) translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3'UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (CCND1) and a 3'UTR consisting of sequences from both the CCND1 3'UTR and myotonic dystrophy kinase-related Cdc42-binding kinase's (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3'UTR with siRNA results in decreased CCND1 levels.

  2. Prognostic significance of cyclin D1 gene (CCND1) polymorphism in epithelial ovarian cancer.

    PubMed

    Dhar, K. K.; Branigan, K.; Howells, R. E. J.; Musgrove, C.; Jones, P. W.; Strange, R. C.; Fryer, A. A.; Redman, C. W. E.; Hoban, P. R.

    1999-07-01

    We have investigated the influence of CCND1 genotype on clinical outcome in 138 women with epithelial ovarian cancer. CCND1 genotypes were identified from peripheral blood DNA by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. Patient CCND1 genotypes were compared with clinical details including FIGO tumor stage, residual tumor volume, tumor histology and differentiation, response to chemotherapy, progression free interval, and survival. We observed no association between patient CCND1 genotypes and tumor characteristics or response to chemotherapy. There was no significant difference in overall survival and progression free interval (PFI) among women with different CCND1 genotypes. However, analysis of data from patients who responded to postoperative chemotherapy revealed that women with CCND1 AA genotype were associated with early disease progression (P = 0.020, HR 4.58, 95% CI 1.27-16.48) and reduced survival (P = 0.026, HR 4.48, 95% CI 1.19-16.79) compared with those with CCND1 AG and GG genotypes. These data show that CCND1 genotype does not influence overall prognosis in a cohort of epithelial ovarian cancer patients, however, it is associated with disease progression in a subgroup of patients following initial response to chemotherapy.

  3. Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

    PubMed

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2016-05-01

    Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes.

  4. Association of CCND1 Gene c.870G>A Polymorphism with Breast Cancer Risk: A Case-ControlStudy and a Meta-Analysis.

    PubMed

    Soleimani, Zahra; Kheirkhah, Davood; Sharif, Mohammad Reza; Sharif, Alireza; Karimian, Mohammad; Aftabi, Younes

    2016-12-21

    Cyclin D1 (CCND1) plays an essential role in regulating the progress of the cell cycle from G1 to S phase. There is a common c.870G>A polymorphism in the CCND1 gene. The aim of this study was to investigate the association of CCND1 gene c.870G>A polymorphism with breast cancer risk in a case-control study, which followed by a meta-analysis and an in silico analysis. Three hundred and thirty-five subjects composed of 174 women with breast cancer and 161 healthy controls were included in the case-control study. CCND1 gene c.870G>A genotyping was performed by PCR-RFLP. Meta-analysis was done for 14 studies composed of 7281 cases and 6820 controls. Some bioinformatics tools were applied to investigate the effects of c.870G>A on the mRNA splicing and structure. Our data obtained from case-control study revealed that GA genotype (OR: 1.89, 95%CI: 1.12-3.17, p = 0.017), AA genotype (OR: 1.95, 95%CI: 1.08-3.53, p = 0.027), and A allele (OR: 1.44, 95%CI: 1.06-1.95, p = 0.019) were significantly associated with breast cancer risk. The results of meta-analysis showed a significant association between CCND1 c.870G>A polymorphism and breast cancer risk, especially in Caucasian population. In silico analysis revealed that c.870G>A transition affect CCND1 mRNA splicing and secondary structure.

  5. Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

    PubMed Central

    Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.

    1996-01-01

    Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images

  6. CCND1 — EDRN Public Portal

    Cancer.gov

    CCND1 is a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance throughout the cell cycle. CCND1 forms a complex with and functions as a regulatory subunit of CDK4 or CDK6, whose activity is required for cell cycle G1/S transition.

  7. 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.

  8. Evolutionary conservation of CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2003-07-01

    The CCND1-ORAOV1-FGF19-FGF4-FGF3-FLJ10261-FADD-PPFIA1-EMS1 locus on human chromosome 11q13 is frequently amplified in esophageal cancer, breast cancer, and bladder tumors. FGF19, FGF4 and FGF3 genes are implicated in embryogenesis and carcinogenesis. We proposed in 2002 the hypothesis that mouse Fgf15 might be the ortholog of human FGF19 based on comparative genomics. Here, we identified zebrafish fgf19 and oraov1 genes by using bioinformatics to demonstrate the hypothesis. Zebrafish fgf19 gene, consisting of three exons, was located around nucleotide position 121802-124963 of zebrafish genome draft sequence AL929586.12 in the reverse orientation. Zebrafish fgf19 (209 aa) was more homologous to chicken fgf19 and human FGF19 than to rodent Fgf15. Zebrafish oraov1 gene, consisting of five exons, was located around nucleotide position 112172-115838 of AL929586.12 in the reverse orientation. Zebrafish oraov1 protein (141 aa) was more homologous to human ORAOV1 than to rodent Oraov1. The CCND1-ORAOV1-FGF19-FGF4 locus was well conserved between human and zebrafish genomes in the order of genes, in the direction of genes, and in the exon-intron structure. Rat Ccnd1-Oraov1-Fgf15-Fgf4 locus was synthenic to mouse Ccnd1-Oraov1 (also known as 2210010N10Rik)-Fgf15-Fgf4 locus. Fgf15, homologous to human FGF19 and zebrafish fgf19, was located on the synthenic locus of human FGF19 and zebrafish fgf19 within rodent genomes. Based on the evolutionary conservation of the CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human, it was concluded that Fgf15 gene is the rodent ortholog of human FGF19 gene.

  9. 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

  10. Contribution of cyclin D1 (CCND1) and E-cadherin (CDH1) alterations to colorectal cancer susceptibility: a case-control study.

    PubMed

    Govatati, Suresh; Singamsetty, Gopi Krishna; Nallabelli, Nayudu; Malempati, Sravanthi; Rao, Pasupuleti Sreenivasa; Madamchetty, Venkata Kranthi Kumar; Govatati, Sowdamani; Kanapuram, Rudramadevi; Narayana, Nagesh; Bhanoori, Manjula; Kassetty, Kondaiah; Nallanchakravarthula, Varadacharyulu

    2014-12-01

    Cyclin D1 (CCND1) and E-cadherin (CDH1) are two important genes of the β-catenin/LEF pathway that is involved in tumorigenesis of various cancers including colorectal cancer (CRC). However, studies of the association between genetic variants of these two genes and CRC have shown conflicting results. We conducted a genetic association study in South Indian population (cases, 103; controls, 107) to assess the association of CCND1 870G/A and CDH1 -160C/A single nucleotide polymorphisms (SNPs) with CRC risk. Genotyping of SNPs was performed by PCR sequencing analysis. Haplotype frequencies for multiple loci and the standardized disequilibrium coefficient (D') for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software. In addition, to better understand the role of CCND1 and CDH1 in the pathophysiology of CRC, the expression pattern was evaluated in analogous tumor and adjacent normal tissues from 23 CRC patients by Western blot analysis. The frequencies of CCND1 870A/A (P = 0.045) genotype, CDH1 -160A allele (P = 0.042), and 870A/-160A haplotype (P = 0.002) were significantly higher in patients as compared with controls. Strong LD was observed between 870G/A and -160C/A SNPs in cases (D' = 0.76) as compared to controls (D' = 0.32). Furthermore, elevated CCND1 and diminished CDH1 expression was observed in tumor tissue as compared with analogous normal tissue of CRC patients. Interestingly, advanced-stage tumors showed wider expression alterations than in early-stage tumors. In conclusion, CCND1 870G/A and CDH1 -160C/A SNPs may modify the risk of CRC susceptibility in South Indian population. In addition, elevated CCND1 and diminished CDH1 expression appears to be useful prognostic markers for CRC.

  11. Over-expression of AGPase genes enhances seed weight and starch content in transgenic maize.

    PubMed

    Li, Ning; Zhang, Shujuan; Zhao, Yajie; Li, Bei; Zhang, Juren

    2011-02-01

    Cereal crops accumulate starch in the seed endosperm as an energy reserve. ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds. The AGPase in the maize endosperm is a heterotetramer of two small subunits, encoded by Brittle2 (Bt2) gene, and two large subunits, encoded by the Shrunken2 (Sh2) gene. The two genes (Bt2, Sh2) from maize were introduced into two elite maize inbred lines, solely and in tandem, and under the control of endosperm-specific promoters for over-expression. PCR, Southern blotting, and real-time RT-PCR analysis indicated that the transgenes were integrated into the genome of transgenic plants and were over-expressed in their progeny. The over-expression of either gene enhanced AGPase activity, seed weight and starch content compared with the WT, but the amounts were lower than plants with over-expression of both Bt2 and Sh2. Developing seeds from co-expression transgenic maize plants had higher cytoplasmic AGPase activity: the 100-grain weight increased 15% over the wild type (WT), and the starch content increased to over 74% compared with the WT of 65%. These results indicate that over-expression of the genes in transgenic maize plants could improve kernel traits. This report provides a feasible approach for increasing starch content and seed weight in maize.

  12. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

    PubMed

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W; Amin, Hesham M; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D; Rosen, Steven T; Pham, Lan V; Ngo, Vu N

    2016-11-08

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5-10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

  13. CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma

    PubMed Central

    Mohanty, Atish; Sandoval, Natalie; Das, Manasi; Pillai, Raju; Chen, Lu; Chen, Robert W.; Amin, Hesham M.; Wang, Michael; Marcucci, Guido; Weisenburger, Dennis D.; Rosen, Steven T.; Pham, Lan V.; Ngo, Vu N.

    2016-01-01

    Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL. PMID:27713153

  14. BAX gene over-expression via nucleofection to induce apoptosis in human lens epithelial cells.

    PubMed

    Fang, Yanwen; Mo, Xiaofen; Luo, Yi; Lu, Yi

    2012-09-01

    Despite significant advances in cataract surgery techniques, posterior capsule opacification (PCO) remains a common complication. In PCO, remaining epithelial cells cloud the lens capsule and impair postoperative vision. This in vitro study was designed to investigate the potential of a gene-based approach, specifically over-expression of the proapoptotic BAX gene, to prevent PCO. Human lens epithelial cells (HLECs) were transfected by nucleofection with a plasmid encoding a fusion protein of green fluorescent protein and human BAX. The expression levels of BAX and its antiapoptotic counterpart BCL2 were determined by realtime reverse transcription polymerase chain reaction, Western blotting and immunofluorescence. BAX over-expression-induced cell death was analyzed by fluorescence-activated cell sorting using the Annexin V antibody. Fluorescence microscopy and transmission electron microscopy were used to assess changes in morphology and ultrastructure. Differential expression of the downstream apoptosis-related factor, caspase 3, was detected by Western blotting. Nucleofection efficiency was high (nearly 80%). BAX-transfected HLECs showed remarkably enhanced BAX gene expression and BAX:BCL2 ratio, but relatively little change in endogenous BCL2 expression. BAX over-expression also led to significant cytotoxicity, induction of apoptosis-related characteristics and activation of caspase 3. In conclusion, our results indicate that BAX gene over-expression can trigger cell death in HLECs via an apoptotic pathway. Thus, BAX may be a promising candidate for human gene therapy to treat PCO.

  15. 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.

  16. Computational identification of gene over-expression targets for metabolic engineering of taxadiene production.

    PubMed

    Boghigian, Brett A; Armando, John; Salas, Daniel; Pfeifer, Blaine A

    2012-03-01

    Taxadiene is the first dedicated intermediate in the biosynthetic pathway of the anticancer compound Taxol. Recent studies have taken advantage of heterologous hosts to produce taxadiene and other isoprenoid compounds, and such ventures now offer research opportunities that take advantage of the engineering tools associated with the surrogate host. In this study, metabolic engineering was applied in the context of over-expression targets predicted to improve taxadiene production. Identified targets included genes both within and outside of the isoprenoid precursor pathway. These targets were then tested for experimental over-expression in a heterologous Escherichia coli host designed to support isoprenoid biosynthesis. Results confirmed the computationally predicted improvements and indicated a synergy between targets within the expected isoprenoid precursor pathway and those outside this pathway. The presented algorithm is broadly applicable to other host systems and/or product choices.

  17. Over-expression of OsDREB genes lead to enhanced drought tolerance in rice.

    PubMed

    Chen, Jian-Qiang; Meng, Xiu-Ping; Zhang, Yun; Xia, Mian; Wang, Xi-Ping

    2008-12-01

    The DREB transcription factors, which specifically interact with C-repeat/DRE (A/GCCGAC), play an important role in plant abiotic stress tolerance by controlling the expression of many cold or/and drought-inducible genes in an ABA-independent pathway. We have isolated three novel rice DREB genes, OsDREB1E, OsDREB1G, and OsDREB2B, which are homologous to Arabidopsis DREB genes. The yeast one-hybrid assay indicated that OsDREB1E, OsDREB1G, and OsDREB2B can specifically bind to the C-repeat/DRE element. To elucidate the function of respective OsDREB genes, we have stably introduced these to rice by Agrobacterium-mediated transformation. Transgenic rice plants analysis revealed that over-expression of OsDREB1G and OsDREB2B in rice significantly improved their tolerance to water deficit stress, while over-expression of OsDREB1E could only slightly improved the tolerance to water deficit stress, suggesting that the OsDREBs might participate in the stress response pathway in different manners.

  18. 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

  19. Suppressing role of miR-520a-3p in breast cancer through CCND1 and CD44

    PubMed Central

    Li, Jun; Wei, Juan; Mei, Zhu; Yin, Yongmei; Li, Yongfei; Lu, Mingjie; Jin, Shidai

    2017-01-01

    Recent studies show that many microRNAs (miRNAs) are found to play important roles in breast cancer, however, most of miRNAs are not investigated completely. In the present study, significant down-regulation of miR-520a-3p was found in the breast cancer tissues and cell lines. The restoration of miR-520a-3p expression in breast cancer cells could inhibit cell proliferation, migration and invasion. In addition, miR-520a-3p was able to induce breast cancer cell apoptosis. Luciferase assay was used to confirm that CCND1 and CD44 were the direct target genes of miR-520a-3p. The ectopic expression of miR-520a-3p repressed CCND1 and CD44 expression on post-transcriptional levels in breast cancer cells. This study suggests that miR-520a-3p may act as an optional method for breast cancer therapy. PMID:28123641

  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. MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2

    PubMed Central

    Shen, Yu-Qing; Wang, Hui-Min; Ding, Lei; Gu, Yu-Chen; Lou, Jia-Tao; Zhao, Xin-Tai; Ma, Zhong-Liang; Jin, You-Xin

    2016-01-01

    Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3′-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2. PMID:27494902

  2. [A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

    PubMed

    Cao, C; Shi, C; Li, P; Ma, Q

    1997-01-01

    A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

  3. CCND1 G870A polymorphism and colorectal cancer risk: An updated meta-analysis

    PubMed Central

    XU, XIAO-MING; NI, XIAO-BING; YANG, GONG-LI; LUO, ZHI-GUO; NIU, YU-MING; SHEN, MING

    2016-01-01

    Molecular epidemiological studies have revealed a closer association between cyclin D1 (CCND1) polymorphism and the risk of colorectal cancer; however, the results were inconsistent. The aim of the present meta-analysis was to investigate the association between CCND1 G870A polymorphism and colorectal cancer risk. Online electronic databases (PubMed and Embase) were searched. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to assess the association between CCND1 G870A polymorphism and the risk of colorectal cancer. In addition, heterogeneity, publication bias and sensitivity analysis were performed to guarantee the statistical power. In total, 23 published case-control studies with 6,320 patients and 8,252 controls were selected. Significantly increased risks were observed in four genetic models (A vs. G: OR=1.09, 95% CI=1.00–1.18, I2=54.3%; GA vs. GG: OR=1.13, 95% CI=1.04–1.24, I2=18.2%; AA vs. GG, OR=1.17: 95% CI=1.00–1.38, I2=52.5%; GA+AA vs. GG: OR=1.14, 95% CI=1.05–1.24, I2=33.8%). Similarly, significant associations were also identified in the stratified analysis in the cancer subtype of sporadic colorectal cancer (GA vs. GG: OR=1.21, 95% CI=1.04–1.42, I2=24.1%; GA+AA vs. GG: OR=1.18, 95% CI=1.02–1.37, I2=35.0%), Caucasian population (GA vs. GG, OR=1.14, 95% CI=1.02–1.28, I2=19.8%; GA+AA vs. GG, OR=1.14, 95% CI=1.02–1.27, I2=37.5%) and other subgroups of control design and genotyping type. The present updated meta-analysis suggested that CCND1 G870A may present an increased risk for developing colorectal cancer, particularly in sporadic colorectal cancer and a Caucasian population. PMID:27284448

  4. RNA interference improves myopathic phenotypes in mice over-expressing FSHD region gene 1 (FRG1).

    PubMed

    Wallace, Lindsay M; Garwick-Coppens, Sara E; Tupler, Rossella; Harper, Scott Q

    2011-11-01

    Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.

  5. Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers.

    PubMed

    Wen, Xiao-Peng; Pang, Xiao-Ming; Matsuda, Narumi; Kita, Masayuki; Inoue, Hiromichi; Hao, Yu-Jin; Honda, Chikako; Moriguchi, Takaya

    2008-04-01

    An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

  6. Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown

    PubMed Central

    Liu, MaFeng; Wang, MengYi; Zhu, DeKang; Wang, MingShu; Jia, RenYong; Chen, Shun; Sun, KunFeng; Yang, Qiao; Wu, Ying; Chen, XiaoYue; Biville, Francis; Cheng, AnChun

    2016-01-01

    Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and knockdown to investigate its function. First, we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters. The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5 × 10−5 antibiotic-resistant transconjugants per recipient cell. Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established. Knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organism’s growth ability in TSB medium but did not affect its hemin utilization. In contrast, over-expression of TbfA in Riemerella anatipestifer ATCC11845ΔtonB1ΔtonB2. Significantly promoted the organism’s growth in TSB medium but significantly inhibited its hemin utilization. Collectively, these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer. PMID:27845444

  7. 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

  8. Over-expression of brain-derived neurotrophic factor in mesenchymal stem cells transfected with recombinant lentivirus BDNF gene.

    PubMed

    Zhang, X; Zhu, J; Zhang, K; Liu, T; Zhang, Z

    2016-12-30

    This study was aimed at investigating the expression of brain-derived neurotrophic factor (BDNF) in mesenchymal stem cells (MSCs) modified with recombinant lentivirus bearing BDNF gene. Lentivirus vectors bearing BDNF gene were constructed. MSCs were isolated from rats and cultured. The lentiviral vectors containing BDNF gene were transfected into the MSCs, and BDNF gene and protein expressions were monitored with enhanced green fluorescent protein (EGFP). RT-PCR and Western blot were used to measure gene and protein expressions, respectibvely in MSCs, MSCs-EGFP and MSCs-EGFP-BDNF groups. Green fluorescence assay confirmed successful transfection of BDNF gene recombinant lentivirus into MSCs. RT-PCR and Western blot revealed that BDNF gene and protein expressions in the MSCs-EGFP-BDNF group were significantly higher than that in MSCs group and MSCs-EGFP group. There were no statistically significant differences in gene expression between MSCs and MSCs-EGFP groups. MSCs can over-express BDNF when transfected with recombinant lentivirus bearing BDNF gene.

  9. Association between cyclin D1 (CCND1) G870A polymorphism and gastric cancer risk: a meta-analysis

    PubMed Central

    Zhang, Yafei; Zeng, Xianling; Lu, Hongwei; Ji, Hong; Zhao, Enfa; Li, Yiming

    2016-01-01

    Published data on the association between cyclin D1 (CCND1) G870A polymorphism and gastric cancer (GC) risk are inconclusive. Thus, we conducted a meta-analysis to evaluate the relationship between CCND1 G870A polymorphism and GC risk. We searched PubMed, EMBASE, Web of science and the Cochrane Library up to June 12, 2015 for relevant studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to estimate the strength of associations. Nine studies published from 2003 to 2014, with a total of 1813 cases and 2173 controls, were included in this meta-analysis. The pooled results showed that there was no association between CCND1 G870A polymorphism and GC risk in any genetic model. The subgroup analysis stratified by ethnicity showed an increased breast cancer risk in Caucasian based on heterozygote comparison (GA vs. GG: OR=1.49, 95% CI=1.06-2.10, P=0.02). We found the same association in population based (PB) stratified analyses by Source of controls (AA vs. GG: OR=1.39, 95% CI=1.01-1.93, 0.05). When stratifying by the type, Sex and H. pylori infection in dominant model, Interestingly, we found the opposite result in Male (AA + GA vs. GG: OR=0.5, 95% CI=0.33-0.76, P=0.001), there were no association between CCND1 G870A polymorphism and GC risk in any other subgroup. This meta-analysis suggests that CCND1 G870A polymorphism is a risk factor for susceptibility to GC in Caucasians and in general populations. While, CCND1 G870A polymorphism plays a possible protective effect in GC in Male. Further large scale multicenter epidemiological studies are warranted to confirm this finding. PMID:27623072

  10. Over-expression of an FT-homologous gene of apple induces early flowering in annual and perennial plants.

    PubMed

    Tränkner, Conny; Lehmann, Sandra; Hoenicka, Hans; Hanke, Magda-Viola; Fladung, Matthias; Lenhardt, Denise; Dunemann, Frank; Gau, Achim; Schlangen, Karin; Malnoy, Mickael; Flachowsky, Henryk

    2010-11-01

    The protein encoded by the FLOWERING LOCUS T (FT) gene from Arabidopsis thaliana seems to be the long-searched florigen, and over-expression of FT orthologues resulted in accelerated flower development in annual and perennial plants. In the present study, we isolated two allelic mRNA sequences of an FT-homologous gene from apple, which was designated as MdFT1. Using a SSR motif this gene was mapped on LG 12 of apple. Over-expression of MdFT1 in Arabidopsis and the commercially important tree species poplar and apple itself using the CaMV 35S or the Arabidopsis Suc2 promoter resulted in significant accelerated flowering compared with wild-type plants. Transgenic T(0) plants of Arabidopsis flowered 4-6 days on average earlier than wild-type Arabidopsis under LD conditions. Under short-day conditions Suc2::MdFT1 plants of the T(1)-generation flowered after 66 ± 18 days, while wild-type plants flowered about 22 days later. All transgenic Arabidopsis plants showed a normal habit except for the early flowering phenotype. Early flowering was detected 6-10 months after transformation in transgenic polar clones containing MdFT1 driven by the CaMV 35S, whereas plants of the transgenic apple clone T780 set up its first flowers during in vitro cultivation. Based on our results we conclude that MdFT1 is responsible for inducing flowering and that the function of the apple FT1 gene is conserved in annual herbaceous species as well as perennial woody species. Furthermore, we discuss the role of MdFT1 in flower development with regard to the findings of genetic studies on apple.

  11. 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.

  12. Detection of t(12;14)(p13;q32) in a patient with IGH-CCND1 negative mantle cell lymphoma resembling ultra-high risk chronic lymphocytic leukemia.

    PubMed

    Miao, Yi; Wang, Rong; Fan, Lei; Qiu, Hairong; Wu, Yujie; Chen, Yaoyu; Xu, Wei; Li, Jianyong

    2015-01-01

    T(12;14)(p13;q32) is a rare recurrent chromosomal translocation, which has only been identified in a small subgroup of mantle cell lymphoma (MCL) without typical t(11;14)(q13;q32). This rearrangement causes aberrant over-expression of cyclin D2 (CCND2), which disrupts the normal cell cycle. Here we report a subtle case of MCL with t(12;14)(p13;q32) that was initially misdiagnosed as ultra-high risk chronic lymphocytic leukemia (CLL). A 60-year-old male patient presented with obvious leukocytosis and progressive weakness. Morphology of peripheral blood and immunophenotyping by flow cytometry pointed to a diagnosis of chronic lymphocytic leukemia. Fluorescence in situ hybridization (FISH) using IGH-CCND1 probe was negative for CCND1 abnormality, but demonstrated IGH breakapart signals. The initial diagnosis of CLL was established and the patient was treated with six courses of immunochemotherpy with fludarabine, cyclophosphamide and rituximab (FCR). Complete remission (CR) was achieved at the end of treatment, but disease relapsed quickly. The patient was transferred to our hospital, flow cytometry using additional markers showed that the clonal cells were CD200+(dim), CD148+(strong), and chromosome analysis revealed a complex karyotype, 47, XY, t(12;14)(p13;q32), +12, del(9p21), which indicated over-expression of CCND2, and immunostaining showed strong positivity of SOX11 further confirming the characteristics of CCND1-negtive MCL. The final diagnosis was revised to rare subtype of MCL with CCND2 translocation and intensive regimens were employed. This confusable MCL case illustrates the importance of cytogenetic analysis and clinicopathologic diagnosis of this rare category of MCL.

  13. Integrative genomics analysis of chromosome 5p gain in cervical cancer reveals target over-expressed genes, including Drosha

    PubMed Central

    Scotto, Luigi; Narayan, Gopeshwar; Nandula, Subhadra V; Subramaniyam, Shivakumar; Kaufmann, Andreas M; Wright, Jason D; Pothuri, Bhavana; Mansukhani, Mahesh; Schneider, Achim; Arias-Pulido, Hugo; Murty, Vundavalli V

    2008-01-01

    Background Copy number gains and amplifications are characteristic feature of cervical cancer (CC) genomes for which the underlying mechanisms are unclear. These changes may possess oncogenic properties by deregulating tumor-related genes. Gain of short arm of chromosome 5 (5p) is the most frequent karyotypic change in CC. Methods To examine the role of 5p gain, we performed a combination of single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and gene expression analyses on invasive cancer and in various stages of CC progression. Results The SNP and FISH analyses revealed copy number increase (CNI) of 5p in 63% of invasive CC, which arises at later stages of precancerous lesions in CC development. We integrated chromosome 5 genomic copy number and gene expression data to identify key target over expressed genes as a consequence of 5p gain. One of the candidates identified was Drosha (RNASEN), a gene that is required in the first step of microRNA (miRNA) processing in the nucleus. Other 5p genes identified as targets of CNI play a role in DNA repair and cell cycle regulation (BASP1, TARS, PAIP1, BRD9, RAD1, SKP2, and POLS), signal transduction (OSMR), and mitochondrial oxidative phosphorylation (NNT, SDHA, and NDUFS6), suggesting that disruption of pathways involving these genes may contribute to CC progression. Conclusion Taken together, we demonstrate the power of integrating genomics data with expression data in deciphering tumor-related targets of CNI. Identification of 5p gene targets in CC denotes an important step towards biomarker development and forms a framework for testing as molecular therapeutic targets. PMID:18559093

  14. Promotion of photosynthesis in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene by nitric oxide donors.

    PubMed

    Chen, Pingbo; Li, Xia; Huo, Kai; Wei, Xiaodong; Dai, Chuanchao; Lv, Chuangen

    2014-03-15

    We determined the effects of exogenous nitric oxide on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC). Seedlings were subjected to treatments with NO donors, an NO scavenger, phospholipase inhibitors, a Ca(2+) chelator, a Ca(2+) channel inhibitor, and a hydrogen peroxide (H2O2) inhibitor, individually and in various combinations. The NO donors significantly increased the net photosynthetic rate (PN) of PC and wild-type (WT), especially that of PC. Treatment with an NO scavenger did inhibit the PN of rice plants. The treatments with phospholipase inhibitors and a Ca(2+) chelator decreased the PN of WT and PC, and photosynthesis was more strongly inhibited in WT than in PC. Further analyses showed that the NO donors increased endogenous levels of NO and PLD activity, but decreased endogenous levels of Ca(2+) both WT and PC. However, there was a greater increase in NO in WT and a greater increase in PLD activity and Ca(2+) level in PC. The NO donors also increased both PEPC activity and pepc gene expression in PC. PEPC activity can be increased by SNP alone. But the expression of its encoding gene in PC might be regulated by SNP, together with PA and Ca(2+).

  15. 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.

  16. 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.

  17. 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.

  18. Strengthening Triterpene Saponins Biosynthesis by Over-Expression of Farnesyl Pyrophosphate Synthase Gene and RNA Interference of Cycloartenol Synthase Gene in Panax notoginseng Cells.

    PubMed

    Yang, Yan; Ge, Feng; Sun, Ying; Liu, Diqiu; Chen, Chaoyin

    2017-04-05

    To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS) was transformed into Panax notoginseng (P. notoginseng) cells in which RNA interference (RNAi) of the cycloartenol synthase (CAS) gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT) cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.

  19. Ischemic preconditioning inhibits over-expression of arginyl-tRNA synthetase gene Rars in ischemia-injured neurons.

    PubMed

    Shen, Yin; Zhao, Hong-Yang; Wang, Hai-Jun; Wang, Wen-Liang; Zhang, Li-Zhi; Fu, Rong

    2016-08-01

    The expression changes of Rars gene in ischemia-injured neurons were investigated by detecting its translational product arginyl-tRNA synthetase (ArgRS), and the inhibitory effects of ischemic preconditioning (IPC) on Rars gene were explored. Both IPC model and prolonged ischemia (PI) model were established by using the classic oxygen glucose deprivation (OGD) method. The primary cultured neurons were assigned into the following groups: the experimental group (IPC+PI group), undergoing PI after a short period of IPC; the conditional control group (PI control group), subjected to PI without IPC; blank control group, the normally cultured neurons. The Rars transcriptional activities and ArgRS expression levels were measured at different time points after re-oxygenation (3 h/6 h/12 h/24 h). Data were collected and statistically analyzed. Compared to the blank control group, the Rars activities and ArgRS levels were significantly increased in PI control group, peaking at the time point of 6 h after re-oxygenation. Rars activities and ArgRS levels were significantly lower in the experimental group than in the PI control group at different time points after re-oxygenation. PI insult can induce an escalating activity of Rars and lead to ArgRS over-expression in primary cultured neurons. IPC can inhibit the increased Rars activity and down-regulate ArgRS expression of ischemia-insulted neurons. This mechanism may confer ischemic tolerance on neurons.

  20. Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit.

    PubMed

    Leclercq, J; Martin, F; Sanier, C; Clément-Vidal, A; Fabre, D; Oliver, G; Lardet, L; Ayar, A; Peyramard, M; Montoro, P

    2012-10-01

    Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.

  1. Over-expression of GSH1 gene and disruption of PEP4 gene in self-cloning industrial brewer's yeast.

    PubMed

    Wang, Zhao-Yue; He, Xiu-Ping; Zhang, Bo-Run

    2007-11-01

    Foam stability is often influenced by proteinase A, and flavor stability is often affected by oxidation during beer storage. In this study, PEP4, the gene coding for proteinase A, was disrupted in industrial brewing yeast. In the meantime, one copy of GSH1 gene increased in the same strain. GSH1 is responsible for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for synthesis of glutathione which is one kind of important antioxidant and beneficial to beer flavor stability. In order to improve the brewer's yeast, plasmid pYPEP, pPC and pPCG1 were firstly constructed, which were recombined plasmids with PEP4 gene, PEP4's disruption and PEP4's disruption+GSH1 gene respectively. These plasmids were verified to be correct by restriction enzymes' assay. By digesting pPCG1 with AatII and PstI, the DNA fragment for homologous recombination was obtained carrying PEP4 sequence in the flank and GSH1 gene internal to the fragment. Since self-cloning technique was applied in the study and the modified genes were from industrial brewing yeast itself, the improved strains, self-cloning strains, were safe to public. The genetic stability of the improved strains was 100%. The results of PCR analysis of genome DNA showed that coding sequence of PEP4 gene had been deleted and GSH1 gene had been inserted into the locus of PEP4 gene in self-cloning strains. The fermentation ability of self-cloning strain, SZ-1, was similar to that of the host. Proteinase A could not be detected in beer brewed with SZ-1, and GSH content in the beer increased 35% compared to that of the host, Z-1.

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

    PubMed

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

    2012-12-19

    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.

  3. 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.

  4. 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.

  5. CCND1-CDK4-mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo.

    PubMed

    Mende, Nicole; Kuchen, Erika E; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico; Waskow, Claudia

    2015-07-27

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1-CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1-CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1-CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

  6. 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.

  7. Over-expression of a rice tau class glutathione s-transferase gene improves tolerance to salinity and oxidative stresses in Arabidopsis.

    PubMed

    Sharma, Raghvendra; Sahoo, Annapurna; Devendran, Ragunathan; Jain, Mukesh

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional proteins encoded by large gene family in plants, which play important role in cellular detoxification of several endobiotic and xenobiotic compounds. Previously, we suggested the diverse roles of rice GST gene family members in plant development and various stress responses based on their differential expression. In this study, we report the functional characterization of a rice tau class GST gene, OsGSTU4. OsGSTU4 fusion protein was found to be localized in nucleus and cytoplasm. The over-expression of OsGSTU4 in E. coli resulted in better growth and higher GST activity under various stress conditions. Further, we raised over-expression transgenic Arabidopsis plants to reveal its in planta function. These transgenic lines showed reduced sensitivity towards plant hormones, auxin and abscisic acid. Various analyses revealed improved tolerance in transgenic Arabidopsis plants towards salinity and oxidative stresses, which may be attributed to the lower accumulation of reactive oxygen species and enhanced GST activity. In addition, microarray analysis revealed up-regulation of several genes involved in stress responses and cellular detoxification processes in the transgenic plants as compared to wild-type. These results suggest that OsGSTU4 can be used as a good candidate for the generation of stress-tolerant crop plants.

  8. Over-Expression of a Rice Tau Class Glutathione S-Transferase Gene Improves Tolerance to Salinity and Oxidative Stresses in Arabidopsis

    PubMed Central

    Sharma, Raghvendra; Sahoo, Annapurna; Devendran, Ragunathan; Jain, Mukesh

    2014-01-01

    Glutathione S-transferases (GSTs) are multifunctional proteins encoded by large gene family in plants, which play important role in cellular detoxification of several endobiotic and xenobiotic compounds. Previously, we suggested the diverse roles of rice GST gene family members in plant development and various stress responses based on their differential expression. In this study, we report the functional characterization of a rice tau class GST gene, OsGSTU4. OsGSTU4 fusion protein was found to be localized in nucleus and cytoplasm. The over-expression of OsGSTU4 in E. coli resulted in better growth and higher GST activity under various stress conditions. Further, we raised over-expression transgenic Arabidopsis plants to reveal its in planta function. These transgenic lines showed reduced sensitivity towards plant hormones, auxin and abscisic acid. Various analyses revealed improved tolerance in transgenic Arabidopsis plants towards salinity and oxidative stresses, which may be attributed to the lower accumulation of reactive oxygen species and enhanced GST activity. In addition, microarray analysis revealed up-regulation of several genes involved in stress responses and cellular detoxification processes in the transgenic plants as compared to wild-type. These results suggest that OsGSTU4 can be used as a good candidate for the generation of stress-tolerant crop plants. PMID:24663444

  9. 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

  10. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

    PubMed

    Jadaun, Jyoti Singh; Sangwan, Neelam S; Narnoliya, Lokesh K; Singh, Neha; Bansal, Shilpi; Mishra, Bhawana; Sangwan, Rajender Singh

    2017-04-01

    Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  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

    2016-08-31

    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.

  17. Over-expression of dopamine D2 receptor and inwardly rectifying potassium channel genes in drug-naive schizophrenic peripheral blood lymphocytes as potential diagnostic markers.

    PubMed

    Zvara, Agnes; Szekeres, György; Janka, Zoltán; Kelemen, János Z; Cimmer, Csongor; Sántha, Miklós; Puskás, László G

    2005-01-01

    Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL) express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3) was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2) and the inwardly rectifying potassium channel (Kir2.3) were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR) using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

  18. Effect of ascorbate oxidase over-expression on ascorbate recycling gene expression in response to agents imposing oxidative stress.

    PubMed

    Fotopoulos, Vasileios; Sanmartin, Maite; Kanellis, Angelos K

    2006-01-01

    Ascorbate oxidase (AO) is a cell wall-localized enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to the unstable radical monodehydroascorbate (MDHA) which rapidly disproportionates to yield dehydroascorbate (DHA) and AA, and thus contributes to the regulation of the AA redox state. Here, it is reported that in vivo lowering of the apoplast AA redox state, through increased AO expression in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi), exerts no effects on the expression levels of genes involved in AA recycling under normal growth conditions, but plants display enhanced sensitivity to various oxidative stress-promoting agents. RNA blot analyses suggest that this response correlates with a general suppression of the plant's antioxidative metabolism as demonstrated by lower expression levels of AA recycling genes. Furthermore, studies using Botrytis cinerea reveal that transgenic plants exhibit increased sensitivity to fungal infection, although the response is not accompanied by a similar suppression of AA recycling gene expression. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in the AA redox state in the leaf apoplast of these transgenic plants results in shifts in their capacity to withstand oxidative stress imposed by agents imposing oxidative stress.

  19. Transcription-dependent radial distribution of TCF7L2 regulated genes in chromosome territories.

    PubMed

    Torabi, Keyvan; Wangsa, Darawalee; Ponsa, Immaculada; Brown, Markus; Bosch, Anna; Vila-Casadesús, Maria; Karpova, Tatiana S; Calvo, Maria; Castells, Antoni; Miró, Rosa; Ried, Thomas; Camps, Jordi

    2017-03-25

    Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/β-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.

  20. 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.

  1. 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.

  2. 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

  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. Mutation in the Arabidopisis thaliana DEK1 calpain gene perturbs endosperm and embryo development while over-expression affects organ development globally.

    PubMed

    Lid, Stein Erik; Olsen, Lene; Nestestog, Ragnhild; Aukerman, Milo; Brown, Roy C; Lemmon, Betty; Mucha, Mark; Opsahl-Sorteberg, Hilde-Gunn; Olsen, Odd-Arne

    2005-06-01

    A T-DNA insertion in the Arabidopsis thaliana DEK1 gene, encoding a calpain-like cysteine proteinase with a predicted membrane anchor, causes unorganized embryo development displaying irregular mitotic divisions in the embryo proper and suspensor. Embryo development is arrested at the globular stage, and the embryo proper lacks a defined protoderm. In the endosperm, the aleurone-like peripheral cell layer is partly or completely lacking. The Arabidopsis DEK1 wild-type transcript is expressed evenly throughout the endosperm and the embryo in developing seed as determined using in situ hybridization. The conclusion that the observed phenotype is caused by a T-DNA insertion in the Arabidopsis DEK1 gene is confirmed by complementation with the Arabidopisis DEK1 genomic sequence, as well as analysis of a second T-DNA insertion allele. Over-expression of the Arabidopsis DEK1 gene coding sequence under the control of the 35S promoter causes a number of developmental phenotypes, including a global lack of trichomes, leaves exhibiting improper dorsiventral symmetry and aberrant cell organization in flowers. We interpret the data to suggest a role for DEK1 in providing cells with positional clues for an appropriate developmental context within plant tissues.

  5. The zinc finger gene ZIC2 has features of an oncogene and its over- expression correlates strongly with the clinical course of epithelial ovarian cancer

    PubMed Central

    Marchini, Sergio; Poynor, Elizabeth; Barakat, Richard R; Clivio, Luca; Cinquini, Michela; Fruscio, Robert; Porcu, Luca; Bussani, Cecilia; D’Incalci, Maurizio; Erba, Eugenio; Romano, Michela; Cattoretti, Giorgio; Katsaros, Dionyssios; Koff, Andrew; Luzzatto, Lucio

    2015-01-01

    Purpose Epithelial ovarian tumors (EOTs) are amongst the most lethal of malignancies in women. We have previously identified ZIC2 as expressed at a higher level in samples of a malignant form (MAL) of EOT than in samples of a form with low malignant potential (LMP). We have now investigated the role of ZIC2 in driving tumor growth and its association with clinical outcomes. Experimental Design ZIC2 expression levels were analysed in two independent tumor tissue collections of LMP and MAL. In vitro experiments aimed to test the role of ZIC2 as a transforming gene. Cox models were used to correlate ZIC2 expression with clinical endpoints. Results ZIC2 expression was about 40-fold in terms of mRNA and about 17-fold in terms of protein in MAL (n = 193) versus LMP (n = 39) tumors. ZIC2 mRNA levels were high in MAL cell lines, but undetectable in LMP cell lines. Over-expression of ZIC2 was localized to the nucleus. ZIC2 over-expression increases the growth rate and foci formation of NIH 3T3 cells, and stimulates anchorage-independent colony formation; down-regulation of ZIC2 decreases the growth rate of MAL cell lines. Zinc finger domains 1 and 2 are required for transforming activity. In stage I MAL ZIC2 expression was significantly associated with overall survival in both univariate (p = 0.046), and multivariate model (p = 0.049). Conclusions ZIC2, a transcription factor related to the sonic hedgehog pathway, is a strong discriminant between MAL and LMP tumors: it may be a major determinant of outcome of EOT. PMID:22733541

  6. Enhanced drought tolerance in transgenic rice over-expressing of maize C4 phosphoenolpyruvate carboxylase gene via NO and Ca(2+).

    PubMed

    Qian, Baoyun; Li, Xia; Liu, Xiaolong; Chen, Pingbo; Ren, Chengang; Dai, Chuanchao

    2015-03-01

    We determined the effects of endogenous nitric oxide and Ca(2+) on photosynthesis and gene expression in transgenic rice plants (PC) over-expressing the maize C4pepc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) under drought. In this study, seedlings were subjected to PEG 6000 treatments using PC and wild type (WT; Kitaake). The results showed that, compared with WT, PC had higher relative water content (RWC) and net photosynthetic rate (Pn) under drought. During a 2-day re-watering treatment, Pn recovered faster in PC than in WT. Further analyses showed that, under the drought treatment, the amount of endogenous hydrogen peroxide (H2O2) increased in WT mainly via NADPH oxidase. While in PC, the endogenous nitric oxide (NO) content increased via nitrate reductase and nitric oxide synthase on day 2 of the drought treatment and day 1 of the re-watering treatment. After 2 days of drought treatment, PC also showed higher PEPC activity, calcium content, phospholipase D (PLD) activity, C4-pepc and NAC6 transcript levels, and protein kinase activity as compared with PC without treatment. These changes did not occur in WT. Correlation analysis also proved NO associated with these indicators in PC. Based on these results, there was a particular molecular mechanism of drought tolerance in PC. The mechanism is related to the signaling processes via NO and Ca(2+) involving the protein kinase and the transcription factor, resulted in up-regulation of PEPC activity and its gene expression, such as C4pepc. Some genes encode antioxidant system, cu/zn-sod as well, which promote antioxidant system to clear MDA and superoxide anion radical, thereby conferring drought tolerance.

  7. MicroRNA-26b attenuates monocrotaline-induced pulmonary vascular remodeling via targeting connective tissue growth factor (CTGF) and cyclin D1 (CCND1)

    PubMed Central

    Zhou, Sijing; Li, Min; Sun, Li; Xu, Xuan; Fei, Guanghe

    2016-01-01

    MicroRNAs are involved in the control of cell growth, and deregulated pulmonary artery smooth muscle cell proliferation plays an essential role in the development of pulmonary hypertension. The objective of this study was to identify differentially expressed microRNA(s) and explore its therapeutic role in treatment of the disease. MicroRNA expression profile analysis showed microRNA-26b was differentially expressed in pulmonary artery smooth muscle cells harvested from monocrotaline-treated rats, and we validated microRNA-26b targets, in vitro and in vivo, CTGF and CCND1, both of which have been shown, in our previous work, to be involved in the pathogenesis of pulmonary hypertension. In vivo experiments demonstrated monocrotaline-induced pulmonary artery remodeling could be almost completely abolished by administration of microRNA-26b, while CTGF or CCND1 shRNA significantly, but only partially, attenuated the remodeling by silencing the designed target. Additionally, exogenous expression of the microRNA-26b substantially downregulated CTGF and CCND1 in human pulmonary artery smooth muscle cells. MicroRNA-26b might be a potent therapeutic tool to treat pulmonary hypertension. PMID:27322082

  8. Microrna-26b attenuates monocrotaline-induced pulmonary vascular remodeling via targeting connective tissue growth factor (CTGF) and cyclin D1 (CCND1).

    PubMed

    Wang, Ran; Ding, Xing; Zhou, Sijing; Li, Min; Sun, Li; Xu, Xuan; Fei, Guanghe

    2016-11-08

    MicroRNAs are involved in the control of cell growth, and deregulated pulmonary artery smooth muscle cell proliferation plays an essential role in the development of pulmonary hypertension. The objective of this study was to identify differentially expressed microRNA(s) and explore its therapeutic role in treatment of the disease. MicroRNA expression profile analysis showed microRNA-26b was differentially expressed in pulmonary artery smooth muscle cells harvested from monocrotaline-treated rats, and we validated microRNA-26b targets, in vitro and in vivo, CTGF and CCND1, both of which have been shown, in our previous work, to be involved in the pathogenesis of pulmonary hypertension. In vivo experiments demonstrated monocrotaline-induced pulmonary artery remodeling could be almost completely abolished by administration of microRNA-26b, while CTGF or CCND1 shRNA significantly, but only partially, attenuated the remodeling by silencing the designed target. Additionally, exogenous expression of the microRNA-26b substantially downregulated CTGF and CCND1 in human pulmonary artery smooth muscle cells. MicroRNA-26b might be a potent therapeutic tool to treat pulmonary hypertension.

  9. 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

  10. 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.

  11. CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway.

    PubMed

    Yang, Yang; Hu, Shuai; Liu, Jie; Cui, Yun; Fan, Yu; Lv, Tianjing; Liu, Libo; Li, Jun; He, Qun; Han, Wenke; Yu, Wei; Sun, Yin; Jin, Jie

    2017-02-20

    Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors.

  12. CD8+ T cells promote proliferation of benign prostatic hyperplasia epithelial cells under low androgen level via modulation of CCL5/STAT5/CCND1 signaling pathway

    PubMed Central

    Yang, Yang; Hu, Shuai; Liu, Jie; Cui, Yun; Fan, Yu; Lv, Tianjing; Liu, Libo; Li, Jun; He, Qun; Han, Wenke; Yu, Wei; Sun, Yin; Jin, Jie

    2017-01-01

    Previous studies by our group have shown that low intra-prostatic dihydrotestosterone (DHT) induced BPH epithelial cells (BECs) to recruit CD8+ T cells. However, the influence of the recruited CD8+ T cells on BECs under a low androgen level is still unknown. Here, we found CD8+ T cells have the capacity to promote proliferation of BECs in low androgen condition. Mechanism dissection revealed that interaction between CD8+ T cells and BECs through secretion of CCL5 might promote the phosphorylation of STAT5 and a higher expression of CCND1 in BECs. Suppressed CCL5/STAT5 signals via CCL5 neutralizing antibody or STAT5 inhibitor Pimozide led to reverse CD8+ T cell-enhanced BECs proliferation. IHC analysis from Finasteride treated patients showed PCNA expression in BECs was highly correlated to the level of CD8+ T cell infiltration and the expression of CCL5. Consequently, our data indicated infiltrating CD8+ T cells could promote the proliferation of BECs in low androgen condition via modulation of CCL5/STAT5/CCND1 signaling. The increased secretion of CCL5 from the CD8+ T cells/BECs interaction might help BECs survive in a low DHT environment. Targeting these signals may provide a new potential therapeutic approach to better treat BPH patients who failed the therapy of 5α-reductase inhibitors. PMID:28216616

  13. CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

    PubMed Central

    Mende, Nicole; Kuchen, Erika E.; Lesche, Mathias; Grinenko, Tatyana; Kokkaliaris, Konstantinos D.; Hanenberg, Helmut; Lindemann, Dirk; Dahl, Andreas; Platz, Alexander; Höfer, Thomas; Calegari, Federico

    2015-01-01

    Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis. PMID:26150472

  14. A rare case of t(11;22) in a mantle cell lymphoma like B-cell neoplasia resulting in a fusion of IGL and CCND1: case report

    PubMed Central

    2011-01-01

    The chromosomal translocation (11;14)(q13;q32) rearranging the locus for cyclin D1 (CCND1) to that of the immunoglobulin heavy chain (IGH) can be found in virtually all cases of mantle cell lymphoma (MCL), while other CCND1 translocations are extremely rare. As CCND1 overexpression and activation is a hallmark of MCL it is regarded as a central biological mechanism in the development and maintenance of this disease. Here we present a patient initially diagnosed with chronic lymphocytic leukemia (CLL) where chromosome banding analysis revealed, among other aberrations, a translocation (11;22)(q13;q11.2). We show by fluorescence in situ hybridization (FISH) analysis that on chromosome 22 the immunoglobulin light chain lambda (IGL) is involved in this cytogenetic aberration. Additionally, we demonstrate the resulting overexpression of CCND1 on the RNA and protein level, thereby consolidating the new diagnosis of a MCL-like B-cell neoplasia. Summing up, we described a rare case of t(11;22)(q13;q11.2) in a MCL-like neoplasia and showed that this aberration leads to an overexpression of CCND1 which is regarded as a key biological feature in MCL. This case underlines the importance of cytogenetic analyses especially in atypical cases of B cell lymphomas. PMID:21457541

  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 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

  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

    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.

  17. 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

  18. Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer.

    PubMed

    Li, Ning; Zhong, Xiaomin; Lin, Xiaojuan; Guo, Jinyi; Zou, Lian; Tanyi, Janos L; Shao, Zhongjun; Liang, Shun; Wang, Li-Ping; Hwang, Wei-Ting; Katsaros, Dionyssios; Montone, Kathleen; Zhao, Xia; Zhang, Lin

    2012-05-18

    The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).

  19. 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.

  20. 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.

  1. Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.

    PubMed Central

    Alefounder, P R; Baldwin, S A; Perham, R N; Short, N J

    1989-01-01

    Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies. Images Fig. 2. PMID:2649077

  2. Engineering drought tolerant tomato plants over-expressing BcZAT12 gene encoding a C₂H₂ zinc finger transcription factor.

    PubMed

    Rai, Avinash Chandra; Singh, Major; Shah, Kavita

    2013-01-01

    Efficient genetic transformation of cotyledonary explants of tomato (Solanum lycopersicum, cv. H-86, Kashi vishesh) was obtained. Disarmed Agrobacterium tumifaciens strain GV 3101 was used in conjugation with binary vector pBinAR containing a construct consisting of the coding sequence of the BcZAT12 gene under the regulatory control of the stress inducible Bclea1a promoter. ZAT12 encodes a C₂H₂ zinc finger protein which confers multiple abiotic stress tolerance to plants. Integration of ZAT12 gene into nuclear genome of individual kanamycin resistant transformed T₀ tomato lines was confirmed by Southern blot hybridization with segregation analysis of T(1) plants showing Mendelian inheritance of the transgene. Expression of ZAT12 in drought-stressed transformed tomato lines was verified in T₂ generation plants using RT-PCR. Of the six transformed tomato lines (ZT1-ZT6) the transformants ZT1 and ZT5 showed maximum expression of BcZAT12 gene transcripts when exposed to 7 days drought stress. Analysis of relative water content (RWC), electrolyte leakage (EL), chlorophyll colour index (CCI), H₂O₂ level and catalase activity suggested that tomato BcZAT12 transformants ZT1 and ZT5 have significantly increased levels of drought tolerance. These results suggest that BcZAT12 transformed tomato cv. H-86 has real potential for molecular breeding programs aimed at augmenting yield of tomato in regions affected with drought stress.

  3. Over-expression of the PaAP1 gene from sweet cherry (Prunus avium L.) causes early flowering in Arabidopsis thaliana.

    PubMed

    Wang, Jing; Zhang, Xiaoming; Yan, Guohua; Zhou, Yu; Zhang, Kaichun

    2013-02-15

    A homologue of SQUAMOSA/APETALA1, designated PaAP1, was isolated from Prunus avium by reverse transcription-PCR (RT-PCR). The full length of PaAP1 cDNA is 753 bp, and it codes for a polypeptide of 250 amino acid residues. Sequence comparison revealed that PaAP1 belongs to the MADS-box gene family. Phylogenetic analysis indicated that PaAP1 shared the highest identity with SQUA/AP1 homologues from Prunus serrulata. Real-time fluorescence quantitative PCR analysis showed that PaAP1 was expressed at high levels in petal, sepal, style, and flower buds, which was slightly different from the expression pattern of AP1 of Arabidopsis thaliana. To characterize the functions of PaAP1, we assessed Arabidopsis transformed with 35S::PaAP1. A total of 8 transgenic T(1) lines with an early flowering phenotype were obtained, and a 3:1 segregation ratio of flowering time was observed in the T(2) generation of 4 lines. This study provides the first functional analysis of an SQUA/AP1 homolog from P. avium and suggests that PaAP1 is potentially useful for shortening the juvenile period in sweet cherry.

  4. Over-expression of the Beclin1 gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells.

    PubMed

    Sun, Yang; Liu, Jia-Hua; Jin, Long; Lin, Sai-Mei; Yang, Yin; Sui, Yu-Xia; Shi, Hong

    2010-08-28

    The purpose of this study was to investigate whether the autophagy-related gene, Beclin1, plays a role in the regulation of chemosensitivity to anti-cancer drugs in cervical cancer CaSki cells. Expression of the Beclin1 protein was up-regulated in pcDNA3.1-Bec transfectants and led to cell arrest in the G(0)/G(1) phase of the cell cycle. The MTT assay indicated that over-expression of Beclin1 sensitized CaSki cells to chemotherapeutic drugs (cisplatin, paclitaxel, 5-fluorouracil, and epirubicin) and induced greater degrees of cytotoxicity than vector-only controls. After treatment with anti-cancer drugs, flow cytometric analysis indicated that the Beclin1-transfected group showed a greater increase in apoptosis than did the non-transfected group. Furthermore, pSUPER-Bec transfectants did not lead to a significant increase of resistance to each of these anti-cancer drugs. These results suggest that Beclin1 plays an important role in the regulation of potent anti-tumor activity, and over-expression of Beclin1 in CaSki cells may enhance apoptosis signaling induced by anti-cancer drugs.

  5. Amplification and overexpression of CTTN and CCND1 at chromosome 11q13 in Esophagus squamous cell carcinoma (ESCC) of North Eastern Chinese Population.

    PubMed

    Hu, Xiaoxia; Moon, Ji Wook; Li, Shibo; Xu, Weihong; Wang, Xianfu; Liu, Yuanyuan; Lee, Ji-Yun

    2016-01-01

    Esophageal squamous cell carcinoma (ESCC) is a genetically complex tumor type and is a major cause of cancer-related mortality. The combination of genetics, diet, behavior, and environment plays an important role in the carcinogenesis of ESCC. To characterize the genomic aberrations of this disease, we investigated the genomic imbalances in 19 primary ESCC cases using high-resolution array comparative genomic hybridization (CGH). All cases showed either loss or gain of whole chromosomes or segments of chromosome(s) with variable genomic sizes. The copy number alterations per case affected the median 34% (~ 1,034Mb/3,000Mb) of the whole genome. Recurrent gains were 1q21.3-qter, 3q13.11-qter, 5pter-p11, 7pter-p15.3, 7p12.1-p11.2, 7q11-q11.2, 8p12-qter, 11q13.2-q13.3, 12pter-p13.31, 17q24.2, 20q11.21-qter, and 22q11.21-q11.22 whereas the recurrent losses were 3pter-p11.1, 4pter-p12, 4q28.3-q31.22, 4q31.3-q32.1, 9pter-p12, 11q22.3-qter and 13q12.11-q22.1. Amplification of 11q13 resulting in overexpression of CTTN/CCND1 was the most prominent finding, which was observed in 13 of 19 ESCC cases. These unique profiles of copy number alteration should be validated by further studies and need to be taken into consideration when developing biomarkers for early detection of ESCC.

  6. 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.

  7. mTOR is involved in 17β-estradiol-induced, cultured immature boar Sertoli cell proliferation via regulating the expression of SKP2, CCND1, and CCNE1.

    PubMed

    Yang, Wei-Rong; Wang, Yong; Wang, Yi; Zhang, Jiao-Jiao; Zhang, Jia-Hua; Lu, Cheng; Wang, Xian-Zhong

    2015-04-01

    Mammalian target of rapamycin (mTOR) is known to be involved in mammalian cell proliferation, while S-phase kinase-associated protein 2 (SKP2) plays a vital role in the cell cycle. Within the testis, estrogen also plays an important role in Sertoli cell proliferation, although it is not clear how. The present study asked if mTOR is involved in 17β-estradiol-dependent Sertoli cell proliferation. We specifically assessed if extracellular signal-regulated kinase 1/2 (ERK1/2) and/or phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) exert convergent effects toward the activation of mTOR signaling, and if this signaling regulates the expression of SKP2 through retinoblastoma (RB) and early mitotic inhibitor 1 (EMI1) protein and on CCNE1 and CCND1 mRNA levels. Treatment with 17β-estradiol for 15-90 min activated mTOR, with mTOR phosphorylation peaking after 30 min. U0126 (5 μM), a specific inhibitor of (MEK1/2), and 10-DEBC (2 μM), a selective inhibitor of AKT, both significantly reduced 17β-estradiol-induced phosphorylation of mTOR. Rapamycin suppressed 17β-estradiol-induced Sertoli cell proliferation, appearing to act by reducing the abundance of SKP2, CCND1, and CCNE1 mRNA as well as RB and EMI1 protein. These data indicated that 17β-estradiol enhances Sertoli cell proliferation via mTOR activation, which involves both ERK1/2 and PI3K/AKT signaling. Activated mTOR subsequently increases SKP2 mRNA and protein expression by enhancing the expression of CCND1 and CCNE1, and inhibits SKP2 protein degradation by increasing EMI1 abundance.

  8. Cortical Astrocytes Acutely Exposed to the Monomethylarsonous Acid (MMA(III)) Show Increased Pro-inflammatory Cytokines Gene Expression that is Consistent with APP and BACE-1: Over-expression.

    PubMed

    Escudero-Lourdes, C; Uresti-Rivera, E E; Oliva-González, C; Torres-Ramos, M A; Aguirre-Bañuelos, P; Gandolfi, A J

    2016-10-01

    Long-term exposure to inorganic arsenic (iAs) through drinking water has been associated with cognitive impairment in children and adults; however, the related pathogenic mechanisms have not been completely described. Increased or chronic inflammation in the brain is linked to impaired cognition and neurodegeneration; iAs induces strong inflammatory responses in several cells, but this effect has been poorly evaluated in central nervous system (CNS) cells. Because astrocytes are the most abundant cells in the CNS and play a critical role in brain homeostasis, including regulation of the inflammatory response, any functional impairment in them can be deleterious for the brain. We propose that iAs could induce cognitive impairment through inflammatory response activation in astrocytes. In the present work, rat cortical astrocytes were acutely exposed in vitro to the monomethylated metabolite of iAs (MMA(III)), which accumulates in glial cells without compromising cell viability. MMA(III) LD50 in astrocytes was 10.52 μM, however, exposure to sub-toxic MMA(III) concentrations (50-1000 nM) significantly increased IL-1β, IL-6, TNF-α, COX-2, and MIF-1 gene expression. These effects were consistent with amyloid precursor protein (APP) and β-secretase (BACE-1) increased gene expression, mainly for those MMA(III) concentrations that also induced TNF-α over-expression. Other effects of MMA(III) on cortical astrocytes included increased proliferative and metabolic activity. All tested MMA(III) concentrations led to an inhibition of intracellular lactate dehydrogenase (LDH) activity. Results suggest that MMA(III) induces important metabolic and functional changes in astrocytes that may affect brain homeostasis and that inflammation may play a major role in cognitive impairment-related pathogenicity in As-exposed populations.

  9. Over-expression of JcDREB, a putative AP2/EREBP domain-containing transcription factor gene in woody biodiesel plant Jatropha curcas, enhances salt and freezing tolerance in transgenic Arabidopsis thaliana.

    PubMed

    Tang, Mingjuan; Liu, Xiaofei; Deng, Huaping; Shen, Shihua

    2011-12-01

    Jatropha curcas L. is an all-purpose biodiesel plant and is widely distributed in tropical and subtropical climates. It can grow well on poor quality soil which is not qualified for crop cultivation. This is very important for relieving land, food and energy crises. However, tropical and subtropical distribution limits the production of J. curcas seed. So it is valuable to know the molecular mechanism of J. curcas response to adverse abiotic environmental factors, especially freezing stress, in order to change the plant's characteristics. Until now there are just a few reports about J. curcas molecular biology. In this paper, we cloned and characterized a DNA binding protein from this plant, designated as JcDREB. Sequence analysis and yeast one-hybrid assays show that JcDREB can effectively function as a transcription factor of DREB protein family belonging to A-6 subgroup member. Expression patterns of JcDREB showed that it was induced by cold, salt and drought stresses, not by ABA. Over-expression of JcDREB in transgenic Arabidopsis exhibited enhanced salt and freezing stresses. Understanding the molecular mechanisms of J. curcas responses to environmental stresses, for example, high salinity, drought and low temperature, is crucial for improving their stress tolerance and productivity. This work provides more information about A-6 subgroup members of DREB subfamily.

  10. 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

  11. Influence of over-expression of the Flowering Promoting Factor 1 gene (FPF1) from Arabidopsis on wood formation in hybrid poplar (Populus tremula L. × P. tremuloides Michx.).

    PubMed

    Hoenicka, Hans; Lautner, Silke; Klingberg, Andreas; Koch, Gerald; El-Sherif, Fadia; Lehnhardt, Denise; Zhang, Bo; Burgert, Ingo; Odermatt, Jürgen; Melzer, Siegbert; Fromm, Jörg; Fladung, Matthias

    2012-02-01

    Constitutive expression of the FPF1 gene in hybrid aspen (Populus tremula L. × P. tremuloides Michx.) showed a strong effect on wood formation but no effect on flowering time. Gene expression studies showed that activity of flowering time genes PtFT1, PtCO2, and PtFUL was not increased in FPF1 transgenic plants. However, the SOC1/TM3 class gene PTM5, which has been related to wood formation and flowering time, showed a strong activity in stems of all transgenic lines studied. Wood density was lower in transgenic plants, despite significantly reduced vessel frequency which was overcompensated by thinner fibre cell walls. Chemical screening of the wood by pyrolysis GC/MS showed that FPF1 transgenics have higher fractions of cellulose and glucomannan products as well as lower lignin content. The latter observation was confirmed by UV microspectrophotometry on a cellular level. Topochemical lignin distribution revealed a slower increase of lignin incorporation in the developing xylem of the transgenics when compared with the wild-type plants. In line with the reduced wood density, micromechanical wood properties such as stiffness and ultimate stress were also significantly reduced in all transgenic lines. Thus, we provide evidence that FPF1 class genes may play a regulatory role in both wood formation and flowering in poplar.

  12. Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses.

    PubMed

    Kalamaki, Mary S; Alexandrou, Dimitris; Lazari, Diamanto; Merkouropoulos, Georgios; Fotopoulos, Vasileios; Pateraki, Irene; Aggelis, Alexandros; Carrillo-López, Armando; Rubio-Cabetas, Maria J; Kanellis, Angelos K

    2009-01-01

    A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified.

  13. Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains

    PubMed Central

    Cairns, T.C.; Sidhu, Y.S.; Chaudhari, Y.K.; Talbot, N.J.; Studholme, D.J.; Haynes, K.

    2015-01-01

    Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen. PMID:26092797

  14. Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.

    PubMed

    Cairns, T C; Sidhu, Y S; Chaudhari, Y K; Talbot, N J; Studholme, D J; Haynes, K

    2015-06-01

    Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.

  15. Absence of effects of Sir2 over-expression on lifespan in C. elegans and Drosophila

    PubMed Central

    Burnett, Camilla; Valentini, Sara; Cabreiro, Filipe; Goss, Martin; Somogyvári, Milán; Piper, Matthew D.; Hoddinott, Matthew; Sutphin, George L.; Leko, Vid; McElwee, Joshua J.; Vazquez, Rafael; Orfila, Anne-Marie; Ackerman, Daniel; Au, Catherine; Vinti, Giovanna; Riesen, Michèle; Howard, Ken; Neri, Christian; Bedalov, Antonio; Kaeberlein, Matt; Söti, Csaba; Partridge, Linda; Gems, David

    2011-01-01

    Over-expression of sirtuins (NAD+-dependent protein deacetylases) has been reported to increase lifespan in budding yeast, Caenorhabditis elegans and Drosophila melanogaster1-3. Studies of gene effects on ageing are vulnerable to confounding effects of genetic background4. We re-examined the reported effects of sirtuin over-expression on ageing and found that standardisation of genetic background and use of appropriate controls abolished the apparent effects in both C. elegans and Drosophila. In C. elegans, outcrossing of a line with high level sir-2.1 over-expression1 abrogated the longevity increase, but not sir-2.1 over-expression. Instead, longevity co-segregated with a second-site mutation affecting sensory neurons. Outcrossing of a line with low copy number sir-2.1 over-expression2 also abrogated longevity. A Drosophila strain with ubiquitous over-expression of dSir2 using the UAS-GAL4 system was long-lived relative to wild-type controls, as previously reported3, but not relative to the appropriate transgenic controls, and nor was a new line with stronger over-expression of dSir2. These findings underscore the importance of controlling for genetic background and the mutagenic effects of transgene insertions in studies of genetic effects on lifespan. The life extending effect of dietary restriction (DR) on ageing in Drosophila has also been reported to be dSir2 dependent3. We found that DR increased fly lifespan independently of dSir2. Our findings do not rule out a role for sirtuins in determination of metazoan lifespan, but they do cast doubt on the robustness of the previously reported effects on lifespan in C. elegans and Drosophila. PMID:21938067

  16. Dlx2 over-expression: a possible mechanism for first branchial arch malformation.

    PubMed

    Dai, Jie-wen; Wang, Xu-dong; Sun, Hao; Jiang, Wen-hui; Lu, Jing-ting; Shen, Guo-fang

    2011-06-01

    The first branchial arch malformation (FBAM) is a rare congenital defect associated with anomalous development of the first and second branchial arches. Cause of FBAM still remains unknown, and is thought in most cases to be multifactorial, involving both genetic and enviromental factors. Dlx2 as a member of the Dlx homeobox gene family, plays a crucial role in the development of the first branchial arch. The tissues regulated mainly by Dlx2 are coincident with the tissues mainly involved in FBAM. Dlx2 over-expression generated by electroporation transfection can disturb the migration and differentiation of cranial neural crest cells (CNCCs), which migrate to the branchial arches and in turn give rise to much of the facial skeleton and connective tissues. Furthermore, Dlx2 over-expression can be found in the first branchial arch spontaneous mutant mice. So we hypothesize that Dlx2 over-expression mutation causes FBAM due to an increase in cell-cell adhesion and inhibiting the migration of CNCC to the first branchial arch in the early stage, or migrating to an incorrect position and can't differentiate into normal tissues. What an exact role of Dlx2 over-expression in FBAM remains to be investigated and Dlx2 over-expression transgenic mouse will be a nice model for further research in FBAM.

  17. Over-expression of microRNA169 confers enhanced drought tolerance to tomato.

    PubMed

    Zhang, Xiaohui; Zou, Zhe; Gong, Pengjuan; Zhang, Junhong; Ziaf, Khurram; Li, Hanxia; Xiao, Fangming; Ye, Zhibiao

    2011-02-01

    Plant miRNA regulates multiple developmental and physiological processes, including drought responses. We found that the accumulation of Sly-miR169 in tomato (Solanum lycopersicum) was induced by drought stress. Consequently, Sly-miR169 targets, namely, three nuclear factor Y subunit genes (SlNF-YA1/2/3) and one multidrug resistance-associated protein gene (SlMRP1), were significantly down-regulated by drought stress. Constitutive over-expression of a miR169 family member, Sly-miR169c, in tomato plant can efficiently down-regulate the transcripts of the target genes. Compared with non-transgenic plants, transgenic plants over-expressing Sly-miR169c displayed reduced stomatal opening, decreased transpiration rate, lowered leaf water loss, and enhanced drought tolerance. Our study is the first to provide evidence that the Sly-miR169c negatively regulates stomatal movement in tomato drought responses.

  18. GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

    PubMed

    Mohan, Sara; Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Gatehouse, Michelle L; Monaghan, Katrina S; Vanga, Bhanupratap R; Pitman, Andrew R; Jones, E Eirian; Conner, Anthony J; Jacobs, Jeanne M E

    2014-03-01

    Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

  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. PMID:27630644

  20. Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase

    SciTech Connect

    Brizio, Carmen; Galluccio, Michele; Wait, Robin; Torchetti, Enza Maria; Bafunno, Valeria; Accardi, Rosita; Gianazza, Elisabetta; Indiveri, Cesare; Barile, Maria . E-mail: m.barile@biologia.uniba.it

    2006-06-09

    FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63 kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60 kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl{sub 2}, as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8 {+-} 1.3 nmol of FAD synthesized/min/mg protein and exhibited a K {sub M} value for FMN of 1.5 {+-} 0.3 {mu}M. This is First report on characterization of human FADS, and First cloning and over-expression of FADS from an organism higher than yeast.

  1. CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis

    PubMed Central

    Wang, Yinjie; Sheng, Liping; Zhang, Huanru; Du, Xinping; An, Cong; Xia, Xiaolong; Chen, Fadi; Jiang, Jiafu; Chen, Sumei

    2017-01-01

    The gene encoding the MYB (v-myb avian myeloblastosis vira l oncogene homolog) transcription factor CmMYB19 was isolated from chrysanthemum. It encodes a 200 amino acid protein and belongs to the R2R3-MYB subfamily. CmMYB19 was not transcriptionally activated in yeast, while a transient expression experiment conducted in onion epidermal cells suggested that the CmMYB19 product localized to the nucleus. CmMYB19 transcription was induced by aphid (Macrosiphoniella sanborni) infestation, and the abundance of transcript was higher in the leaf and stem than in the root. The over-expression of CmMYB19 restricted the multiplication of the aphids. A comparison of transcript abundance of the major genes involved in lignin synthesis showed that CmPAL1 (phenylalanine ammonia lyase 1), CmC4H (cinnamate4 hydroxylase), Cm4CL1 (4-hydroxy cinnamoyl CoA ligase 1), CmHCT (hydroxycinnamoyl CoA-shikimate/quinate hydroxycinnamoyl transferase), CmC3H1 (coumarate3 hydroxylase1), CmCCoAOMT1 (caffeoyl CoA O-methyltransferase 1) and CmCCR1 (cinnamyl CoA reductase1) were all upregulated, in agreement with an increase in lignin content in CmMYB19 over-expressing plants. Collectively, the over-expression of CmMYB19 restricted the multiplication of the aphids on the host, mediated by an enhanced accumulation of lignin. PMID:28287502

  2. A metagenomic alkaline protease from saline habitat: cloning, over-expression and functional attributes.

    PubMed

    Purohit, Megha K; Singh, Satya P

    2013-02-01

    Metagenomics has opened new horizon to unlock the biotechnological potential for novel enzymes. An alkaline protease gene was obtained from the total environmental DNA extracted from a saline habitat. After cloning and sequencing, it was identified that the protease gene related to uncultivable bacteria (HM219181). The protease was over expressed at 6h of induction with optimum induction at 1mM IPTG and 27°C. The purified enzyme was characterized with respect to various factors; temperature, pH, NaCl and chemical denaturant. The sequence analysis indicated a hydrophobic tendency of the protein, while the predicted 3D structure indicated the enzyme as a serine protease.

  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. 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.

  5. 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

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

    PubMed

    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.

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

    PubMed

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

    2014-05-15

    DMRT1 encodes a conserved transcription factor with an essential role in gonadal function. In the chicken, DMRT1 in 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 feminising 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.

  8. A viral over-expression system for the major malaria mosquito Anopheles gambiae

    PubMed Central

    Suzuki, Yasutsugu; Niu, Guodong; Hughes, Grant L.; Rasgon, Jason L.

    2014-01-01

    Understanding pathogen/mosquito interactions is essential for developing novel strategies to control mosquito-borne diseases. Technical advances in reverse-genetics, such as RNA interference (RNAi), have facilitated elucidation of components of the mosquito immune system that are antagonistic to pathogen development, and host proteins essential for parasite development. Forward genetic approaches, however, are limited to generation of transgenic insects, and while powerful, mosquito transgenesis is a resource- and time-intensive technique that is not broadly available to most laboratories. The ability to easily “over-express” genes would enhance molecular studies in vector biology and expedite elucidation of pathogen-refractory genes without the need to make transgenic insects. We developed and characterized an efficient Anopheles gambiae densovirus (AgDNV) over-expression system for the major malaria vector Anopheles gambiae. High-levels of gene expression were detected at 3 days post-infection and increased over time, suggesting this is an effective system for gene induction. Strong expression was observed in the fat body and ovaries. We validated multiple short promoters for gene induction studies. Finally, we developed a polycistronic system to simultaneously express multiple genes of interest. This AgDNV-based toolset allows for consistent transduction of genes of interest and will be a powerful molecular tool for research in Anopheles gambiae mosquitoes. PMID:24875042

  9. 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

  10. Over-expression of HSP47 augments mouse embryonic stem cell smooth muscle differentiation and chemotaxis.

    PubMed

    Wong, Mei Mei; Yin, Xiaoke; Potter, Claire; Yu, Baoqi; Cai, Hao; Di Bernardini, Elisabetta; Xu, Qingbo

    2014-01-01

    In the recent decade, embryonic stem cells (ESC) have emerged as an attractive cell source of smooth muscle cells (SMC) for vascular tissue engineering owing to their unlimited self-renewal and differentiation capacities. Despite their promise in therapy, their efficacy is still hampered by the lack of definitive SMC differentiation mechanisms and difficulties in successful trafficking of the ESC towards a site of injury or target tissue. Heat shock protein 47 (HSP47) is a 47-kDa molecular chaperone that is required for the maturation of various types of collagen and has been shown to be a critical modulator of different pathological and physiological processes. To date, the role of HSP47 on ESC to SMC differentiation or ESC chemotaxis is not known and may represent a potential molecular approach by which ESC can be manipulated to increase their efficacy in clinic. We provide evidence that HSP47 is highly expressed during ESC differentiation into the SMC lineage and that HSP47 reduction results in an attenuation of the differentiation. Our experiments using a HSP47 plasmid transfection system show that gene over-expression is sufficient to induce ESC-SMC differentiation, even in the absence of exogenous stimuli. Furthermore, HSP47 over-expression in ESC also increases their chemotaxis and migratory responses towards a panel of chemokines, likely via the upregulation of chemokine receptors. Our findings provide direct evidence of induced ESC migration and differentiation into SMC via the over-expression of HSP47, thus identifying a novel approach of molecular manipulation that can potentially be exploited to improve stem cell therapy for vascular repair and regeneration.

  11. Response to Multiple Radiation Doses of Fibroblasts Over-Expressing Dominant Negative Ku70

    SciTech Connect

    Urano, Muneyasu Huang Yunhong; He Fuqiu; Minami, Akiko; Ling, C. Clifton; Li, Gloria C.

    2008-06-01

    Purpose: To evaluate the response of cells over-expressing dominant negative (DN) Ku70 to single and multiple small radiation doses. Methods and Materials: Clones of fibroblasts over-expressing DNKu70, DNKu70-7, DNKu70-11, and parental Rat-1 cells were irradiated under oxic or hypoxic conditions with single or multiple doses. Cells were trypsinized 0 or 6 h after irradiation to determine surviving fraction (SF). Results: Oxic DNKu70-7 or -11 cells trypsinized 6 h after irradiation were 1.52 or 1.25 and 1.28 or 1.15 times more sensitive than oxic Rat-1 at SF of 0.5 and 0.1, respectively. Hypoxic DNKu70-7 or -11 cells trypsinized 6 h after irradiation were 1.44 or 1.70 and 1.33 or 1.51 times more sensitive than hypoxic Rat-1 at SF of 0.5 and 0.1, respectively. To the multiple doses, oxic and hypoxic DNKu70-7 or -11 cells were 1.35 or 1.37 and 2.23 or 4.61 times more sensitive than oxic and hypoxic Rat-1, respectively, resulting in very small oxygen enhancement ratios. Namely, enhancement caused by DNKu70 under hypoxia after multiple doses was greater than that under oxic conditions and greater than that after single dose. Conclusions: Over-expression of DNKu70 enhances cells' response to radiation given as a single dose and as multiple small doses. The enhancement after multiple doses was stronger under hypoxic than under oxic conditions. These results encourage the use of DNKu70 fragment in a gene-radiotherapy.

  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. Over-expression of Dof-type transcription factor increases lipid production in Chlamydomonas reinhardtii.

    PubMed

    Ibáñez-Salazar, Alejandro; Rosales-Mendoza, Sergio; Rocha-Uribe, Alejandro; Ramírez-Alonso, Jocelín Itzel; Lara-Hernández, Ignacio; Hernández-Torres, Araceli; Paz-Maldonado, Luz María Teresita; Silva-Ramírez, Ana Sonia; Bañuelos-Hernández, Bernardo; Martínez-Salgado, José Luis; Soria-Guerra, Ruth Elena

    2014-08-20

    The high demand for less polluting, newer, and cheaper fuel resources has increased the search of the most innovative options for the production of the so-called biofuels. Chlamydomonas reinhardtii is a photosynthetic unicellular algae with multiple biotechnological advantages such as easy handling in the laboratory, a simple scale-up to industrial levels, as well as a feasible genetic modification at nuclear and chloroplast levels. Besides, its fatty acids can be used to produce biofuels. Previous studies in plants have found that the over expression of DOF-type transcription factor genes increases the synthesis and the accumulation of total lipids in seeds. In this context, the over-expression of a DOF-type transcription factor in C. reinhardtii was applied as approach to increase the amount of lipids. The results indicate higher amounts (around 2-fold) of total lipids, which are mainly fatty acids, in the genetically C. reinhardtii modified strains when compared with the non-genetically modified strain. In order to elucidate the possible function of the introduced Dof-type transcription factor, we performed a transcription profile of 8 genes involved in fatty acid biosynthesis and 6 genes involved in glycerolipid biosynthesis, by quantitative real time (qRT-PCR). Differential expression profile was observed, which can explain the increase in lipid accumulation. However, these strains did not show notable changes in the fatty acid profile. This work represents an early effort in generating a strategy to increase fatty acids production in C. reinhardtii and their use in biofuel synthesis.

  14. Glucocorticoid-induced p11 over-expression and chromatin remodeling: a novel molecular mechanism of traumatic stress?

    PubMed

    Zhang, Lei; Li, He; Hu, Xianzhang; Li, Xiao Xia; Smerin, Stanley; Ursano, Robert

    2011-06-01

    While the actions of glucocorticoids on brain function have been comprehensively studied, understanding of the underlying genomic mechanisms is advancing slowly. Recently, it was found that p11 is associated with traumatic stress and depression, and glucocorticoids regulate expression of the p11 gene. The ligand-activated glucocorticoid receptor (GR) interacts with two glucocorticoid response elements (GREs) in the p11 promoter region to up-regulate the p11 gene. RU486, a glucocorticoid receptor antagonist, and mutation of GREs both block glucocorticoid-induced p11 over-expression, suggesting that glucocorticoid-induced p11 over-expression is mediated by GR and GREs. Thus, the p11 gene can be transcriptionally activated. There is evidence that this transcriptional activation is mediated by the remodeling of chromatin complexes in response to glucocorticoid receptor-regulated promotors. The regulation of eukaryotic gene expression by chromatin remodeling is complex and is essential for numerous cellular processes. The association of linker-histone, non-histone and heterochromatin-specific proteins plays a key role in the generation of higher-order chromatin structures. Understanding the chromatin remodeling involved in the glucocorticoid-mediated increase of p11 expression by stress may clarify stress-induced over-expression of p11 and also identify a new therapeutic target for post-traumatic disorder and depressive disorders, i.e., chromatin remodeling.

  15. Over-expression of ThpI from Choristoneura fumiferana enhances tolerance to cold in Arabidopsis.

    PubMed

    Zhu, Bo; Xiong, Ai-Sheng; Peng, Ri-He; Xu, Jing; Jin, Xiao-Fen; Meng, Xiu-Rong; Yao, Quan-Hong

    2010-02-01

    Thermal hysteresis proteins (Thps) known as antifreeze proteins for their antifreeze activity, depress the freezing point of water below the melting point in many polar marine fishes, terrestrial arthropods and plants. For the purpose of breeding cold-resistant plants, we designed to introduce the Thp gene into the plants. The physiological and biochemical effect of high-lever expression of the modified Choristoneura fumiferana Thp (ThpI) in Arabidopsis thaliana plants was analyzed. Under low temperature stress, the ThpI transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the ThpI over-expressing plants was confirmed by the changes of electrolyte leakage activity, malonyldialdehyde and proline contents. These results preliminarily showed that the Thp possibly be used to enhance the low temperature-tolerant ability of plants.

  16. ERG deregulation induces PIM1 over-expression and aneuploidy in prostate epithelial cells.

    PubMed

    Magistroni, Vera; Mologni, Luca; Sanselicio, Stefano; Reid, James Frances; Redaelli, Sara; Piazza, Rocco; Viltadi, Michela; Bovo, Giorgio; Strada, Guido; Grasso, Marco; Gariboldi, Manuela; Gambacorti-Passerini, Carlo

    2011-01-01

    The ERG gene belongs to the ETS family of transcription factors and has been found to be involved in atypical chromosomal rearrangements in several cancers. To gain insight into the oncogenic activity of ERG, we compared the gene expression profile of NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG. We found that all three ERG fusions significantly up-regulate PIM1 expression in the NIH-3T3 cell line. PIM1 is a serine/threonine kinase frequently over-expressed in cancers of haematological and epithelial origin. We show here that tERG expression induces PIM1 in the non-malignant prostate cell line RWPE-1, strengthening the relation between tERG and PIM1 up-regulation in the initial stages of prostate carcinogenesis. Silencing of tERG reversed PIM1 induction. A significant association between ERG and PIM1 expression in clinical prostate carcinoma specimens was found, suggesting that such a mechanism may be relevant in vivo. Chromatin Immunoprecipitation experiments showed that tERG directly binds to PIM1 promoter in the RWPE-1 prostate cell line, suggesting that tERG could be a direct regulator of PIM1 expression. The up-regulation of PIM1 induced by tERG over-expression significantly modified Cyclin B1 levels and increased the percentage of aneuploid cells in the RWPE-1 cell line after taxane-based treatment. Here we provide the first evidence for an ERG-mediated PIM1 up-regulation in prostate cells in vitro and in vivo, suggesting a direct effect of ERG transcriptional activity in the alteration of genetic stability.

  17. Improving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters.

    PubMed

    Zhang, Jia; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Hong, Jiong

    2015-01-01

    Three transporter genes including Kluyveromyces marxianus aquaglyceroporin gene (KmFPS1), Candida intermedia glucose/xylose facilitator gene (CiGXF1) or glucose/xylose symporter gene (CiGXS1) were over-expressed in K. marxianus YZJ017 to improve xylitol production at elevated temperatures. The xylitol production of YZJ074 that harbored CiGXF1 was improved to 147.62g/L in Erlenmeyer flask at 42°C. In fermenter, 99.29 and 149.60g/L xylitol were produced from 99.55 and 151.91g/L xylose with productivity of 4.14 and 3.40g/L/h respectively at 42°C. Even at 45°C, YZJ074 could produce 101.30g/L xylitol from 101.41g/L xylose with productivity of 2.81g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074 could produce 312.05g/L xylitol which is the highest yield reported to date. The engineered strains YZJ074 which can produce xylitol at elevated temperatures is an excellent foundation for xylitol bioconversion.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. Cardiac α-actin over-expression therapy in dominant ACTA1 disease.

    PubMed

    Ravenscroft, Gianina; McNamara, Elyshia; Griffiths, Lisa M; Papadimitriou, John M; Hardeman, Edna C; Bakker, Anthony J; Davies, Kay E; Laing, Nigel G; Nowak, Kristen J

    2013-10-01

    More than 200 mutations in the skeletal muscle α-actin gene (ACTA1) cause either dominant or recessive skeletal muscle disease. Currently, there are no specific therapies. Cardiac α-actin is 99% identical to skeletal muscle α-actin and the predominant actin isoform in fetal muscle. We previously showed cardiac α-actin can substitute for skeletal muscle α-actin, preventing the early postnatal death of Acta1 knock-out mice, which model recessive ACTA1 disease. Dominant ACTA1 disease is caused by the presence of 'poison' mutant actin protein. Experimental and anecdotal evidence nevertheless indicates that the severity of dominant ACTA1 disease is modulated by the relative amount of mutant skeletal muscle α-actin protein present. Thus, we investigated whether transgenic over-expression of cardiac α-actin in postnatal skeletal muscle could ameliorate the phenotype of mouse models of severe dominant ACTA1 disease. In one model, lethality of ACTA1(D286G). Acta1(+/-) mice was reduced from ∼59% before 30 days of age to ∼12%. In the other model, Acta1(H40Y), in which ∼80% of male mice die by 5 months of age, the cardiac α-actin transgene did not significantly improve survival. Hence cardiac α-actin over-expression is likely to be therapeutic for at least some dominant ACTA1 mutations. The reason cardiac α-actin was not effective in the Acta1(H40Y) mice is uncertain. We showed that the Acta1(H40Y) mice had endogenously elevated levels of cardiac α-actin in skeletal muscles, a finding not reported in dominant ACTA1 patients.

  3. Cortactin gene amplification and expression in breast cancer: a chromogenic in situ hybridisation and immunohistochemical study.

    PubMed

    Dedes, Konstantin J; Lopez-Garcia, Maria-Angeles; Geyer, Felipe C; Lambros, Maryou B K; Savage, Kay; Vatcheva, Radost; Wilkerson, Paul; Wetterskog, Daniel; Lacroix-Triki, Magali; Natrajan, Rachael; Reis-Filho, Jorge S

    2010-12-01

    Amplification of 11q13 is found in approximately 15% of breast cancers. Cyclin D1 (CCND1) has been reported to be the 'driver' of this amplicon, however, multiple genes map to the smallest region of amplification of 11q13. Out of these genes, cortactin (CTTN) has been shown to be consistently overexpressed at the mRNA level in tumours harbouring 11q13 amplification. The aims of this study are to define whether CTTN is consistently co-amplified with the main core of the 11q13 amplicon, whether it is consistently overexpressed when amplified and to determine correlations between CTTN amplification and overexpression with clinicopathological features of breast cancers and survival of breast cancer patients. CTTN and CCND1 chromogenic in situ hybridisation (CISH) probes and a validated monoclonal antibody against CTTN were applied to a tissue microarray of a cohort of breast cancers from patients treated with anthracycline-based chemotherapy. CTTN and CCND1 amplifications were found in 12.3 and 12.4% of cases, respectively. All cases harbouring CTTN amplification also displayed CCND1 amplification. High expression of CTTN was found in 10.8% of cases and was associated with CTTN amplification, expression of 'basal' markers and topoisomerase IIα. Exploratory subgroup analysis of tumours devoid of 11q13 amplification revealed that high expression of CTTN in the absence of CTTN gene amplification was associated with lymph node negative disease, lack of hormone receptors and FOXA1, expression of 'basal' markers, high Ki-67 indices, p53 nuclear expression, and basal-like and triple negative phenotypes. CTTN expression and CTTN gene amplification were not associated with disease-, metastasis-free and overall survival. In conclusion, CTTN is consistently co-amplified with CCND1 and expressed at higher levels in breast cancers harbouring 11q13 amplification, suggesting that CTTN may also constitute one of the drivers of this amplicon. CTTN expression is not associated with the

  4. 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

  5. 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.

  6. Over-expression of Multi-heme C-type Cytochromes

    SciTech Connect

    Shi, Liang; Lin, Chiann Tso; Markillie, Lye Meng; Squier, Thomas C.; Hooker, Brian S.

    2005-02-01

    ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

  7. Over-expression of microRNA-1 causes arrhythmia by disturbing intracellular trafficking system

    PubMed Central

    Su, Xiaomin; Liang, Haihai; Wang, He; Chen, Guizhi; Jiang, Hua; Wu, Qiuxia; Liu, Tianyi; Liu, Qiushuang; Yu, Tong; Gu, Yunyan; Yang, Baofeng; Shan, Hongli

    2017-01-01

    Dysregulation of intracellular trafficking system plays a fundamental role in the progression of cardiovascular disease. Up-regulation of miR-1 contributes to arrhythmia, we sought to elucidate whether intracellular trafficking contributes to miR-1-driven arrhythmia. By performing microarray analyses of the transcriptome in the cardiomyocytes-specific over-expression of microRNA-1 (miR-1 Tg) mice and the WT mice, we found that these differentially expressed genes in miR-1 Tg mice were significantly enrichment with the trafficking-related biological processes, such as regulation of calcium ion transport. Also, the qRT-PCR and western blot results validated that Stx6, Braf, Ube3a, Mapk8ip3, Ap1s1, Ccz1 and Gja1, which are the trafficking-related genes, were significantly down-regulated in the miR-1 Tg mice. Moreover, we found that Stx6 was decreased in the heart of mice after myocardial infarction and in the hypoxic cardiomyocytes, and further confirmed that Stx6 is a target of miR-1. Meanwhile, knockdown of Stx6 in cardiomyocytes resulted in the impairments of PLM and L-type calcium channel, which leads to the increased resting ([Ca2+]i). On the contrary, overexpression of Stx6 attenuated the impairments of miR-1 or hypoxia on PLM and L-type calcium channel. Thus, our studies reveals that trafficking-related gene Stx6 may regulate intracellular calcium and is involved in the occurrence of cardiac arrhythmia, which provides new insights in that miR-1 participates in arrhythmia by regulating the trafficking-related genes and pathway.

  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. 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.

  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.

  11. Lysosomal acid lipase over-expression disrupts lamellar body genesis and alveolar structure in the lung.

    PubMed

    Li, Yuan; Qin, Yulin; Li, Huimin; Wu, Renliang; Yan, Cong; Du, Hong

    2007-12-01

    The functional role of neutral lipids in the lung is poorly understood. Lysosomal acid lipase (LAL) is a critical enzyme in hydrolysis of cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Human LAL was over-expressed in a doxycycline-controlled system in mouse respiratory epithelial cells to accelerate intracellular neutral lipid degradation and perturb the surfactant homeostasis in the lung. In this animal system, neutral lipid concentrations of pulmonary surfactant were reduced in bronchoalveolar lavage fluid (BALF) in association with decrease of surfactant protein C (SP-C) gene expression. The size and the number of lamellar bodies in alveolar type II epithelial cells (AT II cells) were significantly reduced accordingly. The number of macrophages required for surfactant recycling in BALF was also significantly reduced. As a result of these combinatory effects, emphysema of the alveolar structure was observed. Taken together, neutral lipid homeostasis is essential for maintenance of lamellar body genesis and the alveolar structure in the lung.

  12. Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

    PubMed

    Li, Zhiqian; Zeng, Baosheng; Ling, Lin; Xu, Jun; You, Lang; Aslam, Abu F M; Tan, Anjiang; Huang, Yongping

    2015-01-01

    RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.

  13. Crebbp loss cooperates with Bcl2 over-expression to promote lymphoma in mice.

    PubMed

    García-Ramírez, Idoia; Tadros, Saber; González-Herrero, Inés; Martín-Lorenzo, Alberto; Rodríguez-Hernández, Guillermo; Moore, Dalia; Ruiz-Roca, Lucía; Blanco, Oscar; Alonso-López, Diego; De Las Rivas, Javier; Hartert, Keenan; Duval, Romain; Klinkebiel, David; Bast, Martin; Vose, Julie; Lunning, Matthew; Fu, Kai; Greiner, Timothy; Rodrigues-Lima, Fernando; Jiménez, Rafael; García Criado, Francisco Javier; García Cenador, María Begoña; Brindle, Paul; Vicente-Dueñas, Carolina; Alizadeh, Ash; Sánchez-García, Isidro; Green, Michael R

    2017-03-13

    CREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 over-expression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B-cells we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B-cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these two diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared to FL. Together our data therefore provide important links between Crebbp inactivation and Bcl2 dependence, and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.

  14. A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

    PubMed Central

    Martins, Marta; McCusker, Matthew P; Viveiros, Miguel; Couto, Isabel; Fanning, Séamus; Pagès, Jean-Marie; Amaral, Leonard

    2013-01-01

    It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to

  15. Over-expression of pcvA involved in vesicle-vacuolar fusion affects the conidiation and penicillin production in Penicillium chrysogenum.

    PubMed

    Xu, Xinxin; Yang, Jing; An, Yang; Pan, Yuanyuan; Liu, Gang

    2012-03-01

    Rab GTPase is required for vesicle-vacuolar fusion during the vacuolar biogenesis in fungi. Rab GTPase-encoding gene, pcvA, was cloned from Penicillium chrysogenum: it contained five introns and its predicted protein contained the conserved Rab GTPase domain involved in GTP-binding and hydrolysis. Over-expression of pcvA significantly stimulated the vesicle-vacuolar fusion but repressed the conidiation and decreased conidial tolerance against thermal stress. Penicillin production was decreased in the pcvA over-expressed strain suggesting that pcvA is involved in vesicle-vacuolar fusion participates in the penicillin biosynthesis in P. chrysogenum.

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

    PubMed

    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

    2015-08-28

    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.

  17. 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).

  18. 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

  19. 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.

  20. Increased impulsive behavior and risk proneness following lentivirus-mediated dopamine transporter over-expression in rats' nucleus accumbens.

    PubMed

    Adriani, W; Boyer, F; Gioiosa, L; Macrì, S; Dreyer, J-L; Laviola, G

    2009-03-03

    Multiple theories have been proposed for sensation seeking and vulnerability to impulse-control disorders [Zuckerman M, Kuhlman DM (2000) Personality and risk-taking: Common biosocial factors. J Pers 68:999-1029], and many of these rely on a dopamine system deficit. Available animal models reproduce only some behavioral symptoms and seem devoid of construct validity. We used lentivirus tools for over-expressing or silencing the dopamine transporter (DAT) and we evaluated the resulting behavioral profiles in terms of motivation and self-control. Wistar adult rats received stereotaxic inoculation of a lentivirus that allowed localized intra-accumbens delivery of a DAT gene enhancer/silencer, or the green fluorescent protein, GFP. These animals were studied for intolerance to delay, risk proneness and novelty seeking. As expected, controls shifted their demanding from a large reward toward a small one when the delivery of the former was increasingly delayed (or uncertain). Interestingly, in the absence of general locomotor effects, DAT over-expressing rats showed increased impulsivity (i.e. a more marked shift of demanding from the large/delayed toward the small/soon reward), and increased risk proneness (i.e. a less marked shift from the large/uncertain toward the small/sure reward), compared with controls. Rats with enhanced or silenced DAT expression did not show any significant preference for a novel environment. In summary, consistent with literature on comorbidity between attention-deficit/hyperactivity disorder and pathological gambling, we demonstrate that DAT over-expression in rats' nucleus accumbens leads to impulsive and risk prone phenotype. Thus, a reduced dopaminergic tone following altered accumbal DAT function may subserve a sensation-seeker phenotype and the vulnerability to impulse-control disorders.

  1. Abnormalities of Endocytosis, Phagocytosis, and Development Process in Dictyostelium Cells That Over-Express Acanthamoeba castellanii Metacaspase Protein

    PubMed Central

    SAHEB, Entsar; TRZYNA, Wendy; MARINGER, Katherine; BUSH, John

    2015-01-01

    Background: Acanthamoeba castellanii forms a resistant cyst that protects the parasite against the host’s immune response. Acanthamoeba Type-I metacaspase (Acmcp) is a caspase-like protein that has been found to be expressed during the encystations. Dictyostelium discoideum is an organism closely related to Acanthamoeba useful for studying the molecular function of this protozoan caspase-like protein. Methods: The full length of Acmcp and a mutated version of the same gene, which lacks the proline rich N-terminal region (Acmcp-dpr), were cloned into the pDneo2a-GFP vector separately. The pDneo2a-GFP-Acmcp and pDneo2a-GFPAcmcp-dpr were electro-transfected into wild type D. discoideum cells to create cell lines that over-expressed Acmcp or Acmcp-dpr. Results: Both cell lines that over-expressed Acmcp and Acmcp-dpr showed a significant increase in the fluid phase internalization and phagocytosis rate compared to the control cells. Additionally, the cells expressing the Acmcp-dpr mutant were unable to initiate early development and failed to aggregate or form fruiting bodies under starvation conditions, whereas Acmcp over-expressing cells showed the opposite phenomena. Quantitative cell death analysis provided additional support for these findings. Conclusion: Acmcp is involved in the processes of endocytosis and phagocytosis. In addition, the proline rich region in Acmcp is important for cellular development in Dictyostelium. Given its important role in the development process, metacaspase protein is proposed as a candidate drug target against infections caused by A. castellanii. PMID:26246819

  2. 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.

  3. Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice.

    PubMed

    Qi, Yaocheng; Wang, Hongjuan; Zou, Yu; Liu, Cheng; Liu, Yanqi; Wang, Ying; Zhang, Wei

    2011-01-03

    In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H(2)O(2)-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ(m)) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ(m) relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ(m) and inhibiting the amplification of ROS.

  4. 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...

  5. Promotor Regions Determining Over-Expression of Metalloproteinase Genes in Breast Cancer

    DTIC Science & Technology

    1999-06-01

    enlarged right popliteal lymph nodes (Table 1). Histological exami- the extraction of RNA, by the addition of cell-lysis solution to the metaplastic...The right and left popliteal lymph nodes were removed by blunt dissecting and 6). along the midline of the biceps femoris muscle, which encloses the...established from the popliteal lymph node metastases of dissected into one-third and two-third portions. The one-third portion was fixed five of the

  6. Promoter Regions Determining Over-Expression of Metalloproteinase Genes in Breast Cancer

    DTIC Science & Technology

    2000-07-01

    The two cell fractions were then processed for enlarged right popliteal lymph nodes (Table 1). Histological exami- the extraction of RNA, by the...cells (Fig. 2A, Lanes 5 The right and left popliteal lymph nodes were removed by blunt dissecting and 6). along the midline of the biceps femoris muscle... lymph node was lines were established from the popliteal lymph node metastases of dissected into one-third and two-third portions. The one-third portion

  7. Over expression of miR-200c suppresses invasion and restores methotrexate sensitivity in lung cancer A549 cells.

    PubMed

    Shan, Wulin; Zhang, Xiaolei; Li, Ming; Deng, Fang; Zhang, Jing

    2016-11-30

    MicroRNAs have become recognized as key players in the development of malignancy. MiR-200c can function as a tumor suppressor gene. However, the effect of miR-200c on methotrexate resistance remains unclear to date. This study aims to evaluate the function of miR-200c in lung cancer A549 cells. The data presented in our study demonstrated that the expression of miR-200c was down-regulated in methotrexate-resistant A549 cells. Over expression of miR-200c could significantly inhibit cell proliferation, induce G0/G1 cell cycle arrest and induce cell apoptosis. RT-PCR and Western blot assays showed that the expression of P53 and P21 were significantly increased with miR-200c overexpression. These results indicated that over expression of miR-200c might enhance the sensitivity of A549 cells to methotrexate through the P53/P21 pathway. Furthermore, miR-200c overexpression significantly inhibited cell migration and invasion with increasing the expression of E-cad and decreasing the expression of EZH2. In consequence, we provide a mechanism of acquired resistance to methotrexate that is caused by the loss of miR-200c in lung cancer cells. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance.

  8. PGC-1α over-expression suppresses the skeletal muscle atrophy and myofiber-type composition during hindlimb unloading.

    PubMed

    Wang, Jing; Wang, Fei; Zhang, Peng; Liu, Hongju; He, Jian; Zhang, Chenyu; Fan, Ming; Chen, Xiaoping

    2017-03-01

    Disuse leads to severe muscle atrophy and a slow-to-fast myofiber-type transition. PGC-1α (Peroxisome proliferator-activated receptor γ coactivator 1α) is documented to play an important role in muscle atrophy and slow-twitch myofiber determination. Transcription of atrophy-related Atrogin-1 by FoxO3 can be reduced by PGC-1α. While Smad3 augments FoxO3-induced Atrogin-1 and MuRF1 promoter activity. So PGC-1α, as a transcription co-activator, may regulate hindlimb unloading (HU)-induced myofiber-type transition and muscle atrophy through Smad3. Our results showed that transgenic PGC-1α mice resisted HU-induced muscle loss, atrophy-related genes expression, and slow-to-fast myofiber-type transition. Furthermore, over-expression of PGC-1α resisted the increase in pSmad3 during muscle atrophy in vivo and in vitro. And, PGC-1α over-expression inhibited the expression of atrogenes via suppressing the phosphorylation of Smad3 in vitro. Thus, PGC-1α is effective in regulating myofiber-type transition during HU, and it alleviates skeletal muscle atrophy partially through suppressing the activation of Smad3.

  9. 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.

  10. 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.

  11. Biochemical mechanisms of imidacloprid resistance in Nilaparvata lugens: over-expression of cytochrome P450 CYP6AY1.

    PubMed

    Ding, Zhiping; Wen, Yucong; Yang, Baojun; Zhang, Yixi; Liu, Shuhua; Liu, Zewen; Han, Zhaojun

    2013-11-01

    Imidacloprid is a key insecticide extensively used for control of Nilaparvata lugens, and its resistance had been reported both in the laboratory selected strains and field populations. A target site mutation Y151S in two nicotinic acetylcholine receptor subunits and enhanced oxidative detoxification have been identified in the laboratory resistant strain, contributing importantly to imidacloprid resistance in N. lugens. To date, however, imidacloprid resistance in field population is primarily attributable to enhanced oxidative detoxification by over-expressed P450 monooxygenases. A resistant strain (Res), originally collected from a field population and continuously selected in laboratory with imidacloprid for more than 40 generations, had 180.8-fold resistance to imidacloprid, compared to a susceptible strain (Sus). Expression of different putative P450 genes at mRNA levels was detected and compared between Res and Sus strains, and six genes were found expressed significantly higher in Res strain than in Sus strain. CYP6AY1 was found to be the most different expressed P450 gene and its mRNA level in Res strain was 17.9 times of that in Sus strain. By expressing in E. coli cells, CYP6AY1 was found to metabolize imidacloprid efficiently with initial velocity calculated of 0.851 ± 0.073 pmol/min/pmol P450. When CYP6AY1 mRNA levels in Res strain was reduced by RNA interference, imidacloprid susceptibility was recovered. In four field populations with different resistance levels, high levels of CYP6AY1 transcript were also found. In vitro and in vivo studies provided evidences that the over-expression of CYP6AY1 was one of the key factors contributing to imidacloprid resistance in the laboratory selected strain Res, which might also be the important mechanism for imidacloprid resistance in field populations, when the target site mutation was not prevalent at present.

  12. Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by over-expression of an Arabidopsis thaliana transcription factor AtPAP1.

    PubMed

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

    2014-08-22

    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.

  13. 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

  14. Transgenic over-expression of YY1 induces pathologic cardiac hypertrophy in a sex-specific manner

    PubMed Central

    Stauffer, Brian L.; Dockstader, Karen; Russell, Gloria; Hijmans, Jamie; Walker, Lisa; Cecil, Mackenzie; Demos-Davies, Kimberly; Medway, Allen; McKinsey, Timothy A.; Sucharov, Carmen C.

    2015-01-01

    YY1 can activate or repress transcription of various genes. In cardiac myocytes in culture YY1 has been shown to regulate expression of several genes involved in myocyte pathology. YY1 can also acutely protect the heart against detrimental changes in gene expression. In this study we show that cardiac over-expression of YY1 induces pathologic cardiac hypertrophy in male mice, measured by changes in gene expression and lower ejection fraction/fractional shortening. In contrast, female animals are protected against pathologic gene expression changes and cardiac dysfunction. Furthermore, we show that YY1 regulates, in a sex-specific manner, the expression of mammalian enable (Mena), a factor that regulates cytoskeletal actin dynamics and whose expression is increased in several models of cardiac pathology, and that Mena expression in humans with heart failure is sex-dependent. Finally, we show that sex differences in YY1 expression are also observed in human heart failure. In summary, this is the first work to show that YY1 has a sex-specific effect in the regulation of cardiac pathology. PMID:25935483

  15. Rhomboid-7 over-expression results in Opa1-like processing and malfunctioning mitochondria.

    PubMed

    Rahman, Mokhlasur; Kylsten, Per

    2011-10-22

    Rhomboid-7 (rho-7) is a mitochondrial-specific intramembranous protease. The loss-of-function mutation rho-7 results in semi-lethality, while escapers have a reduced lifespan with several neurological disorders [1]. Here we show that general, or CNS-specific expression of rho-7 can rescue the lethality of rho-7. General, or CNS-specific over-expression of rho-7 in otherwise wild-type animals caused semi-lethality, with approximately 50% of the animals escaping this lethality, developing into adults displaying a shortened life span with larval locomotory problem. On a cellular level, over-expression resulted in severe depression of ATP levels and cytochrome c oxidase subunit II mRNA levels, a lowered number of mitochondria in neurons and aggregation of mitochondria in the brain indicating mitochondrial malfunction. Over-expression of rho-7 in developing eye discs resulted in an elevated apoptotic index. In the CNS, elevated levels of rho-7 were accompanied by both isoforms of Opa1-like, a dynamin-like GTPase, a mitochondrial component involved in regulating mitochondrial dynamics and function, including apoptosis. Most, but not all, of rho-7 over-expression phenotypes were suppressed by introducing a heterozygous mutation for Opa1-like. Our results suggest that rho-7 and Opa1-like function in a common molecular pathway affecting mitochondrial function and apoptosis in Drosophila melanogaster.

  16. p130Cas over-expression impairs mammary branching morphogenesis in response to estrogen and EGF.

    PubMed

    Camacho Leal, Maria del Pilar; Pincini, Alessandra; Tornillo, Giusy; Fiorito, Elisa; Bisaro, Brigitte; Di Luca, Elisa; Turco, Emilia; Defilippi, Paola; Cabodi, Sara

    2012-01-01

    p130Cas adaptor protein regulates basic processes such as cell cycle control, survival and migration. p130Cas over-expression has been related to mammary gland transformation, however the in vivo consequences of p130Cas over-expression during mammary gland morphogenesis are not known. In ex vivo mammary explants from MMTV-p130Cas transgenic mice, we show that p130Cas impairs the functional interplay between Epidermal Growth Factor Receptor (EGFR) and Estrogen Receptor (ER) during mammary gland development. Indeed, we demonstrate that p130Cas over-expression upon the concomitant stimulation with EGF and estrogen (E2) severely impairs mammary morphogenesis giving rise to enlarged multicellular spherical structures with altered architecture and absence of the central lumen. These filled acinar structures are characterized by increased cell survival and proliferation and by a strong activation of Erk1/2 MAPKs and Akt. Interestingly, antagonizing the ER activity is sufficient to re-establish branching morphogenesis and normal Erk1/2 MAPK activity. Overall, these results indicate that high levels of p130Cas expression profoundly affect mammary morphogenesis by altering epithelial architecture, survival and unbalancing Erk1/2 MAPKs activation in response to growth factors and hormones. These results suggest that alteration of morphogenetic pathways due to p130Cas over-expression might prime mammary epithelium to tumorigenesis.

  17. 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.

  18. 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

  19. 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

  20. Over-expression of phage HK022 Nun protein is toxic for Escherichia coli

    PubMed Central

    Uc-Mass, Augusto; Khodursky, Arkady; Brown, Lewis; Gottesman, Max E.

    2008-01-01

    The Nun protein of coliphage HK022 excludes superinfecting λ phage. Nun recognizes and binds to the N utilization (nut) sites on phage λ nascent RNA and induces transcription termination. Over-expression of Nun from a high-copy plasmid is toxic for E.coli, despite the fact that nut sites are not encoded in the E.coli genome. Cells expressing Nun cannot exit stationary phase. Toxicity is related to transcription termination, since host and nun mutations that block termination also suppress cell killing. Nun inhibits expression of wild-type lacZ, but not lacZ expressed from the Crp/cAMP–independent lacUV5 promoter. Microarray and proteomics analyses show Nun down-regulates crp and tnaA. Crp over-expression and high indole concentrations partially reverse Nun-mediated toxicity and restore lacZ expression. PMID:18571198

  1. 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

  2. MicroRNA 218 mediates the effects of Tbx5a over-expression on zebrafish heart development.

    PubMed

    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.

  3. 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

  4. 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

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

    PubMed Central

    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. PMID:26745629

  6. 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.

  7. Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa.

    PubMed

    Raharjo, Tri J; Chang, Wen-Te; Verberne, Marianne C; Peltenburg-Looman, Anja M G; Linthorst, Huub J M; Verpoorte, Robert

    2004-04-01

    A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L. This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C. sativa was identified. The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04. The gene shares a high homology with a chalcone synthase gene of H. lupulus, 85% and 94% homology on the level of DNA and protein, respectively. Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein. The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity. Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

  8. 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.

  9. Tobacco seeds simultaneously over-expressing Cu/Zn-superoxide dismutase and ascorbate peroxidase display enhanced seed longevity and germination rates under stress conditions.

    PubMed

    Lee, Young Pyo; Baek, Kwang-Hyun; Lee, Haeng-Soon; Kwak, Sang-Soo; Bang, Jae-Woog; Kwon, Suk-Yoon

    2010-05-01

    Reactive oxygen species (ROS) are produced during seed desiccation, germination, and ageing, leading to cellular damage and seed deterioration and, therefore, decreased seed longevity. The effects of simultaneous over-expression of two antioxidant enzymes on seed longevity and seed germination under stressful conditions were investigated. Transgenic tobacco simultaneously over-expressing the Cu/Zn-superoxide dismutase (CuZnSOD) and ascorbate peroxidase (APX) genes in plastids showed normal growth and seed development. Furthermore, the transgenic seeds displayed increased CuZnSOD and APX enzymatic activities during seed development and maintained antioxidant enzymatic activity after two years of dried storage at room temperature. The two-year stored non-transgenic seeds (aged NT seeds) had higher levels of ion leakage than the two-year stored transgenic seeds (aged CA seeds), indicating membrane damage caused by ROS was more severe in the aged NT seeds than the aged CA seeds. The aged CA seeds decreased germination rates as compared to newly harvested transgenic and non-transgenic seeds. The aged CA seeds, however, significantly increased germination rates under various abiotic stress conditions as compared to aged NT seeds. These data strongly suggest that simultaneous over-expression of the CuZnSOD and APX genes in plastids improves seed longevity and germination under various environmental stress conditions by attenuating the effects of oxidative stress produced by elongated storage conditions and harsh environmental stresses.

  10. Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain.

    PubMed

    Lewis, Jo E; Brameld, John M; Hill, Phil; Cocco, Cristina; Noli, Barbara; Ferri, Gian-Luca; Barrett, Perry; Ebling, Francis J P; Jethwa, Preeti H

    2017-01-01

    VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well

  11. Hypothalamic over-expression of VGF in the Siberian hamster increases energy expenditure and reduces body weight gain

    PubMed Central

    Brameld, John M.; Hill, Phil; Cocco, Cristina; Noli, Barbara; Ferri, Gian-Luca; Barrett, Perry; Ebling, Francis J. P.; Jethwa, Preeti H.

    2017-01-01

    VGF (non-acronymic) was first highlighted to have a role in energy homeostasis through experiments involving dietary manipulation in mice. Fasting increased VGF mRNA in the Arc and levels were subsequently reduced upon refeeding. This anabolic role for VGF was supported by observations in a VGF null (VGF-/-) mouse and in the diet-induced and gold-thioglucose obese mice. However, this anabolic role for VGF has not been supported by a number of subsequent studies investigating the physiological effects of VGF-derived peptides. Intracerebroventricular (ICV) infusion of TLQP-21 increased resting energy expenditure and rectal temperature in mice and protected against diet-induced obesity. Similarly, ICV infusion of TLQP-21 into Siberian hamsters significantly reduced body weight, but this was due to a decrease in food intake, with no effect on energy expenditure. Subsequently NERP-2 was shown to increase food intake in rats via the orexin system, suggesting opposing roles for these VGF-derived peptides. Thus to further elucidate the role of hypothalamic VGF in the regulation of energy homeostasis we utilised a recombinant adeno-associated viral vector to over-express VGF in adult male Siberian hamsters, thus avoiding any developmental effects or associated functional compensation. Initially, hypothalamic over-expression of VGF in adult Siberian hamsters produced no effect on metabolic parameters, but by 12 weeks post-infusion hamsters had increased oxygen consumption and a tendency to increased carbon dioxide production; this attenuated body weight gain, reduced interscapular white adipose tissue and resulted in a compensatory increase in food intake. These observed changes in energy expenditure and food intake were associated with an increase in the hypothalamic contents of the VGF-derived peptides AQEE, TLQP and NERP-2. The complex phenotype of the VGF-/- mice is a likely consequence of global ablation of the gene and its derived peptides during development, as well

  12. 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.

  13. Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium.

    PubMed

    Zhang, Dongsheng; Fan, Guo-Chang; Zhou, Xiaoyang; Zhao, Tiemin; Pasha, Zeeshan; Xu, Meifeng; Zhu, Yi; Ashraf, Muhammad; Wang, Yigang

    2008-02-01

    Bone marrow mesenchymal stem cells (MSCs) participate in myocardial repair following myocardial infarction. However, their in vivo reparative capability is limited due to lack of their survival in the infarcted myocardium. To overcome this limitation, we genetically engineered male rat MSCs overexpressing CXCR4 in order to maximize the effect of stromal cell-derived factor-1alpha (SDF-1alpha) for cell migration and regeneration. MSCs were isolated from adult male rats and cultured. Adenoviral transduction was carried out to over-express either CXCR4/green fluorescent protein (Ad-CXCR4/GFP) or Ad-null/GFP alone (control). Flow cytometry was used to identify and isolate GFP/CXCR4 over-expressing MSCs for transplantation. Female rats were assigned to one of four groups (n=8 each) to receive GFP-transduced male MSCs (2 x 10(6)) via tail vein injection 3 days after ligation of the left anterior descending (LAD) coronary artery: GFP-transduced MSCs (Ad-null/GFP-MSCs, group 1) or MSCs over-expressing CXCR4/GFP (Ad-CXCR4/GFP-MSCs, group 2), or Ad-CXCR4/GFP-MSCs plus SDF-1alpha (50 ng/microl) (Ad-CXCR4/GFP-MSCs/SDF-1alpha, group 3), or Ad-miRNA targeting CXCR4 plus SDF-1alpha (Ad-miRNA/GFP-MSCs+SDF-1alpha treatment, group 4). Cardiodynamic data were obtained 4 weeks after induction of regional myocardial infarction (MI) using echocardiography after which hearts were harvested for immunohistochemical studies. The migration of GFP and Y-chromosome positive cells increased significantly in the peri- and infarct areas of groups 2 and 3 compared to control group (p<0.05), or miRNA-CXCR4 group (p<0.01). The number of CXCR4 positive cells in groups 2, 3 was intimately associated with angiogenesis and myogenesis. MSCs engraftment was blocked by pretreatment with miRNA (group 4). Cardiac function was significantly improved in rats receiving MSCs over-expressing CXCR4 alone or with SDF-1alpha. The up-regulation of matrix metalloproteinases (MMPs) by CXCR4 overexpressing MSCs perhaps

  14. Diet-induced over-expression of flightless-I protein and its relation to flightlessness in Mediterranean fruit fly, Ceratitis capitata.

    PubMed

    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.

  15. 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.

  16. 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

  17. Ectopic over-expression of oncogene Pim-2 induce malignant transformation of nontumorous human liver cell line L02.

    PubMed

    Ren, Ke; Duan, Wentao; Shi, Yujun; Li, Bo; Liu, Zuojin; Gong, Jiangping

    2010-07-01

    In order to prove that ectopic over-expression of Pim-2 could induce malignant transformation of human liver cell line L02, three groups of cells were set up including human liver cell line L02 (L02), L02 cells transfected with Pim-2 gene (L02/Pim-2) and L02 cells transfected with empty-vector (L02/Vector). Pim-2 expression levels were detected. The morphology, proliferation level, apoptosis rate and migration ability of the cells were detected respectively. Then the cells were subcutaneously inoculated into athymic mice and the microstructures of the neoplasm were observed. Compared with the controls, Pim-2 expression levels were significantly higher in L02/Pim-2 cells (P<0.05), and their morphology had obvious malignant changes. They also showed a significantly increased proliferation rate (P<0.05) and migration capacity (P<0.05), as well as a significantly decreased apoptosis rate (P<0.05). Only the athymic mice inoculated with L02/Pim-2 cells could generate neoplasm, and the morphology of the neoplasm coincided with that of the hepatoma. The results manifest that ectopic Pim-2 gene could be stably expressed in L02/Pim-2 cells. Both the morphological and biological changes of L02/Pim-2 cells demonstrate the trend of malignant transformation. L02/Pim-2 cells could generate hepatoma in athymic mice. In conclusion, Pim-2 could induce malignant transformation of human liver cell line L02.

  18. Over-expression of STP13, a hexose transporter, improves plant growth and nitrogen use in Arabidopsis thaliana seedlings.

    PubMed

    Schofield, R A; Bi, Y-M; Kant, S; Rothstein, S J

    2009-03-01

    In Arabidopsis thaliana, the regulation of hexose levels by the large monosaccharide transporter (MST) gene family influences many aspects of plant growth. The cloning and transgenic expression of one family member (STP13) enabled the manipulation of carbon (C) and nitrogen (N) metabolism in Arabidopsis. Transgenic seedlings constitutively over-expressing STP13 (STP13OX) had increased rates of glucose uptake, higher endogenous sucrose levels and accumulated more total C and biomass per plant when grown on soil-less media supplemented with 55 mM glucose and sufficient N (9 mM nitrate). Furthermore, STP13OX seedlings acquired 90% more total N than the Col-0 seedlings, and had higher levels of expression of the nitrate transporter NRT2.2. In addition, STP13OX seedlings were larger and had higher biomass than Col-0 seedlings when grown under a limiting N condition (3 mM nitrate). Transgene analysis of STP13 reveals that its gene product is localized to the plasma membrane (PM) in tobacco BY-2 suspension cells, that it encodes a functional MST in planta, and that the STP13 promoter directs GUS expression to the vasculature and to leaf mesophyll cells. This work highlights the link between C and N metabolism, demonstrating that a plant's N use may be improved by increasing the availability of C.

  19. Lipocalin 2 over-expression facilitates progress of castration-resistant prostate cancer via improving androgen receptor transcriptional activity

    PubMed Central

    Feng, Chenchen; Jiang, Haowen; Xu, Jianfeng; Ding, Qiang

    2016-01-01

    Background Castration-resistant prostate cancer (CRPC) is the lethal phenotype of prostate cancer. Lipocalin 2 (LCN2) is aberrantly expressed in many cancers including primary prostate cancer (PCa), but its role in CRPC has not been reported. Results LCN2 expression was upregulated in human primary PCa and CRPC tissues. Overexpression of LCN2 promoted C4-2B and 22RV1 cell proliferation while knockdown of LCN2 markedly inhibited C4-2B and 22RV1 cell growth. LCN2 overexpression led to increased AR downstream gene SLC45A3 without upregulating AR expression. In the xenograft model, overexpression of LCN2 significantly promoted tumor growth. Methods LCN2 expression was detected in primary PCa and CRPC tissues and cell lines C4-2B and 22RV1 using immunohistochemistry and western blotting, respectively. Serum LCN2 level was detected vi ELISA. Lentiviruses-mediated over-expression of LCN2 and LCN2 knockdown were performed in CRPC cell lines. Expressions of androgen receptor (AR) downstream genes was examined in cell lines, in CRPC tissues, and in animal models. Conclusion LCN2 could facilitate cell proliferation of CRPC via AR transcriptional activity. LCN2 could be a novel target in CRPC. PMID:27602760

  20. ABCB1 C3435T gene polymorphism as a potential biomarker of clinical outcomes in HER2-positive breast cancer patients.

    PubMed

    Madrid-Paredes, Adela; Cañadas-Garre, Marisa; Sánchez-Pozo, Antonio; Segura-Pérez, Ana María; Chamorro-Santos, Clara; Vergara-Alcaide, Esther; Castillo-Portellano, Lucía; Calleja-Hernández, Miguel Ángel

    2016-04-30

    HER2-positive breast cancer patients treated with trastuzumab schemes have good initial clinical outcomes. Despite this beneficial effect, many patients experiment resistance to these drugs. Several gene polymorphisms in ABCB1, HER2, and CCND1 have been proposed as potential predictors of clinical outcomes of trastuzumab schemes. The aim of this study was to evaluate the association between 4 gene polymorphisms potentially responsible for bad prognosis (HER2-Ile655Val, CCND1-A870G and ABCB1C1236T, C3435T) and clinical outcomes in HER2-positive BC patients. A retrospective cohorts study was performed. Eighty-four HER2-positive BC patients treated with trastuzumab schemes were included. The four gene polymorphisms were analyzed by PCR Real-Time with Taqman(®) probes. Genotypes were investigated for their association with tumor response, survival and resistance. Patients with CC genotype of ABCB1-C3435T presented higher risk of resistance to chemotherapy/trastuzumab schemes, compared to those carrying the T-allele (RR: 2.71; CI95%:1.29-5.68; p=0.013888), progression (RR: 1.89; p=0.017964); and exitus (RR: 2.09; p=0.03276). Multivariate logistic regression analysis considering clinical variables and ABCB1-C3435T revealed that the only independent factor associated to resistance to therapy was ABCB1-C3435T gene polymorphism (ORCT/CC: 0.25; p=0.0123; ORTT/CC: 0.09; p=0.0348. The protective effect of ABCB1-C3435T T-allele was confirmed in the multivariate Cox regression analysis for PFS (HRCT/CC: 0.41; p=0.00806; HRTT/CC: 0.22; p=0.01982) and OS (HRCT/CC: 0.49; p=0.0555; HRTT/CC: 0.12; p=0.0398). ABCB1-C1236T, CCND1-A870G and HER2-Ile655Val polymorphisms were not associated to resistance, PFS or OS (p>0.05). The A-allele for CCND1-rs9344 was associated with higher response rates (RR: 3.44; uncorrected p-value: 0.03816) in the bivariate analysis, but no statically association was found after Bonferroni correction (p=0.15264). ABCB1-C3435T, ABCB1-C1236T and HER2-Ile655Val

  1. Enhanced polyamine accumulation alters carotenoid metabolism at the transcriptional level in tomato fruit over-expressing spermidine synthase.

    PubMed

    Neily, Mohamed Hichem; Matsukura, Chiaki; Maucourt, Mickaël; Bernillon, Stéphane; Deborde, Catherine; Moing, Annick; Yin, Yong-Gen; Saito, Takeshi; Mori, Kentaro; Asamizu, Erika; Rolin, Dominique; Moriguchi, Takaya; Ezura, Hiroshi

    2011-02-15

    Polyamines are involved in crucial plant physiological events, but their roles in fruit development remain unclear. We generated transgenic tomato plants that show a 1.5- to 2-fold increase in polyamine content by over-expressing the spermidine synthase gene, which encodes a key enzyme for polyamine biosynthesis. Pericarp-columella and placental tissue from transgenic tomato fruits were subjected to (1)H-nuclear magnetic resonance (NMR) for untargeted metabolic profiling and high-performance liquid chromatography-diode array detection for carotenoid profiling to determine the effects of high levels of polyamine accumulation on tomato fruit metabolism. A principal component analysis of the quantitative (1)H NMR data from immature green to red ripe fruit showed a clear discrimination between developmental stages, especially during ripening. Quantification of 37 metabolites in pericarp-columella and 41 metabolites in placenta tissues revealed distinct metabolic profiles between the wild type and transgenic lines, particularly at the late ripening stages. Notably, the transgenic tomato fruits also showed an increase in carotenoid accumulation, especially in lycopene (1.3- to 2.2-fold), and increased ethylene production (1.2- to 1.6-fold) compared to wild-type fruits. Genes responsible for lycopene biosynthesis, including phytoene synthase, phytoene desaturase, and deoxy-d-xylulose 5-phosphate synthase, were significantly up-regulated in ripe transgenic fruits, whereas genes involved in lycopene degradation, including lycopene-epsilon cyclase and lycopene beta cyclase, were down-regulated in the transgenic fruits compared to the wild type. These results suggest that a high level of accumulation of polyamines in the tomato regulates the steady-state level of transcription of genes responsible for the lycopene metabolic pathway, which results in a higher accumulation of lycopene in the fruit.

  2. Over-expression of ST3Gal-I promotes mammary tumorigenesis.

    PubMed

    Picco, Gianfranco; Julien, Sylvain; Brockhausen, Inka; Beatson, Richard; Antonopoulos, Aristotelis; Haslam, Stuart; Mandel, Ulla; Dell, Anne; Pinder, Sarah; Taylor-Papadimitriou, Joyce; Burchell, Joy

    2010-10-01

    Changes in glycosylation are common in malignancy, and as almost all surface proteins are glycosylated, this can dramatically affect the behavior of tumor cells. In breast carcinomas, the O-linked glycans are frequently truncated, often as a result of premature sialylation. The sialyltransferase ST3Gal-I adds sialic acid to the galactose residue of core 1 (Galbeta1,3GalNAc) O-glycans and this enzyme is over-expressed in breast cancer resulting in the expression of sialylated core 1 glycans. In order to study the role of ST3Gal-I in mammary tumor development, we developed transgenic mice that over-express the sialyltransferase under the control of the human membrane-bound mucin 1 promoter. These mice were then crossed with PyMT mice that spontaneously develop mammary tumors. As expected, ST3Gal-I transgenic mice showed increased activity and expression of the enzyme in the pregnant and lactating mammary glands, the stomach, lungs and intestine. Although no obvious defects were observed in the fully developed mammary gland, when these mice were crossed with PyMT mice, a highly significant decrease in tumor latency was observed compared to the PyMT mice on an identical background. These results indicate that ST3Gal-I is acting as a tumor promoter in this model of breast cancer. This, we believe, is the first demonstration that over-expression of a glycosyltransferase involved in mucin-type O-linked glycosylation can promote tumorigenesis.

  3. Over-expression of TRESK K(+) channels reduces the excitability of trigeminal ganglion nociceptors.

    PubMed

    Guo, Zhaohua; Cao, Yu-Qing

    2014-01-01

    TWIK-related spinal cord K(+) (TRESK) channel is abundantly expressed in trigeminal ganglion (TG) and dorsal root ganglion neurons and is one of the major background K(+) channels in primary afferent neurons. Mutations in TRESK channels are associated with familial and sporadic migraine. In rats, both chronic nerve injury and inflammation alter the expression level of TRESK mRNA. Functional studies indicate that reduction of endogenous TRESK channel activity results in hyper-excitation of primary afferent neurons, suggesting that TRESK is a potential target for the development of new analgesics. However, whether and how enhancing TRESK channel activity would decrease the excitability of primary afferent neurons has not been directly tested. Here, we over-expressed TRESK subunits in cultured mouse TG neurons by lipofectamine-mediated transfection and investigated how this altered the membrane properties and the excitability of the small-diameter TG population. To account for the heterogeneity of neurons, we further divided small TG neurons into two groups, based on their ability to bind to fluorescently-labeled isolectin B (IB4). The transfected TG neurons showed a 2-fold increase in the level of TRESK proteins. This was accompanied by a significant increase in the fraction of lamotrigine-sensitive persistent K(+) currents as well as the size of total background K(+) currents. Consequently, both IB4-positive and IB4-negative TG neurons over-expressing TRESK subunits exhibited a lower input resistance and a 2-fold increase in the current threshold for action potential initiation. IB4-negative TG neurons over-expressing TRESK subunits also showed a significant reduction of the spike frequency in response to supra-threshold stimuli. Importantly, an increase in TRESK channel activity effectively inhibited capsaicin-evoked spikes in TG neurons. Taken together, our results suggest that potent and specific TRESK channel openers likely would reduce the excitability of

  4. Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis

    PubMed Central

    Albini, Sonia; Coutinho Toto, Paula; Dall’Agnese, Alessandra; Malecova, Barbora; Cenciarelli, Carlo; Felsani, Armando; Caruso, Maurizia; Bultman, Scott J; Puri, Pier Lorenzo

    2015-01-01

    Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex—Brahma (Brm) and Brg1—are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes. PMID:26136374

  5. Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

    PubMed

    Uchida, D; Kawamata, H; Omotehara, F; Miwa, Y; Hino, S; Begum, N M; Yoshida, H; Sato, M

    2000-06-01

    We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p < 0.01; one-way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

  6. Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

    PubMed

    Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

    2015-01-01

    Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

  7. APN/CD13 Is Over-expressed by Psoriatic Fibroblasts and Is Modulated by CGRP and IL-4 But not by Retinoic Acid Treatment.

    PubMed

    Gerbaud, Pascale; Guibourdenche, Jean; Jarray, Rafika; Conti, Marc; Palmic, Patricia; Leclerc-Mercier, Stéphanie; Bruneau, Julie; Hermine, Olivier; Lepelletier, Yves; Raynaud, Françoise

    2017-04-07

    Psoriasis vulgaris is a common skin inflammatory disease characterized by recurrent flare episodes associated with scaly well-demarcated skin plaques. Skin biopsies from psoriatic patients with high PASI score (22.67 ± 8.67) and from HD were used to study APN/CD13. APN/CD13 is over-expressed in LP and nLP compare to HD skins and fibroblasts. This over-expression is positively correlated with specific enzymatic activity enhancement. However, discrepancies between APN/CD13 expression in LP and nLP prompt us to focus our study on APN/CD13 modulation. Calcitonin Gene Related Peptide (CGRP), a neuropeptide, positively modulated expression and activity of APN/CD13. CGRP consistently induced IL4 secretion, which is also involved in the increase of APN/CD13 expression and activity, which is significantly reversed using IL-4 blocking antibody. Surprisingly, retinoic acid altered the APN/CD13 enzymatic activity only in nLP fibroblasts without modification of APN/CD13 expression. APN/CD13 is over-expressed on psoriatic fibroblasts and exerted high level of activity compare to HD fibroblasts. Taken together, several factors such as CGRP and IL-4 acted on positive regulation of APN/CD13 expression and activity. This study highlighted the interest of APN/CD13 as a new potential target, which should be investigated in psoriasis. This article is protected by copyright. All rights reserved.

  8. Simultaneous Over-Expression of PaSOD and RaAPX in Transgenic Arabidopsis thaliana Confers Cold Stress Tolerance through Increase in Vascular Lignifications

    PubMed Central

    Shafi, Amrina; Dogra, Vivek; Gill, Tejpal; Ahuja, Paramvir Singh; Sreenivasulu, Yelam

    2014-01-01

    Antioxidant enzymes play a significant role in eliminating toxic levels of reactive oxygen species (ROS), generated during stress from living cells. In the present study, two different antioxidant enzymes namely copper-zinc superoxide dismutase derived from Potentilla astrisanguinea (PaSOD) and ascorbate peroxidase (RaAPX) from Rheum austral both of which are high altitude cold niche area plants of Himalaya were cloned and simultaneously over-expressed in Arabidopsis thaliana to alleviate cold stress. It was found that the transgenic plants over-expressing both the genes were more tolerant to cold stress than either of the single gene expressing transgenic plants during growth and development. In both single (PaSOD, RaAPX) and double (PaSOD + RaAPX) transgenic plants higher levels of total antioxidant enzyme activities, chlorophyll content, total soluble sugars, proline content and lower levels of ROS, ion leakage were recorded when compared to the WT during cold stress (4°C), besides increase in yield. In the present study, Confocal and SEM analysis in conjunction with qPCR data on the expression pattern of lignin biosynthetic pathway genes revealed that the cold stress tolerance of the transgenic plants might be because of the peroxide induced up-regulation of lignin by antioxidant genes mediated triggering. PMID:25330211

  9. Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants*

    PubMed Central

    He, Cuiwen H.; Xie, Letian X.; Allan, Christopher M.; Tran, UyenPhuong C.; Clarke, Catherine F.

    2014-01-01

    Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. PMID:24406904

  10. Effects of BCL-2 over-expression on B cells in transgenic rats and rat hybridomas.

    PubMed

    Iscache, Anne-Laure; Ménoret, Séverine; Tesson, Laurent; Rémy, Séverine; Usal, Claire; Pedros, Christophe; Saoudi, Abdelhadi; Buelow, Roland; Anegon, Ignacio

    2011-10-01

    The rat is an important biomedical experimental model that benefited from the recent development of new transgenic and knockout techniques. With the goal to optimize rat mAb production and to analyze the impact of Bcl-2 on B-cell development, we generated bcl-2 transgenic rats. Transgenic rats showed Bcl-2 over-expression in B cells, increased B cell numbers in lymphoid organs, elevated production of immunoglobulins (Igs) and prolonged B-cell survival in vitro. Transgenic rats remained healthy, reproduced normally and did not develop autoimmunity. Fusions with bcl-2 transgenic splenocytes did not result in increased hybridoma generation. A comparison of on- and off-rates of 39 mAbs generated with bcl-2 transgenic and wild-type animals revealed no significant differences. Over-expression of Bcl-2 in hybridomas did not change cell proliferation but resulted in increased Ig production. Bcl-2 transgenic rats will be a useful tool for the generation of rat mAbs, the analysis of B cells in different pathophysiological models, such as autoimmunity, cancer or organ transplantation, and the study of rat B-cell biology.

  11. 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.

  12. 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.

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

    PubMed

    Zhang, Xiaolu; Li, Bingnan; de Jonge, Nick; Björkholm, Magnus; Xu, Dawei

    2015-03-10

    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.

  14. Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina.

    PubMed

    Hao, Guangfei; Chen, Haiqin; Du, Kai; Huang, Xiaoyun; Song, Yuanda; Gu, Zhennan; Wang, Lei; Zhang, Hao; Chen, Wei; Chen, Yong Q

    2014-09-01

    Malic enzyme (ME) catalyses the oxidative decarboxylation of L-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n-6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina.

  15. Over-Expression of TRESK K+ Channels Reduces the Excitability of Trigeminal Ganglion Nociceptors

    PubMed Central

    Guo, Zhaohua; Cao, Yu-Qing

    2014-01-01

    TWIK-related spinal cord K+ (TRESK) channel is abundantly expressed in trigeminal ganglion (TG) and dorsal root ganglion neurons and is one of the major background K+ channels in primary afferent neurons. Mutations in TRESK channels are associated with familial and sporadic migraine. In rats, both chronic nerve injury and inflammation alter the expression level of TRESK mRNA. Functional studies indicate that reduction of endogenous TRESK channel activity results in hyper-excitation of primary afferent neurons, suggesting that TRESK is a potential target for the development of new analgesics. However, whether and how enhancing TRESK channel activity would decrease the excitability of primary afferent neurons has not been directly tested. Here, we over-expressed TRESK subunits in cultured mouse TG neurons by lipofectamine-mediated transfection and investigated how this altered the membrane properties and the excitability of the small-diameter TG population. To account for the heterogeneity of neurons, we further divided small TG neurons into two groups, based on their ability to bind to fluorescently-labeled isolectin B (IB4). The transfected TG neurons showed a 2-fold increase in the level of TRESK proteins. This was accompanied by a significant increase in the fraction of lamotrigine-sensitive persistent K+ currents as well as the size of total background K+ currents. Consequently, both IB4-positive and IB4-negative TG neurons over-expressing TRESK subunits exhibited a lower input resistance and a 2-fold increase in the current threshold for action potential initiation. IB4-negative TG neurons over-expressing TRESK subunits also showed a significant reduction of the spike frequency in response to supra-threshold stimuli. Importantly, an increase in TRESK channel activity effectively inhibited capsaicin-evoked spikes in TG neurons. Taken together, our results suggest that potent and specific TRESK channel openers likely would reduce the excitability of primary

  16. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  17. 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

  18. Over-Expression of Leptin Receptors in Hypothalamic POMC Neurons Increases Susceptibility to Diet-Induced Obesity

    PubMed Central

    Gamber, Kevin M.; Huo, Lihong; Ha, Sangdeuk; Hairston, Joyce E.; Greeley, Sarah; Bjørbæk, Christian

    2012-01-01

    Diet-induced obesity (DIO) in rodents is characterized by impaired activation of signal-transducer and activator of transcription 3 (STAT3) by leptin receptors (LepRb) within the hypothalamic arcuate nucleus. This signaling defect likely plays an important role in development of DIO. However, the neuro-chemical identity of the leptin-STAT3 resistant arcuate neurons has not been determined and the underlying mechanisms responsible for development of cellular leptin resistance remain unclear. To investigate this, we first measured arcuate gene expression of known key signaling components of the LepRb signaling pathway and tested whether specifically the critical arcuate pro-opiomelanocortin (POMC) neurons are resistant to LepRb-STAT3 signaling in mice given a high-fat-diet (HFD) compared to mice provided a low-fat control diet (LFD). We found that leptin-dependent STAT3 phosphorylation was decreased within POMC neurons of HFD mice. In addition, Leprb mRNA and suppressor of cytokine signaling 3 (Socs3) mRNA were elevated in the arcuate of HFD mice. To investigate whether increased LepRb expression per se in POMC neurons can influence development of DIO and Socs3 expression, we created mice that over-express LepRb selectively in POMC neurons (POMC-LepRb). No differences in body weight, fat mass or food intake were found between LFD POMC-LepRb mice and LFD controls. Surprisingly, body weight, fat mass and caloric intake of HFD POMC-LepRb mice was markedly higher than HFD control mice. In addition, arcuate Socs3 mRNA was increased in HFD POMC-LepRb mice compared to HFD controls. These data show that specifically POMC neurons of DIO mice are resistant to STAT3 activation by leptin, indicating that those cells might play a role in development of DIO. Furthermore, over-expression of LepRb selectively in POMC neurons increases susceptibility to the development of DIO. We propose a model where over-reactivity of the leptin-LepRb signaling system in arcuate neurons may play

  19. COX-2 over-expression correlates with VEGF and tumour angiogenesis in canine mammary cancer.

    PubMed

    Queiroga, Felisbina L; Pires, Isabel; Parente, Margarida; Gregório, Hugo; Lopes, Carlos S

    2011-07-01

    This study was designed to investigate the possible roles of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) in canine mammary cancer angiogenesis. Immunohistochemistry was performed on 70 tumours (28 benign and 42 malignant) in order to detect COX-2 and VEGF expression. Microvessel density (MVD) was determined by CD31 immunolabelling to assess tumour angiogenesis. There was a significantly higher expression of COX-2 (P<0.001), VEGF (P<0.001) and MVD (P<0.001) in malignant compared to benign tumours. In the malignant group, the MVD of COX-2 positive tumours was significantly higher than that of COX-2 negative tumours (P=0.026). A similar association was observed for VEGF (P<0.001) positive tumours. The results from this study suggested that over-expression of COX-2 and VEGF may contribute to increased angiogenesis and aggression in malignant tumours.

  20. Properties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice

    SciTech Connect

    Cho, Woosung; Messing, Albee

    2009-04-15

    Alexander disease is a fatal leukoencephalopathy caused by dominantly-acting coding mutations in GFAP. Previous work has also implicated elevations in absolute levels of GFAP as central to the pathogenesis of the disease. However, identification of the critical astrocyte functions that are compromised by mis-expression of GFAP has not yet been possible. To provide new tools for investigating the nature of astrocyte dysfunction in Alexander disease, we have established primary astrocyte cultures from two mouse models of Alexander disease, a transgenic that over-expresses wild type human GFAP, and a knock-in at the endogenous mouse locus that mimics a common Alexander disease mutation. We find that mutant GFAP, as well as excess wild type GFAP, promotes formation of cytoplasmic inclusions, disrupts the cytoskeleton, decreases cell proliferation, increases cell death, reduces proteasomal function, and compromises astrocyte resistance to stress.

  1. Angiotensin converting enzyme over expression in myelocytes enhances the immune response

    PubMed Central

    Bernstein, Kenneth E.; Gonzalez-Villalobos, Romer A.; Giani, Jorge F.; Shah, Kandarp; Bernstein, Ellen; Janjulia, Tea; Koronyo, Yosef; Shi, Peng D.; Koronyo-Hamaoui, Maya; Fuchs, Sebastien; Shen, Xiao Z.

    2015-01-01

    Angiotensin converting enzyme (ACE) plays an important role in blood pressure control. ACE also has effects on renal function, reproduction, hematopoiesis and several aspects of the immune response. ACE 10/10 mice over express ACE in monocytic cells; macrophages from ACE 10/10 mice demonstrate increased polarization towards a proinflammatory phenotype. As a result, ACE 10/10 mice have a highly effective immune response following challenge with either melanoma, bacterial infection or Alzheimer’s disease. The ACE 10/10 mice suggest that enhanced monocytic function greatly contributes to the ability of the immune response to defend against a wide variety of antigenic and non-antigenic challenges. PMID:24633750

  2. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

    PubMed Central

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-01-01

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed. PMID:19660100

  3. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression.

    PubMed

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-08-06

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

  4. Neuronal Over-expression of ACE2 Protects Brain from Ischemia-induced Damage

    PubMed Central

    Chen, Ji; Zhao, Yuhui; Chen, Shuzhen; Wang, Jinju; Xiao, Xiang; Ma, Xiaotang; Penchikala, Madhuri; Xia, Huijing; Lazartigues, Eric; Zhao, Bin; Chen, Yanfang

    2014-01-01

    Angiotensin (Ang) II exaggerates cerebral injury in ischemic damage. Angiotensin-converting enzyme type 2 (ACE2) converts Ang II into Ang (1–7) and thus, may protect against the effects of Ang II. We hypothesized that neuronal ACE2 over-expression decreases ischemic stroke in mice with Ang II overproduction. Human renin and angiotensinogen double transgenic (RA) mice and RA mice with neuronal over-expression of ACE2 (SARA) were used for the study. The mean arterial pressure (MAP) was calculated from telemetry-recorded blood pressure (BP). SARA mice were infused peripherally with Norepinephrine to “clamp” the BP, or intracerebroventricularly-infused with a Mas receptor antagonist (A-779). Middle cerebral artery occlusion (MCAO) surgery was performed to induce permanent focal ischemic stroke. Cerebral blood flow (CBF) and neurological function were determined. Two days after surgery, brain samples were collected for various analyses. Results showed: 1) When compared to chronically hypertensive RA mice, SARA mice had lower basal MAP, less MCAO-induced infarct volume, and increased CBF, neurological function and cerebral microvascular density in the peri-infarct area; 2) These changes in SARA mice were not altered after MAP “clamping”, but partially reversed by brain infusion of A-779; 3) Ang (1–7)/Ang II ratio, angiogenic factors, endothelial nitric oxide synthase (eNOS) expression and nitric oxide production were increased, whereas, NADPH oxidase subunits and reactive oxygen species were decreased in the brain of SARA mice. ACE2 protects brain from ischemic injury via the regulation of NADPH oxidase/eNOS pathways by changing Ang (1–7)/Ang II ratio, independently of MAP changes. PMID:24440367

  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. Dopamine D2 receptor over-expression alters behavior and physiology in Drd2-EGFP mice

    PubMed Central

    Kramer, Paul F.; Christensen, Christine H.; Hazelwood, Lisa A.; Dobi, Alice; Bock, Roland; Sibley, David R.; Mateo, Yolanda; Alvarez, Veronica A.

    2011-01-01

    BAC transgenic mice expressing the fluorescent reporter protein EGFP under the control of the D1 and D2 dopamine receptor promoters (Drd1-EGFP and Drd2-EGFP) have been widely used to study striatal function and have contributed to our understanding of the physiological and pathological function of the basal ganglia. These tools were produced and promptly made available to address questions in a cell-specific manner that has transformed the way we frame hypotheses in neuroscience. However, these mice have not been fully characterized until now. We found that Drd2-EGFP mice display a ~40% increase in membrane expression of the dopamine D2 receptor (D2R) and a two-fold increase in D2R mRNA levels in the striatum when compared to wild-type and Drd1-EGFP mice D2R over-expression was accompanied by behavioral hypersensitivity to D2R-like agonists, as well as enhanced electrophysiological responses to D2R activation in midbrain dopaminergic neurons. DA transients evoked by stimulation in the nucleus accumbens showed slower clearance in Drd2-EGFP mice and cocaine actions on DA clearance were impaired in these mice. Thus, it was not surprising to find that Drd2-EGFP mice were hyperactive when exposed to a novel environment and locomotion was suppressed by acute cocaine administration. All together, this study demonstrates that Drd2-EGFP mice over-express D2R and have altered dopaminergic signaling that fundamentally differentiates them from wild-type and Drd1-EGFP mice. PMID:21209197

  7. HER2 over-expressing high grade endometrial cancer expresses high levels of p95HER2 variant

    PubMed Central

    Growdon, Whitfield B.; Groeneweg, Jolijn; Byron, Virginia; DiGloria, Celeste; Borger, Darrell R.; Tambouret, Rosemary; Foster, Rosemary; Chenna, Ahmed; Sperinde, Jeff; Winslow, John; Rueda, Bo R.

    2015-01-01

    Background Subsets of high grade endometrial cancer (EnCa) over-express HER2 (ERBB2), yet clinical trials have failed to demonstrate any anti-tumor activity utilizing trastuzumab, an approved platform for HER2 positive breast cancer (BrCa). A truncated p95HER2 variant lacking the trastuzumab binding site may confer resistance. The objective of this investigation was to characterize the expression of the p95HER2 truncated variant in EnCa. Materials and Methods With institutional approval, 86 high grade EnCa tumors were identified with tumor specimens from surgeries performed between 2000-2011. Clinical data were collected and all specimens underwent tumor genotyping, HER2 immunohistochemistry (IHC, HercepTest®), HER2 fluorescent in situ hybridization (FISH), along with total HER2 (H2T) and p95HER2 assessment with VeraTag® testing. Regression models were used to compare a cohort of 86 breast tumors selected for equivalent HER2 protein expression. Results We identified 44 high grade endometrioid and 42 uterine serous carcinomas (USC). IHC identified high HER2 expression (2+ or 3+) in 59% of the tumors. HER2 gene amplification was observed in 16 tumors (12 USC, 4 endometrioid). Both HER2 gene amplification and protein expression correlated with H2T values. High p95HER2 expression above 2.8 RF/mm2 was observed in 53% (n = 54) with significant correlation with H2T levels. When matched to a cohort of 107 breast tumors based on HercepTest HER2 expression, high grade EnCa presented with higher p95 levels (p < 0.001). Conclusions: These data demonstrate that compared to BrCa, high grade EnCa expresses higher levels of p95HER2 possibly providing rationale for the trastuzumab resistance observed in EnCa. PMID:25602714

  8. 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

  9. Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy.

    PubMed

    Allen, Robert S; Miller, James A C; Chitty, Julie A; Fist, Anthony J; Gerlach, Wayne L; Larkin, Philip J

    2008-01-01

    We demonstrate that both over-expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7-O-acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over-expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry-weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA-encoded hairpin RNA-mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high-performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.

  10. 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.

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

    PubMed

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

    2007-12-05

    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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. Predominant D1 Receptors Involvement in the Over-expression of CART Peptides after Repeated Cocaine Administration

    PubMed Central

    Hu, Zhenzhen; Oh, Eun-Hye; Chung, Yeon Bok; Hong, Jin Tae

    2015-01-01

    The aim of this study was to investigate the involvement of dopaminergic receptors (DR) in behavioral sensitization, as measured by locomotor activity, and the over-expression of cocaine- and amphetamine-regulated transcript (CART) peptides after repeated administration of cocaine in mice. Repeated administrations of cocaine induced behavioral sensitization and CART over-expression in mice. The levels of striatal CART mRNA were significantly increased on the 3rd day. CART peptides were over-expressed on the 5th day in the striata of behaviorally sensitized mice. A higher proportion of CART+ cells in the cocaine-treated mice were present in the nucleus accumbens (NAc) shell than in the dorsolateral (DL) part of caudate putamen (CP). The concomitant administration of both D1R and D2R antagonists, SCH 23390 (D1R selective) and raclopride (D2R selective), blocked cocaine induced-behavioral sensitization, CART over-expression, and cyclic adenosine 5'-monophosphate (cAMP)/protein kinase A (PKA)/phospho-cAMP response element-binding protein (pCREB) signal pathways. SCH 23390 more predominantly inhibited the locomotor activity, CART over-expression, pCREB and PKA activity than raclopride. Cocaine induced-behavioral sensitization was also attenuated in the both D1R and D2R knockout (KO) mice, respectively. CART over-expression and activated cAMP/PKA/pCREB signal pathways were inhibited in the D1R-KO mice, but not in the D2R-KO mice. It is suggested that behavioral sensitization, CART over-expression and activated cAMP/PKA/pCREB signal pathways induced by repeated administration of cocaine could be more predominantly mediated by D1R. PMID:25729269

  17. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo

    PubMed Central

    McDole, Brittnee; Isgor, Ceylan; Pare, Christopher; Guthrie, Kathleen

    2015-01-01

    Olfactory bulb granule cells are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on granule cell spines. These contacts are established in the distal apical dendritic compartment, while granule cell basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong granule cell neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb granule cell spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF produces a marked increase in granule cell spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on granule cells, suggesting a role for this factor in modulating granule cell functional connectivity within adult olfactory circuitry. PMID:26211445

  18. 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

  19. Apoptotic effect of tannic acid on fatty acid synthase over-expressed human breast cancer cells.

    PubMed

    Nie, Fangyuan; Liang, Yan; Jiang, Bing; Li, Xiabing; Xun, Hang; He, Wei; Lau, Hay Tong; Ma, Xiaofeng

    2016-02-01

    Breast cancer is one of the most common cancers and is the second leading cause of cancer mortality in women worldwide. Novel therapies and chemo-therapeutic drugs are urgently needed to be developed for the treatment of breast cancer. Increasing evidence suggests that fatty acid synthase (FAS) plays an important role in breast cancer, for the expression of FAS is significantly higher in human breast cancer cells than in normal cells. Tannic acid (TA), a natural polyphenol, possesses significant biological functions, including bacteriostasis, hemostasis, and anti-oxidant. Our previous studies demonstrated that TA is a natural FAS inhibitor whose inhibitory activity is stronger than that of classical FAS inhibitors, such as C75 and cerulenin. This study further assessed the effect and therapeutic potential of TA on FAS over-expressed breast cancer cells, and as a result, TA had been proven to possess the functions of inhibiting intracellular FAS activity, down-regulating FAS expression in human breast cancer MDA-MB-231 and MCF-7 cells, and inducing cancer cell apoptosis. Since high-expressed FAS is recognized as a molecular marker for breast cancer and plays an important role in cancer prognosis, these findings suggest that TA is a potential drug candidate for treatment of breast cancer.

  20. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo.

    PubMed

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry.

  1. 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

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

    PubMed

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

    2001-07-20

    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.

  3. Reduced expression of pain mediators and pain sensitivity in amyloid precursor protein over-expressing CRND8 transgenic mice.

    PubMed

    Shukla, M; Quirion, R; Ma, W

    2013-10-10

    β-Amyloid (Aβ) peptides are derived from the sequential cleavage of the amyloid precursor protein (APP). They are enriched in plaques present in Alzheimer's brains and thus play important roles in the pathogenesis of this disease. APP is also known to be expressed in the neurons of dorsal root ganglion (DRG) and contributes to neuronal survival and axonal growth during development. However, whether APP and Aβ peptides are involved in nociception and pathological pain states is mostly unknown. In the present study, we have used behavioral, biochemical and morphological approaches to address this issue in both adult rats and APP over-expressing CRND8 transgenic mice. We observed that the Aβ peptide (17-24) was predominantly expressed in small-sized DRG neurons of rats. Following intraplantar (i.pl.) injection of complete Freud's adjuvant (CFA), the levels of APP and Aβ peptides were significantly reduced in the ipsilateral lumbar 4-6 rat DRG. In 3-, 12- and 24-month-old CRND8 mice, pain sensitivity in response to heat and mechanical stimulation was significantly dampened compared to their age-matched wild-type littermates. In parallel with reduced pain sensitivity, the expression of pain mediators such as substance P, calcitonin gene-related peptide and transient receptor potential vanilloid-1 was significantly reduced in L4-6 DRG of CRND8 mice. Although i.pl. injection of CFA induced a rather similar pattern of inflammatory pain in 3-month-old CRND8 mice and their wild-type littermates, recovery from inflammatory pain seemed faster in 12-month-old CRND8 mice than wild-type mice. These findings suggest that APP and Aβ peptides suppress both nociception and inflammatory pain and are likely involved in blunt pain perception of Alzheimer's patients in clinical settings.

  4. 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.

  5. 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

  6. Over-expression of a human chromosome 22q11.2 segment including TXNRD2, COMT and ARVCF developmentally affects incentive learning and working memory in mice.

    PubMed

    Suzuki, Go; Harper, Kathryn M; Hiramoto, Takeshi; Funke, Birgit; Lee, MoonSook; Kang, Gina; Buell, Mahalah; Geyer, Mark A; Kucherlapati, Raju; Morrow, Bernice; Männistö, Pekka T; Agatsuma, Soh; Hiroi, Noboru

    2009-10-15

    Duplication of human chromosome 22q11.2 is associated with elevated rates of mental retardation, autism and many other behavioral phenotypes. However, because duplications cover 1.5-6 Mb, the precise manner in which segments of 22q11.2 causally affect behavior is not known in humans. We have now determined the developmental impact of over-expression of an approximately 190 kb segment of human 22q11.2, which includes the genes TXNRD2, COMT and ARVCF, on behaviors in bacterial artificial chromosome (BAC) transgenic (TG) mice. BAC TG mice and wild-type (WT) mice were tested for their cognitive capacities, affect- and stress-related behaviors and motor activity at 1 and 2 months of age. An enzymatic assay determined the impact of BAC over-expression on the activity level of COMT. BAC TG mice approached a rewarded goal faster (i.e. incentive learning), but were impaired in delayed rewarded alternation during development. In contrast, BAC TG and WT mice were indistinguishable in rewarded alternation without delays, spontaneous alternation, prepulse inhibition, social interaction, anxiety-, stress- and fear-related behaviors and motor activity. Compared with WT mice, BAC TG mice had an approximately 2-fold higher level of COMT activity in the prefrontal cortex, striatum and hippocampus. These data suggest that over-expression of this 22q11.2 segment enhances incentive learning and impairs the prolonged maintenance of working memory, but has no apparent effect on working memory per se, affect- and stress-related behaviors or motor capacity. High copy numbers of this 22q11.2 segment might contribute to a highly selective set of phenotypes in learning and cognition during development.

  7. Over-expression of major colonization factors of enterotoxigenic Escherichia coli, alone or together, on non-toxigenic E. coli bacteria.

    PubMed

    Tobias, Joshua; Holmgren, Jan; Hellman, Maria; Nygren, Erik; Lebens, Michael; Svennerholm, Ann-Mari

    2010-10-08

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveller's diarrhea. Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. We have recently described the development of recombinant strains over-expressing CFA/I; the most prevalent CF among human clinical ETEC isolates. Here, non-toxigenic recombinant E. coli strains over-expressing Coli surface antigen 2 (CS2), CS4, CS5, and CS6, either alone, or each in combination with CFA/I were constructed by cloning the genes required for expression and assembly of each CF into expression vectors harboring a strong promoter. Immunological assays showed that recombinant strains expressing single CFs produced those in significantly larger amounts than did corresponding naturally high producing reference strains. Recombinant strains co-expressing CFA/I together with another CF also expressed significantly larger amounts of both CFs compared with the corresponding references strains. Further, when tested in mice, oral immunization with formalin-killed recombinant bacteria co-expressing one such double-expression CF pair, CFA/I+CS2, induced specific serum IgG+IgM and fecal IgA antibody responses against both CFs exceeding the responses induced by immunizations with natural reference strains expressing CFA/I and CS2, respectively. We conclude that the described type of recombinant bacteria over-expressing major CFs of ETEC, alone or in combination, may be useful as candidate strains for use in an oral whole-cell CF-ETEC vaccine.

  8. CRLF2 over-expression is a poor prognostic marker in children with high risk T-cell acute lymphoblastic leukemia

    PubMed Central

    Palmi, Chiara; Savino, Angela M.; Silvestri, Daniela; Bronzini, Ilaria; Cario, Gunnar; Paganin, Maddalena; Buldini, Barbara; Galbiati, Marta; Muckenthaler, Martina U.; Bugarin, Cristina; Mina, Pamela Della; Nagel, Stefan; Barisone, Elena; Casale, Fiorina; Locatelli, Franco; Nigro, Luca Lo; Micalizzi, Concetta; Parasole, Rosanna; Pession, Andrea; Putti, Maria C.; Santoro, Nicola; Testi, Anna M.; Ziino, Ottavio; Kulozik, Andreas E.; Zimmermann, Martin; Schrappe, Martin; Villa, Antonello; Gaipa, Giuseppe; Basso, Giuseppe; Biondi, Andrea; Valsecchi, Maria G.; Stanulla, Martin; Conter, Valentino; te Kronnie, Geertruy; Cazzaniga, Giovanni

    2016-01-01

    Pediatric T-ALL patients have a worse outcome compared to BCP-ALL patients and they could benefit from new prognostic marker identification. Alteration of CRLF2 gene, a hallmark correlated with poor outcome in BCP-ALL, has not been reported in T-ALL. We analyzed CRLF2 expression in 212 T-ALL pediatric patients enrolled in AIEOP-BFM ALL2000 study in Italian and German centers. Seventeen out of 120 (14.2%) Italian patients presented CRLF2 mRNA expression 5 times higher than the median (CRLF2-high); they had a significantly inferior event-free survival (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and overall survival (47.1%±12.1 vs. 73.8%±4.3, p=0.009) and an increased cumulative incidence of relapse/resistance (52.9%±12.1 vs. 26.2%±4.3, p=0.007) compared to CRLF2-low patients. The prognostic value of CRLF2 over-expression was validated in the German cohort. Of note, CRLF2 over-expression was associated with poor prognosis in the high risk (HR) subgroup where CRLF2-high patients were more frequently allocated. Interestingly, although in T-ALL CRLF2 protein was localized mainly in the cytoplasm, in CRLF2-high blasts we found a trend towards a stronger TSLP-induced pSTAT5 response, sensitive to the JAK inhibitor Ruxolitinib. In conclusion, CRLF2 over-expression is a poor prognostic marker identifying a subset of HR T-ALL patients that could benefit from alternative therapy, potentially targeting the CRLF2 pathway. PMID:27449287

  9. Altered stomatal dynamics in ascorbate oxidase over-expressing tobacco plants suggest a role for dehydroascorbate signalling.

    PubMed

    Fotopoulos, Vasileios; De Tullio, Mario C; Barnes, Jeremy; Kanellis, Angelos K

    2008-01-01

    Control of stomatal aperture is of paramount importance for plant adaptation to the surrounding environment. Here, we report on several parameters related to stomatal dynamics and performance in transgenic tobacco plants (Nicotiana tabacum L., cv. Xanthi) over-expressing cucumber ascorbate oxidase (AO), a cell wall-localized enzyme of uncertain biological function that oxidizes ascorbic acid (AA) to monodehydroascorbic acid which dismutates yielding AA and dehydroascorbic acid (DHA). In comparison to WT plants, leaves of AO over-expressing plants exhibited reduced stomatal conductance (due to partial stomatal closure), higher water content, and reduced rates of water loss on detachment. Transgenic plants also exhibited elevated levels of hydrogen peroxide and a decline in hydrogen peroxide-scavenging enzyme activity. Leaf ABA content was also higher in AO over-expressing plants. Treatment of epidermal strips with either 1 mM DHA or 100 microM hydrogen peroxide resulted in rapid stomatal closure in WT plants, but not in AO-over-expressing plants. This suggests that signal perception and/or transduction associated with stomatal closure is altered by AO over-expression. These data support a specific role for cell wall-localized AA in the perception of environmental cues, and suggest that DHA acts as a regulator of stomatal dynamics.

  10. Role of platelet derived growth factor receptor (PDGFR) over-expression and angiogenesis in ependymoma.

    PubMed

    Moreno, Lucas; Popov, Sergey; Jury, Alexa; Al Sarraj, Saffa; Jones, Chris; Zacharoulis, Stergios

    2013-01-01

    New molecularly targeted therapies are needed for childhood ependymoma. Angiogenesis and the PDGFR pathway could be potential therapeutic targets. This study aimed to screen ependymomas for the expression and clinicopathological correlates of angiogenic factors and potential therapeutic targets including VEGFR, endoglin (CD105), CD34, CD31, c-Kit, PDGFR-α and PDGFR-β. Immunohistochemistry for angiogenesis factors and PDGFR-α and β was performed in 24 archival tumor samples from children and adults treated for ependymoma at our institution. CD31 density, CD105 density and pericyte coverage index (PCI) were calculated. These findings were correlated with clinical outcome. VEGFR2 was overexpressed in tumor cells in only one out of 24 cases, but was found overexpressed in the vessels in 6 cases. PDGFR-α and β were found to be over-expressed in the ependymoma tumor cells in seven out of 24 cases (29.2 %). CD31 density, CD105 density and PCI did not correlate with expression of PDGFRs. Overexpression of PDGFR-α and β in tumor cells and overexpression of PDGFR-α in tumor endothelium had prognostic significance and this was maintained in multivariate analysis for overexpression of PDGFR-α in tumor cells (2 year progression free survival was 16.7 ± 15.2 for cases with overexpression of PDGFR-α in the tumor vs. 74.5 ± 15.2 for those with low/no expression, hazard ratio = 5.78, p = 0.04). A number of angiogenic factors are expressed in ependymoma tumor cells and tumor endothelium. Preliminary evidence suggests that the expression of PDGFRs could have a prognostic significance in ependymoma. This data suggests that PDGFRs should be further evaluated as targets using novel PDGFR inhibitors.

  11. PAR1 is selectively over expressed in high grade breast cancer patients: a cohort study

    PubMed Central

    Hernández, Norma A; Correa, Elma; Avila, Esther P; Vela, Teresa A; Pérez, Víctor M

    2009-01-01

    Background The protease-activated receptor (PAR1) expression is correlated with the degree of invasiveness in cell lines. Nevertheless it has never been directed involved in breast cancer patients progression. The aim of this study was to determine whether PAR1 expression could be used as predictor of metastases and mortality. Methods In a cohort of patients with infiltrating ductal carcinoma studied longitudinally since 1996 and until 2007, PAR1 over-expression was assessed by immunoblotting, immunohistochemistry, and flow citometry. Chi-square and log rank tests were used to determine whether there was a statistical association between PAR1 overexpression and metastases, mortality, and survival. Multivariate analysis was performed including HER1, stage, ER and nodes status to evaluate PAR1 as an independent prognostic factor. Results Follow up was 95 months (range: 2–130 months). We assayed PAR1 in a cohort of patients composed of 136 patients; we found PAR1 expression assayed by immunoblotting was selectively associated with high grade patients (50 cases of the study cohort; P = 0.001). Twenty-nine of 50 (58%) patients overexpressed PAR1, and 23 of these (46%) developed metastases. HER1, stage, ER and PAR1 overexpression were robustly correlated (Cox regression, P = 0.002, P = 0.024 and P = 0.002 respectively). Twenty-one of the 50 patients (42%) expressed both receptors (PAR1 and HER1 P = 0.0004). We also found a statistically significant correlation between PAR1 overexpression and increased mortality (P = 0.0001) and development of metastases (P = 0.0009). Conclusion Our data suggest PAR1 overexpression may be involved in the development of metastases in breast cancer patient and is associated with undifferentiated cellular progression of the tumor. Further studies are needed to understand PAR1 mechanism of action and in a near future assay its potential use as risk factor for metastasis development in high grade breast cancer patients. PMID:19538737

  12. Improving starch yield in cereals by over-expression of ADPglucose pyrophosphorylase: expectations and unanticipated outcomes.

    PubMed

    Tuncel, Aytug; Okita, Thomas W

    2013-10-01

    Significant improvements in crop productivity are required to meet the nutritional requirements of a growing world population. This challenge is magnified by an increased demand for bioenergy as a means to mitigate carbon inputs into the environment. Starch is a major component of the harvestable organs of many crop plants, and various endeavors have been taken to improve the yields of starchy organs through the manipulation of starch synthesis. Substantial efforts have centered on the starch regulatory enzyme ADPglucose pyrophosphorylase (AGPase) due to its pivotal role in starch biosynthesis. These efforts include over-expression of this enzyme in cereal plants such as maize, rice and wheat as well as potato and cassava, as they supply the bulk of the staple food worldwide. In this perspective, we describe efforts to increase starch yields in cereal grains by first providing an introduction about the importance of source-sink relationship and the motives behind the efforts to alter starch biosynthesis and turnover in leaves. We then discuss the catalytic and regulatory properties of AGPase and the molecular approaches used to enhance starch synthesis by manipulation of this process during grain filling using seed-specific promoters. Several studies have demonstrated increases in starch content per seed using endosperm-specific promoters, but other studies have demonstrated an increase in seed number with only marginal impact on seed weight. Potential mechanisms that may be responsible for this paradoxical increase in seed number will also be discussed. Finally, we describe current efforts and future prospects to improve starch yield in cereals. These efforts include further enhancement of starch yield in rice by augmenting the process of ADPglucose transport into amyloplast as well as other enzymes involved in photoassimilate partitioning in seeds.

  13. Cyclooxygenase-2 over-expression inhibits liver apoptosis induced by hyperglycemia.

    PubMed

    Francés, Daniel E A; Ingaramo, Paola I; Mayoral, Rafael; Través, Paqui; Casado, Marta; Valverde, Ángela M; Martín-Sanz, Paloma; Carnovale, Cristina E

    2013-03-01

    Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.

  14. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    PubMed

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  15. [Effects of ggpS over-expression on glycosylglycerol and glycerol biosynthesis of Synechocystis sp. PCC 6803].

    PubMed

    Ma, Peizhen; Tan, Xiaoming; Lü, Xuefeng; Tian, Jiyuan

    2016-03-01

    To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.

  16. 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.

  17. Over-expression of Runx1 transcription factor impairs the development of thymocytes from the double-negative to double-positive stages.

    PubMed

    Wong, Won F; Nakazato, Megumi; Watanabe, Toshio; Kohu, Kazuyoshi; Ogata, Takehiro; Yoshida, Naomi; Sotomaru, Yusuke; Ito, Mamoru; Araki, Kimi; Telfer, Janice; Fukumoto, Manabu; Suzuki, Daisuke; Sato, Takehito; Hozumi, Katsuto; Habu, Sonoko; Satake, Masanobu

    2010-06-01

    Runx1 transcription factor is highly expressed at a CD4/CD8-double-negative (DN) stage of thymocyte development but is down-regulated when cells proceed to the double-positive (DP) stage. In the present study, we examined whether the down-regulation of Runx1 is necessary for thymocyte differentiation from the DN to DP stage. When Runx1 was artificially over-expressed in thymocytes by Lck-driven Cre, the DN3 population was unaffected, as exemplified by proper pre-T-cell receptor expression, whereas the DN4 population was perturbed as shown by the decrease in the CD27(hi) sub-fraction. In parallel, the growth rate of DN4 cells was reduced by half, as measured by bromodeoxyuridine incorporation. These events impaired the transition of DN4 cells to the DP stage, resulting in the drastic reduction of the number of DP thymocytes. The Runx1 gene has two promoters, a proximal and a distal promoter; and, in thymocytes, endogenous Runx1 was mainly transcribed from the distal promoter. Interestingly, only distal, but not proximal, Runx1 over-expression exhibited an inhibitory effect on thymocyte differentiation, suggesting that the distal Runx1 protein may fulfil a unique function. Our collective results indicate that production of the distal Runx1 protein must be adequately down-regulated for thymocytes to transit from the DN to the DP stage, a critical step in the massive expansion of the T-cell lineage.

  18. The over-expression of an Arabidopsis B3 transcription factor, ABS2/NGAL1, leads to the loss of flower petals.

    PubMed

    Shao, Jingxia; Liu, Xiayan; Wang, Rui; Zhang, Gaisheng; Yu, Fei

    2012-01-01

    Transcriptional regulations are involved in many aspects of plant development and are mainly achieved through the actions of transcription factors (TF). To investigate the mechanisms of plant development, we carried out genetic screens for mutants with abnormal shoot development. Taking an activation tagging approach, we isolated a gain-of-function mutant abs2-1D (abnormal shoot 2-1D). abs2-1D showed pleiotropic growth defects at both the vegetative and reproductive developmental stages. We cloned ABS2 and it encodes a RAV sub-family of plant B3 type of transcriptional factors. Phylogenetic analysis showed that ABS2 was closely related to NGATHA (NGA) genes that are involved in flower development and was previously named NGATHA-Like 1 (NGAL1). NGAL1 was expressed mainly in the root and the filament of the stamen in flower tissues and sub-cellular localization assay revealed that NGAL1 accumulated in the nucleus. Interestingly, over-expression of NGAL1 driven by the constitutive 35S promoter led to transgenic plants with conspicuous flower defects, particularly a loss-of-petal phenotype. A loss-of-function ngal1-1 mutant did not show obvious phenotype, suggesting the existence of redundant activities and also the utility of gain-of-function genetic screens. Our results show that the over-expression of NGAL1 is capable of altering flower petal development, as well as shoot development.

  19. Ectopic over-expression of BhHsf1, a heat shock factor from the resurrection plant Boea hygrometrica, leads to increased thermotolerance and retarded growth in transgenic Arabidopsis and tobacco.

    PubMed

    Zhu, Yan; Wang, Zhi; Jing, Yanjun; Wang, Lili; Liu, Xia; Liu, Yongxiu; Deng, Xin

    2009-11-01

    Plant heat shock transcription factors (Hsfs) are commonly found to be involved in various stress responses. Several Hsfs displayed dwarf phenotype while conferred stress tolerance when over-expressed. However, the underlying mechanisms were not fully understood. Here we report the cloning and characterization of an Hsf (BhHsf1) from the resurrection plant Boea hygrometrica. Drought, heat and wound can induce BhHsf1 expression. The over-expression of BhHsf1 conferred growth retardation and stress tolerance in both Arabidopsis and tobacco. Evidence was presented to show that the growth retardation of aerial organs in the transgenic plants was resulted from the reduction of cell proliferation. Gene expression profiling using microarray hybridization and pathway analysis showed that Hsps and stress-associated genes were induced whereas the genes related to DNA replication and mitotic cell cycle were down-regulated in BhHsf1 over-expression Arabidopsis, which was in consistence with the observation of the impaired nuclear endoreduplication. Taking together, our results suggest that BhHsf1 may play dual roles in mediating the processes in heat stress tolerance and growth retardation via regulation of target genes related to stress protection and mitotic cell cycle.

  20. Lentivirus-mediated Persephin over-expression in Parkinson's disease rats

    PubMed Central

    Yin, Xiao-feng; Xu, Hua-min; Jiang, Yun-xia; Zhi, Yun-lai; Liu, Yu-xiu; Xiang, Heng-wei; Liu, Kai; Ding, Xiao-dong; Sun, Peng

    2015-01-01

    Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson's disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson's disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson's disease through protecting dopaminergic neurons. PMID:26807117

  1. Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma

    PubMed Central

    Caffarel, Maria M; Chattopadhyay, Anasuya; Araujo, Angela M; Bauer, Julien; Scarpini, Cinzia G; Coleman, Nicholas

    2013-01-01

    Oncostatin M receptor (OSMR) is commonly over-expressed in advanced cervical squamous cell carcinoma (SCC), producing a significantly worse clinical outcome. Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness. Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells. TGM2 expression correlated with disease progression and with OSMR levels in clinical samples of cervical and oral SCC. TGM2 depletion in cervical SCC cells abrogated OSM-induced migration on fibronectin-coated surfaces and invasiveness through extracellular matrix, while ectopic expression of TGM2 increased cell motility and invasiveness. Confocal microscopy and co-immunoprecipitation assays showed that TGM2 interacted with integrin–α5β1 in the presence of fibronectin in cervical SCC cells, with OSM treatment strengthening the interaction. Importantly, integrin–α5β1 and fibronectin were also over-expressed in cervical and oral SCC, where levels correlated with those of OSMR and TGM2. This combined tissue and in vitro study demonstrates for the first time that stimulation of over-expressed OSMR in cervical SCC cells activates TGM2/integrin-α5β1 interactions and induces pro-malignant changes. We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting. Copyright © 2013 Pathological Society of Great Britain and Ireland. © 2013 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:23765377

  2. Genetic variants in PARP1 (rs3219090) and IRF4 (rs12203592) genes associated with melanoma susceptibility in a Spanish population

    PubMed Central

    2013-01-01

    Background Few high penetrance genes are known in Malignant Melanoma (MM), however, the involvement of low-penetrance genes such as MC1R, OCA2, ASIP, SLC45A2 and TYR has been observed. Lately, genome-wide association studies (GWAS) have been the ideal strategy to identify new common, low-penetrance susceptibility loci. In this case–control study, we try to validate in our population nine melanoma associated markers selected from published GWAS in melanoma predisposition. Methods We genotyped the 9 markers corresponding to 8 genes (PARP1, MX2, ATM, CCND1, NADSYN1, CASP8, IRF4 and CYP2R1) in 566 cases and 347 controls from a Spanish population using KASPar probes. Genotypes were analyzed by logistic regression and adjusted by phenotypic characteristics. Results We confirm the protective role in MM of the rs3219090 located on the PARP1 gene (p-value 0.027). Additionally, this SNP was also associated with eye color (p-value 0.002). A second polymorphism, rs12203592, located on the IRF4 gene was associated with protection to develop MM for the dominant model (p-value 0.037). We have also observed an association of this SNP with both lentigines (p-value 0.014) and light eye color (p-value 3.76 × 10-4). Furthermore, we detected a novel association with rs1485993, located on the CCND1 gene, and dark eye color (p-value 4.96 × 10-4). Finally, rs1801516, located on the ATM gene, showed a trend towards a protective role in MM similar to the one firstly described in a GWAS study. Conclusions To our knowledge, this is the first time that these SNPs have been associated with MM in a Spanish population. We confirmed the proposed role of rs3219090, located on the PARP1 gene, and rs12203592, located on the IRF4 gene, as protective to MM along the same lines as have previous genome-wide associated works. Finally, we have seen associations between IRF4, PARP1, and CCND1 and phenotypic characteristics, confirming previous results for the IRF4 gene and presenting novel data

  3. [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.

  4. 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.

  5. 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

  6. Over-Expression of CDC25B and LAMC2 mRNA and Protein in Esophageal Squamous Cell Carcinomas and Pre-Malignant Lesions in Subjects from a High-Risk Population in China

    PubMed Central

    Shou, Jian-Zhong; Hu, Nan; Takikita, Mikiko; Roth, Mark J; Johnson, Laura Lee; Giffen, Carol; Wang, Quan-Hong; Wang, Chaoyu; Wang, Yuan; Su, Hua; Kong, Li-Hui; Emmert-Buck, Michael R; Goldstein, Alisa M; Hewitt, Stephen M; Taylor, Philip R

    2009-01-01

    Molecular events associated with the initiation and progression of esophageal squamous cell carcinoma (ESCC) remain poorly understood, but likely hold the key to effective early detection approaches for this almost invariably fatal cancer. CDC25B and LAMC2 are two promising early detection candidates emerging from new molecular studies of ESCC. To further elucidate the role of these two genes in esophageal carcinogenesis, we performed a series of studies to: (i) confirm RNA over-expression; (ii) establish the prevalence of protein over-expression; (iii) relate protein over-expression to survival; and (iv) explore their potential as early detection biomarkers. Results of these studies indicated that CDC25B mRNA was over-expressed (≥2-fold over-expression in tumor compared to normal) in 64% of the 73 ESCC cases evaluated, while LAMC2 mRNA was over-expressed in 89% of cases. CDC25B protein expression was categorized as positive in 59% (144/243) of ESCC cases on a tumor tissue microarray, and non-negative LAMC2 patterns of protein expression were observed in 82% (225/275) of cases. Multivariate-adjusted proportional hazard regression models showed no association between CDC25B protein expression score and risk of death (Hazard Ratio [HR] for each unit increase in expression score = 1.00, P=0.90), however, several of the LAMC2 protein expression patterns strongly predicted survival. Using the cytoplasmic pattern as the reference (the pattern with the lowest mortality), cases with a diffuse pattern had a 254% increased risk of death (HR=3.52, P=0.007), cases with no LAMC2 expression had a 169% increased risk of death (HR=2.69, P=0.009), and cases with a peripheral pattern had a 130% greater risk of death (HR=2.30, P=0.02). CDC25B protein expression scores in subjects with esophageal biopsies diagnosed as normal (n=35), dysplastic (n=23), or ESCC (n=32) increased significantly with morphologic progression. For LAMC2, all normal and dysplastic patients had a continuous

  7. Differentiation of human embryonic stem cells into osteogenic or hematopoietic lineages: a dose-dependent effect of osterix over-expression.

    PubMed

    Kärner, Elerin; Unger, Christian; Cerny, Radim; Ahrlund-Richter, Lars; Ganss, Bernhard; Dilber, M Sirac; Wendel, Mikael

    2009-02-01

    Enhanced differentiation of human embryonic stem cells (HESCs), induced by genetic modification could potentially generate a vast number of diverse cell types. Such genetic modifications have frequently been achieved by over-expression of individual regulatory proteins. However, careful evaluation of the expression levels is critical, since this might have important implications for the differentiation potential of HESCs. To date, attempts to promote osteogenesis by means of gene transfer into HESCs using the early bone "master" transcription factor osterix (Osx) have not been reported. In this study, we attained HESC subpopulations expressing two significantly different levels of Osx, following lentiviral gene transfer. Both subpopulations exhibited spontaneous differentiation and reduced expression of markers characteristic of the pluripotent phenotype, such as SSEA3, Tra1-60, and Nanog, In order to promote bone differentiation, the cells were treated with ascorbic acid, beta-glycerophosphate and dexamethasone. The high level of Osx, compared to endogenous levels found in primary human osteoblasts, did not enhance osteogenic differentiation, and did not up-regulate collagen I expression. We show that the high Osx levels instead induced the commitment towards the hematopoietic-endothelial lineage-by up-regulating the expression of CD34 and Gata1. However, low levels of Osx up-regulated collagen I, bone sialoprotein and osteocalcin. Conversely, forced high level expression of the homeobox transcription factor HoxB4, a known regulator for early hematopoiesis, promoted osteogenesis in HESCs, while low levels of HoxB4 lead to hematopoietic gene expression.

  8. GroEL-GroES assisted folding of multiple recombinant proteins simultaneously over-expressed in Escherichia coli.

    PubMed

    Goyal, Megha; Chaudhuri, Tapan K

    2015-07-01

    Folding of aggregation prone recombinant proteins through co-expression of chaperonin GroEL and GroES has been a popular practice in the effort to optimize preparation of functional protein in Escherichia coli. Considering the demand for functional recombinant protein products, it is desirable to apply the chaperone assisted protein folding strategy for enhancing the yield of properly folded protein. Toward the same direction, it is also worth attempting folding of multiple recombinant proteins simultaneously over-expressed in E. coli through the assistance of co-expressed GroEL-ES. The genesis of this thinking was originated from the fact that cellular GroEL and GroES assist in the folding of several endogenous proteins expressed in the bacterial cell. Here we present the experimental findings from our study on co-expressed GroEL-GroES assisted folding of simultaneously over-expressed proteins maltodextrin glucosidase (MalZ) and yeast mitochondrial aconitase (mAco). Both proteins mentioned here are relatively larger and aggregation prone, mostly form inclusion bodies, and undergo GroEL-ES assisted folding in E. coli cells during over-expression. It has been reported that the relative yield of properly folded functional forms of MalZ and mAco with the exogenous GroEL-ES assistance were comparable with the results when these proteins were overexpressed alone. This observation is quite promising and highlights the fact that GroEL and GroES can assist in the folding of multiple substrate proteins simultaneously when over-expressed in E. coli. This method might be a potential tool for enhanced production of multiple functional recombinant proteins simultaneously in E. coli.

  9. Regulation of cell growth and apoptosis through lactate dehydrogenase C over-expression in Chinese hamster ovary cells.

    PubMed

    Fu, Tuo; Zhang, Cunchao; Jing, Yu; Jiang, Cheng; Li, Zhenhua; Wang, Shengyu; Ma, Kai; Zhang, Dapeng; Hou, Sheng; Dai, Jianxin; Kou, Geng; Wang, Hao

    2016-06-01

    Lactate has long been credited as a by-product, which jeopardizes cell growth and productivity when accumulated over a certain concentration during the manufacturing process of therapeutic recombinant proteins by Chinese hamster ovary (CHO) cells. A number of efforts to decrease the lactate concentration have been developed; however, the accumulation of lactate is still a critical issue by the late stage of fed-batch culture. Therefore, a lactate-tolerant cell line was developed through over-expression of lactate dehydrogenase C (LDH-C). In fed-batch culture, sodium lactate or sodium pyruvate was supplemented into the culture medium to simulate the environment of lactate accumulation, and LDH-C over-expression increased the highest viable cell density by over 30 and 50 %, respectively, on day 5, meanwhile the viability was also improved significantly since day 5 compared with that of the control. The percentages of cells suffering early and late apoptosis decreased by 3.2 to 12.5 and 2.0 to 4.3 %, respectively, from day 6 onwards in the fed-batch culture when 40 mM sodium pyruvate was added compared to the control. The results were confirmed by mitochondrial membrane potential assay. In addition, the expression of cleaved caspases 3 and 7 decreased in cells over-expressing LDH-C, suggesting the mitochondrial pathway was involved in the LDH-C regulated anti-apoptosis. In conclusion, a novel cell line with higher lactate tolerance, lowered lactate production, and alleviated apoptosis response was developed by over-expression of LDH-C, which may potentially represent an efficient and labor-saving approach in generating recombinant proteins.

  10. 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.

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

    PubMed

    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.

  12. Functional response to SDF1 alpha through over-expression of CXCR4 on adult subventricular zone progenitor cells.

    PubMed

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

    2008-08-21

    The chemokine receptor CXCR4 and its ligand, stromal cell derived factor-1 alpha (SDF1 alpha) regulate neuroblast migration towards the ischemic boundary after stroke. Using loss- and gain-function, we investigated the biological effect of CXCR4/SDF1 alpha 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 SDF1 alpha (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/SDF1 alpha primarily regulates adult neural progenitor cell motility but not differentiation, while over-expression of CXCR4 in the absence of SDF1 alpha decreases neural progenitor cell proliferation.

  13. The Contributions of HIF-Target Genes to Tumor Growth in RCC

    PubMed Central

    Zhang, Ting; Niu, Xiaohua; Liao, Lili; Cho, Eun-Ah; Yang, Haifeng

    2013-01-01

    Somatic mutations or loss of expression of tumor suppressor VHL happen in the vast majority of clear cell Renal Cell Carcinoma, and it’s causal for kidney cancer development. Without VHL, constitutively active transcription factor HIF is strongly oncogenic and is essential for tumor growth. However, the contribution of individual HIF-responsive genes to tumor growth is not well understood. In this study we examined the contribution of important HIF-responsive genes such as VEGF, CCND1, ANGPTL4, EGLN3, ENO2, GLUT1 and IGFBP3 to tumor growth in a xenograft model using immune-compromised nude mice. We found that the suppression of VEGF or CCND1 impaired tumor growth, suggesting that they are tumor-promoting genes. We further discovered that the lack of ANGPTL4, EGLN3 or ENO2 expression did not change tumor growth. Surprisingly, depletion of GLUT1 or IGFBP3 significantly increased tumor growth, suggesting that they have tumor-inhibitory functions. Depletion of IGFBP3 did not lead to obvious activation of IGFIR. Unexpectedly, the depletion of IGFIR protein led to significant increase of IGFBP3 at both the protein and mRNA levels. Concomitantly, the tumor growth was greatly impaired, suggesting that IGFBP3 might suppress tumor growth in an IGFIR-independent manner. In summary, although the overall transcriptional activity of HIF is strongly tumor-promoting, the expression of each individual HIF-responsive gene could either enhance, reduce or do nothing to the kidney cancer tumor growth. PMID:24260413

  14. 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.

  15. hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias.

    PubMed

    Sáez, C; Japón, M A; Ramos-Morales, F; Romero, F; Segura, D I; Tortolero, M; Pintor-Toro, J A

    1999-09-23

    The role of oncogenes in pituitary tumorigenesis remains elusive since few genetic changes have been identified so far in pituitary tumors. Pituitary tumor-transforming gene (pttg) has been recently cloned from rat GH4 pituitary tumor cells. We have previously isolated and characterized hpttg from human thymus. In the present study, we analyse the expression of hpttg mRNA in a series of human pituitary adenomas. We show that hpttg is highly expressed in the majority of pituitary adenomas while only very low levels of mRNA can be detected in normal pituitary gland by Northern blot analysis. hPTTG protein was immunolocalized mainly in the cytoplasm of adenoma cells. Other common extra-cranial malignant tumors were also analysed by immunohistochemistry. Interestingly, strong hPTTG immunoreactivity was detected in most adenocarcinomas of mammary and pulmonary origins.

  16. In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24

    PubMed Central

    Yan, Lan; Zhang, Qizhong

    2017-01-01

    Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis. PMID:28358920

  17. The EAR motif controls the early flowering and senescence phenotype mediated by over-expression of SlERF36 and is partly responsible for changes in stomatal density and photosynthesis.

    PubMed

    Upadhyay, Rakesh Kumar; Gupta, Asmita; Ranjan, Sanjay; Singh, Ruchi; Pathre, Uday V; Nath, Pravendra; Sane, Aniruddha P

    2014-01-01

    The EAR motif is a small seven amino acid motif associated with active repression of several target genes. We had previously identified SlERF36 as an EAR motif containing gene from tomato and shown that its over-expression results in early flowering and senescence and a 25-35% reduction of stomatal density, photosynthesis and stomatal conductance in transgenic tobacco. In order to understand the role of the EAR motif in governing the phenotypes, we have expressed the full-length SlERF36 and a truncated form, lacking the EAR motif under the CaMV35S promoter, in transgenic Arabidopsis. Plants over-expressing the full-length SlERF36 show prominent early flowering under long day as well as short day conditions. The early flowering leads to an earlier onset of senescence in these transgenic plants which in turn reduces vegetative growth, affecting rosette, flower and silique sizes. Stomatal number is reduced by 38-39% while photosynthesis and stomatal conductance decrease by about 30-40%. Transgenic plants over-expressing the truncated version of SlERF36 (lacking the C-terminal EAR motif), show phenotypes largely matching the control with normal flowering and senescence indicating that the early flowering and senescence is governed by the EAR motif. On the other hand, photosynthetic rates and stomatal number were also reduced in plants expressing SlERF36ΔEAR although to a lesser degree compared to the full- length version indicating that these are partly controlled by the EAR motif. These studies show that the major phenotypic changes in plant growth caused by over-expression of SlERF36 are actually mediated by the EAR motif.

  18. Over-expression of myosin7A in cochlear hair cells of circling mice

    PubMed Central

    Kim, Yoo Yeon; Nam, Hajin; Jung, Harry; Kim, Boyoung

    2017-01-01

    Circling mouse (C57BL/6J-cir/cir) deleted the transmembrane inner ear (Tmie) gene is an animal model for human non-syndromic recessive deafness, DFNB6. In circling mouse, hair cells in the cochlea have degenerated and hair bundles have become irregularity as time goes on. Tmie protein carries out a function of the mechanoelectrical transduction channel in cochlear hair cells. Myosin7a (MYO7A) protein has key roles in development of the cochlear hair bundles as well as in the function of cochlear hair cells. To find whether Tmie protein interacts with MYO7A proteins in the cochlea postnatal developmental stage, we investigated expression of the MYO7A proteins in the cochlear hair cells of circling mice by western blot analysis and whole mount immunofluorescence at postnatal day 5 (P5). The expression of MYO7A showed statistically significant increase in the cochlea of C57BL/6J-+/cir and C57BL/6J-cir/cir mice than that of C57BL/6J-+/+ mice. The MYO7A intensity of the cochlear hair cells also increased in C57BL/6J-+/cir and C57BL/6J-cir/cir mice compared with those of C57BL/6J-+/+ mice. Taken together, the results indicate that Tmie protein may have an important role with MYO7A protein in the development and maintenance of the stereociliary bundles during postnatal developmental stage of the cochlea.

  19. c-Fos over-expression promotes radioresistance and predicts poor prognosis in malignant glioma

    PubMed Central

    Feng, Guokai; Chen, Furong; Tu, Ziwei; Liu, Guiyun; Zhao, Yu; Peng, Ming-Jing; He, Zheng-Wen; Chen, Xiao-Yan; Lindsay, Holly; Xia, Yun-Fei; Li, Xiao-Nan

    2016-01-01

    c-Fos is a major component of activator protein (AP)-1 complex. It has been implicated in cell differentiation, proliferation, angiogenesis, invasion, and metastasis. To investigate the role of c-Fos in glioma radiosensitivity and to understand the underlying molecular mechanisms, we downregulated c-Fos gene expression by lentivirus-mediated shRNA in glioma cell lines and subsequently analyzed the radiosensitivity, DNA damage repair capacity, and cell cycle distribution. Finally, we explored its prognostic value in 41 malignant glioma patients by immunohistochemistry. Our results showed that silencing c-Fos sensitized glioma cells to radiation by increasing radiation-induced DNA double strand breaks (DSBs), disturbing the DNA damage repair process, promoting G2/M cell cycle arrest, and enhancing apoptosis. c-Fos protein overexpression correlated with poor prognosis in malignant glioma patients treated with standard therapy. Our findings provide new insights into the mechanism of radioresistance in malignant glioma and identify c-Fos as a potentially novel therapeutic target for malignant glioma patients. PMID:27602752

  20. 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

  1. 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.

  2. α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons.

    PubMed

    Ortega, Richard; Carmona, Asuncion; Roudeau, Stéphane; Perrin, Laura; Dučić, Tanja; Carboni, Eleonora; Bohic, Sylvain; Cloetens, Peter; Lingor, Paul

    2016-04-01

    Parkinson's disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson's disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson's disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

  3. 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.

  4. Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors.

    PubMed

    Ferré, Sergi; Sebastião, Ana Maria

    2016-03-01

    This Editorial highlights a study by Chiodi et al. () showing that the effects mediated by cannabinoid CB1 receptor (CB1R) activation in the striatum are significantly reduced in rats with neuronal over-expression of adenosine A2A receptors (A2AR). Two hypotheses are derived from that study. Hypothesis A: two subpopulations of pre-synaptic CB1R in corticostriatal glutamatergic terminals exist, one forming and another not forming heteromers with A2AR. Hypothesis B: CB1R are predominantly forming heteromers with A2AR. In the case of hypothesis A, the A2AR might be required for CB1R-A2AR heteromeric signaling, whereas non-heteromeric CB1R activity is inhibited by A2ARs. In the case of hypothesis B, up-regulation of A2ARs may perturb heteromeric stoichiometry, thus reducing CB1R functioning. In any case, pre-synaptic striatal A2AR-CB1R heteromers emerge as important targets of the effects of cannabinoids demonstrated at the neuronal and behavioral level. Read the highlighted article 'Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors' on page 907.

  5. Over-expression of inducible HSP70 chaperone suppresses neuropathology and improves motor function in SCA1 mice.

    PubMed

    Cummings, C J; Sun, Y; Opal, P; Antalffy, B; Mestril, R; Orr, H T; Dillmann, W H; Zoghbi, H Y

    2001-07-01

    Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.

  6. Passive transfer of interferon-γ over-expressing macrophages enhances resistance of SCID mice to Mycobacterium tuberculosis infection.

    PubMed

    Pasula, Rajamouli; Martin, William J; Kesavalu, Banu Rekha; Abdalla, Maher Y; Britigan, Bradley E

    2017-02-23

    Infection with Mycobacterium tuberculosis (M.tb) is associated with increased deaths worldwide. Alveolar macrophages (AMs) play a critical role in host defense against infection with this pathogen. In this work we tested the hypothesis that passive transfer of normal AMs, IFN-γ activated AMs, or macrophages transduced to over-express IFN-γ into the lungs of immunosuppressed SCID mice, where resident macrophages are present but not functional, would enhance alveolar immunity and increase clearance of pulmonary M.tb infection. Accordingly, SCID mice were infected with M.tb intratracheally (I.T.), following which they received either control macrophages or macrophages overexpressing IFN-γ (J774A.1). The extent of M.tb infection was assessed at 30days post-M.tb infection. SCID mice administered macrophages over-expressing IFN-γ showed a significant decrease in M.tb burden and increased survival compared to J774A.1 control macrophages or untreated mice. This was further associated with a significant increase in IFN-γ and TNF-α mRNA and protein expression, as well as NF-κB (p65) mRNA, in the lungs. The increase in IFN-γ and TNF-α lung levels was inversely proportional to the number of M.tb organisms recovered. These results provide evidence that administration of macrophages overexpressing IFN-γ inhibit M.tb growth in vivo and may enhance host defense against M.tb infection.

  7. Enhanced hippocampus-dependent memory and reduced anxiety in mice over-expressing human catalase in mitochondria.

    PubMed

    Olsen, Reid H J; Johnson, Lance A; Zuloaga, Damian G; Limoli, Charles L; Raber, Jacob

    2013-04-01

    Oxidative stress (OS) and reactive oxygen species (ROS) play a modulatory role in synaptic plasticity and signaling pathways. Mitochondria (MT), a major source of ROS because of their involvement in energy metabolism, are important for brain function. MT-generated ROS are proposed to be responsible for a significant proportion of OS and are associated with developmental abnormalities and aspects of cellular aging. The role of ROS and MT function in cognition of healthy individuals is relatively understudied. In this study, we characterized behavioral and cognitive performance of 5- to 6-month-old mice over-expressing mitochondrial catalase (MCAT). MCAT mice showed enhancements in hippocampus-dependent spatial learning and memory in the water maze and contextual fear conditioning, and reduced measures of anxiety in the elevated zero maze. Catalase activity was elevated in MCAT mice in all brain regions examined. Measures of oxidative stress (glutathione, protein carbonyl content, lipid peroxidation, and 8-hydroxyguanine) did not significantly differ between the groups. The lack of differences in these markers of oxidative stress suggests that the differences observed in this study may be due to altered redox signaling. Catalase over-expression might be sufficient to enhance cognition and reduce measures of anxiety even in the absence of alteration in levels of OS.

  8. 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.

  9. Paracrine Engineering of Human Explant-Derived Cardiac Stem Cells to Over-Express Stromal-Cell Derived Factor 1α Enhances Myocardial Repair.

    PubMed

    Tilokee, Everad L; Latham, Nicholas; Jackson, Robyn; Mayfield, Audrey E; Ye, Bin; Mount, Seth; Lam, Buu-Khanh; Suuronen, Erik J; Ruel, Marc; Stewart, Duncan J; Davis, Darryl R

    2016-07-01

    First generation cardiac stem cell products provide indirect cardiac repair but variably produce key cardioprotective cytokines, such as stromal-cell derived factor 1α, which opens the prospect of maximizing up-front paracrine-mediated repair. The mesenchymal subpopulation within explant derived human cardiac stem cells underwent lentiviral mediated gene transfer of stromal-cell derived factor 1α. Unlike previous unsuccessful attempts to increase efficacy by boosting the paracrine signature of cardiac stem cells, cytokine profiling revealed that stromal-cell derived factor 1α over-expression prevented lv-mediated "loss of cytokines" through autocrine stimulation of CXCR4+ cardiac stem cells. Stromal-cell derived factor 1α enhanced angiogenesis and stem cell recruitment while priming cardiac stem cells to readily adopt a cardiac identity. As compared to injection with unmodified cardiac stem cells, transplant of stromal-cell derived factor 1α enhanced cells into immunodeficient mice improved myocardial function and angiogenesis while reducing scarring. Increases in myocardial stromal-cell derived factor 1α content paralleled reductions in myocyte apoptosis but did not influence long-term engraftment or the fate of transplanted cells. Transplantation of stromal-cell derived factor 1α transduced cardiac stem cells increased the generation of new myocytes, recruitment of bone marrow cells, new myocyte/vessel formation and the salvage of reversibly damaged myocardium to enhance cardiac repair after experimental infarction. Stem Cells 2016;34:1826-1835.

  10. 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.

  11. Purification and characterization of an extracellular alpha-L-arabinosidase from a novel isolate Bacillus pumilus ARA and its over-expression in Escherichia coli.

    PubMed

    Pei, Jianjun; Shao, Weilan

    2008-02-01

    The alpha-L-arabinosidase, AraB, was induced when Bacillus pumilus ARA was grown at 50 degrees C in a minimal medium containing xylan. A 56-kDa protein with alpha-L-arabinosidase activity was purified from culture supernatant to gel electrophoretic homogeneity. The optimal activity was at pH 6.4 and 60 degrees C over a 10-min assay. The purified enzyme was stable over a pH range of 5.2-7.6 and had a 1-h half life at 70 degrees C. The enzyme released arabinose from oat spelt xylan. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as substrate gave a K (m), and V (max) of 1.05 mM and 240 U per mg of protein. The NH(2)-terminal amino acid sequence of the enzyme was determined, and its gene araB was subsequently cloned, sequenced, and over-expressed in Escherichia coli. The open reading frame of araB consists of a 1,479-bp fragment encoding a protein of 472 amino acids, which belonged to family 51 of the glycoside hydrolases with an identity of 67% to the protein encoded by abfB of Bacillus subtilis 168.

  12. 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.

  13. Identification of FGF19 as a prognostic marker and potential driver gene of lung squamous cell carcinomas in Chinese smoking patients.

    PubMed

    Tan, Qiang; Li, Fan; Wang, Guan; Xia, Weiliang; Li, Ziming; Niu, Xiaomin; Ji, Wenxiang; Yuan, Hong; Xu, Qiang; Luo, Qingquan; Zhang, Jie; Lu, Shun

    2016-04-05

    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.

  14. 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

  15. SOS1 over-expression in genital skin fibroblasts from hirsute women: a putative role of the SOS1/RAS pathway in the pathogenesis of hirsutism.

    PubMed

    Minella, D; Wannenes, F; Biancolella, M; Amati, F; Testa, B; Nardone, A; Bueno, S; Fabbri, A; Lauro, D; Novelli, G; Moretti, C

    2011-01-01

    Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over-expression

  16. Over-expression of GTP-cyclohydrolase 1 feedback regulatory protein attenuates LPS and cytokine-stimulated nitric oxide production.

    PubMed

    Nandi, Manasi; Kelly, Peter; Vallance, Patrick; Leiper, James

    2008-02-01

    GTP-cyclohydrolase 1 (GTP-CH1) catalyses the first and rate-limiting step for the de novo production of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS). The GTP-CH1-BH(4) pathway is emerging as an important regulator in a number of pathologies associated with over-production of nitric oxide (NO) and hence a more detailed understanding of this pathway may lead to novel therapeutic targets for the treatment of certain vascular diseases. GTP-CH1 activity can be inhibited by BH(4) through its protein-protein interactions with GTP-CH1 regulatory protein (GFRP), and transcriptional and post-translational modification of both GTP-CH1 and GFRP have been reported in response to proinflammatory stimuli. However, the functional significance of GFRP/GTP-CH1 interactions on NO pathways has not yet been demonstrated. We aimed to investigate whether over-expression of GFRP could affect NO production in living cells. Over-expression of N-terminally Myc-tagged recombinant human GFRP in the murine endothelial cell line sEnd 1 resulted in no significant effect on basal BH(4) nor NO levels but significantly attenuated the rise in BH(4) and NO observed following lipopolysaccharide and cytokine stimulation of cells. This study demonstrates that GFRP can play a direct regulatory role in iNOS-mediated NO synthesis and suggests that the allosteric regulation of GTP-CH1 activity by GFRP may be an important mechanism regulating BH(4) and NO levels in vivo.

  17. 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.

  18. Angiopoietin-Like 4 Is Over-Expressed in Rheumatoid Arthritis Patients: Association with Pathological Bone Resorption

    PubMed Central

    Swales, Catherine; Athanasou, Nicholas A.; Knowles, Helen J.

    2014-01-01

    Introduction Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA). The secreted adipokine angiopoietin-like 4 (ANGPTL4) specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA). Results Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL). Conclusions Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA. PMID:25289668

  19. 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.

  20. Exploration of inhibitory mechanisms of curcumin in lung cancer metastasis using a miRNA- transcription factor-target gene network

    PubMed Central

    Jiao, De-min; Yan, Li; Wang, Li-shan; Hu, Hui-zhen; Tang, Xia-li; Chen, Jun; Wang, Jian; Li, You; Chen, Qing-yong

    2017-01-01

    The present study was aimed to unravel the inhibitory mechanisms of curcumin for lung cancer metastasis via constructing a miRNA-transcription factor (TF)-target gene network. Differentially expressed miRNAs between human high-metastatic non-small cell lung cancer 95D cells treated with and without curcumin were identified using a TaqMan human miRNA array followed by real-time PCR, out of which, the top 6 miRNAs (miR-302b-3p, miR-335-5p, miR-338-3p, miR-34c-5p, miR-29c-3p and miR-34a-35p) with more verified target genes and TFs than other miRNAs as confirmed by a literature review were selected for further analysis. The miRecords database was utilized to predict the target genes of these 6 miRNAs, TFs of which were identified based on the TRANSFAC database. The findings of the above procedure were used to construct a miRNA-TF-target gene network, among which miR-34a-5p, miR-34c-5p and miR-302b-3p seemed to regulate CCND1, WNT1 and MYC to be involved in Wnt signaling pathway through the LEF1 transcription factor. Therefore, we suggest miR-34a-5p/miR-34c-5p/miR-302b-3p —LEF1—CCND1/WNT1/MYC axis may be a crucial mechanism in inhibition of lung cancer metastasis by curcumin. PMID:28231299

  1. Alternating expression levels of WWOX tumor suppressor and cancer-related genes in patients with bladder cancer

    PubMed Central

    PŁUCIENNIK, ELŻBIETA; NOWAKOWSKA, MAGDALENA; STĘPIEN, ANNA; WOŁKOWICZ, MATEUSZ; STAWIŃSKI, ADAM; RÓŻAŃSKI, WALDEMAR; LIPIŃSKI, MAREK; BEDNAREK, ANDRZEJ K

    2014-01-01

    The aim of the present study was to determine the roles of the WWOX tumor suppressor and cancer-related genes in bladder tumor carcinogenesis. Reverse transcription-quantitative polymerase chain reaction was used to analyze the status of WWOX promoter methylation (using MethylScreen™ technology) and loss of heterozygosity (LOH) in papillary urothelial cancer tissues. The associations between the expression levels of the following tumorigenesis-related genes were also assessed: The WWOX tumor suppressor gene, the MKI67 proliferation gene, the BAX, BCL2 and BIRC5 apoptotic genes, the EGFR signal transduction gene, the VEGF vascular endothelial growth factor gene, and the CCND1 and CCNE1 cell cycle genes. The results reveal a high frequency of LOH in intron 1 in the WWOX gene, as well as an association between reduced WWOX expression levels and increased promoter methylation. In addition, the present study demonstrates that in bladder tumors, apoptosis is inhibited by increased expression levels of the BCL2 gene. A correlation between the proliferation indices of the MKI67 and the BIRC5 genes was also revealed. Furthermore, the expression levels of VEGF were identified to be positively associated with those of the EGFR gene. PMID:25295115

  2. 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

  3. Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9.

    PubMed

    Fragkouli, Apostolia; Papatheodoropoulos, Costas; Georgopoulos, Spiros; Stamatakis, Antonios; Stylianopoulou, Fotini; Tsilibary, Effie C; Tzinia, Athina K

    2012-04-01

    Evidence accumulating during the past few years points to a significant role of matrix metalloproteinase 9 (MMP9) enzymatic activity in synaptic plasticity and cognitive processes. We have previously demonstrated that MMP9 is involved in receptor-mediated α-secretase-like cleavage of APP in vitro, resulting in increased secretion of sAPPα, the soluble N-terminal product of the non-amyloidogenic pathway known to be involved in neuronal plasticity and memory formation. To study the in vivo role of MMP9, we have generated transgenic mice over-expressing MMP9 in the brain. Herein, we demonstrate that MMP9 transgenic animals display enhanced performance in the non-spatial novel object recognition and the spatial water-maze task and that their enhanced performance was accompanied by increased dendritic spine density in the hippocampus and cortex following behavioural testing. Consistent with the above observations, the electrophysiological analysis revealed prolonged maintenance of long-term synaptic potentiation in hippocampal slices from MMP9 transgenic mice. Moreover, elevated sAPPα levels in the hippocampus and cortex of MPP9 transgenic animals were also observed. Overall, our results extend previous findings on the physiological role of MMP9 in neuronal plasticity and furthermore reveal that, APP may be one of the physiological proteolytic targets of MMP9 in vivo.

  4. Characterization of cerebral microvasculature in transgenic mice with endothelium targeted over-expression of GTP-cyclohydrolase I

    PubMed Central

    Santhanam, Anantha Vijay R.; d’Uscio, Livius V.; Katusic, Zvonimir S.

    2015-01-01

    Tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by nitric oxide synthase (NOS) in the vascular endothelium and its biosynthesis is regulated by the enzymatic activity of GTP-cyclohydrolase I (GTPCH I). The present study was designed to determine the effects of endothelium-targeted overexpression of GTPCH I (eGCH-Tg) on murine cerebral vascular function. Endothelium targeted over-expression of GTPCH I was associated with a significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2) in cerebral microvessels. Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in eGCH-Tg mice. However, expression of endothelial NOS, levels of nitrate/nitrite - indicative of NO production - remained unchanged between cerebral microvessels of wild-type and eGCH-Tg mice. Furthermore, increased BH4 biosynthesis neither affected production of superoxide anion nor expression of antioxidant proteins. Moreover, endothelium-specific GTPCH I overexpression did not alter intracellular levels of cGMP, reflective of NO signaling in cerebral microvessels. The obtained results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGCH-Tg mice. We conclude that under physiological conditions the levels of BH4 in the cerebral microvessels are optimal for activation of endothelial NOS and NO/cGMP signaling. PMID:26343845

  5. Over expression of hRad9 protein correlates with reduced chemosensitivity in breast cancer with administration of neoadjuvant chemotherapy.

    PubMed

    Yun, Haiqin; Shi, Ranran; Yang, Qingrui; Zhang, Xiaofang; Wang, Yan; Zhou, Xingchen; Mu, Kun

    2014-12-18

    Human Rad 9 (hRad9), part of the Rad9-Hus1-Rad1 complex plays an important role in DNA damage repair as an up-stream regulator of checkpoint signaling, however little is known about its role in response to chemotherapy of breast cancer and whether hRad9 inhibition can potentiate the cytotoxic effects of chemotherapy on breast cancer cells remains to be elucidated. Fifty cases of breast cancer receiving neoadjuvant therapy were collected. All these cases were revised and classified into chemotherapy sensitive (CS) or chemotherapy resistant (CR) group according to the Miller and Payne (MP) grading system. Immunohistochemically, hRad9 positive tumours showed nuclear and/or cytoplasmic staining. hRad9 over-expression was associated with an impaired neoadjuvant chemotherapy response. A significant correlation was found between expression of hRad9 and Cyclin D1. In vitro, hRad9 was knocked down using siRNA in breast cancer cell line MCF-7 and MDA-MB-231. Deregulated expression of Rad9 accompanied by down expression of chk1 enhanced the sensitivity of human breast cancer cells to doxorubicin. Our work suggests that hRad9 might be a potential predictor for the response to chemotherapy in patients with breast cancer and its clinical value as a target for improving chemosensitivity needs further exploration.

  6. 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.

  7. Avermectin induced global DNA hypomethylation and over-expression of heat shock proteins in cardiac tissues of pigeon.

    PubMed

    Liu, Ci; Cao, Ye; Zhou, Shuo; Khoso, Pervez Ahmed; Li, Shu

    2017-01-01

    Despite increasing evidences pointing to residues of avermectin (AVM) pose toxic effects on non-target organisms in environment, but the data in pigeon is insufficient. The alteration of global DNA methylation and response of heat shock proteins (Hsps) are important for assessing the AVM toxicity in cardiac tissues of pigeon (Columba livia). To investigate the effects of AVM exposure in cardiac tissues of pigeon, we detected the expression levels of DNA methyltransferases (Dnmts), methylated DNA-binding domain protein 2 (MBD2), and Hsp 60, 70 and 90. Pigeons were exposed to feed containing AVM (0, 20, 40 and 60mg/kg diet) for 30, 60, 90days respectively, and cardiac tissues were collected and analyzed. We found the transcriptional levels of Dnmt1, Dnmt3a and Dnmt3b mRNA were down-regulated, but the transcriptional levels of MBD2 mRNA were up-regulated by AVM exposure in cardiac tissues of pigeon. Necrocytosis, hemorrhage, infiltration of inflammatory cells and abundant vacuoles appeared in cardiac tissues after AVM exposure. Accompanying this phenotype, the mRNA transcriptional and/or protein levels of Hsp30, Hsp60, Hsp70 and Hsp90 increased. In conclusion, these results underscored AVM exposure caused DNA methylation machinery malfunctions, and induced over-expression of Hsps to improve the protective function against cardiac injury.

  8. Over expression of hRad9 protein correlates with reduced chemosensitivity in breast cancer with administration of neoadjuvant chemotherapy

    PubMed Central

    Yun, Haiqin; Shi, Ranran; Yang, Qingrui; Zhang, Xiaofang; Wang, Yan; Zhou, Xingchen; Mu, Kun

    2014-01-01

    Human Rad 9 (hRad9), part of the Rad9-Hus1-Rad1 complex plays an important role in DNA damage repair as an up-stream regulator of checkpoint signaling, however little is known about its role in response to chemotherapy of breast cancer and whether hRad9 inhibition can potentiate the cytotoxic effects of chemotherapy on breast cancer cells remains to be elucidated. Fifty cases of breast cancer receiving neoadjuvant therapy were collected. All these cases were revised and classified into chemotherapy sensitive (CS) or chemotherapy resistant (CR) group according to the Miller and Payne (MP) grading system. Immunohistochemically, hRad9 positive tumours showed nuclear and/or cytoplasmic staining. hRad9 over-expression was associated with an impaired neoadjuvant chemotherapy response. A significant correlation was found between expression of hRad9 and Cyclin D1. In vitro, hRad9 was knocked down using siRNA in breast cancer cell line MCF-7 and MDA-MB-231. Deregulated expression of Rad9 accompanied by down expression of chk1 enhanced the sensitivity of human breast cancer cells to doxorubicin. Our work suggests that hRad9 might be a potential predictor for the response to chemotherapy in patients with breast cancer and its clinical value as a target for improving chemosensitivity needs further exploration. PMID:25520248

  9. Platelet-derived growth factor over-expression in retinal progenitors results in abnormal retinal vessel formation.

    PubMed

    Edqvist, Per-Henrik D; Niklasson, Mia; Vidal-Sanz, Manuel; Hallböök, Finn; Forsberg-Nilsson, Karin

    2012-01-01

    Platelet-derived growth factor (PDGF) plays an important role in development of the central nervous system, including the retina. Excessive PDGF signaling is associated with proliferative retinal disorders. We reported previously that transgenic mice in which PDGF-B was over-expressed under control of the nestin enhancer, nes/tk-PdgfB-lacZ, exhibited enhanced apoptosis in the developing corpus striatum. These animals display enlarged lateral ventricles after birth as well as behavioral aberrations as adults. Here, we report that in contrast to the relatively mild central nervous system phenotype, development of the retina is severely disturbed in nes/tk-PdgfB-lacZ mice. In transgenic retinas all nuclear layers were disorganized and photoreceptor segments failed to develop properly. Since astrocyte precursor cells did not populate the retina, retinal vascular progenitors could not form a network of vessels. With time, randomly distributed vessels resembling capillaries formed, but there were no large trunk vessels and the intraocular pressure was reduced. In addition, we observed a delayed regression of the hyaloid vasculature. The prolonged presence of this structure may contribute to the other abnormalities observed in the retina, including the defective lamination.

  10. 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

  11. Losses of expression of the antigens A, Lea and Lex and over-expression of Ley in carcinomas and HG-SIL of the uterine cervix

    PubMed Central

    Moro-Rodríguez, Ernesto; Álvarez-Fernández, Emilio

    2008-01-01

    Background The glycosylation of a great number of molecules, glyco-protein or glycolipids, has been of interest for decades. Objective To compare the expressive patterns of the isoantigenic determinants of histo-blood groups ABH and Lewis in squamous and simple epithelium and in precursors and cancers of the cervix. Methods A total of 36 lesions and neoplasms (10 LG-SIL, 16 HG-SIL and 10 invasive carcinomas) have been studied with immunohistochemical techniques, using monoclonal antibodies (MoAb BG1 to BG8) for precursor chains, blood-group ABH and Lewis group Lea, Leb, Lex, and Ley, and four types of lectins. In addition, we have studied the expression of p53 protein and PCNA, establishing the rate of proliferation of each lesion. Using PCR techniques, we have also detected part of the intron of the E6 gene of HPV-16. Results In the invasive cervical carcinomas, we observed a loss of expression of the Lex antigen (p < 0.01). With regard to the progression of the different lesions studied, we found alterations in the patterns of expression of the antigens of the ABH and Lewis blood groups. There was a tendency towards a loss of expression and heterogeneous patterns in the more advanced lesions, as well as over-expression of the Ley antigens. With PCNA, we established a proliferative rate which tended to be greater in relation to the progression of the cervix neoplasms. Conclusion These results indicate that there is a relation between the losses of histo-blood groups and the progression of the squamous intraepithelial lesions. PMID:18786253

  12. Expressed gene sequences from adult ovary and adult female gut genes over-expressed upon Babesia bovis infection of Rhipcephalus (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...

  13. Over-expression of Skp2 is associated with resistance to preoperative doxorubicin-based chemotherapy in primary breast cancer

    PubMed Central

    Davidovich, Shirly; Ben-Izhak, Ofer; Shapira, Ma'anit; Futerman, Boris; Hershko, Dan D

    2008-01-01

    Introduction Preoperative chemotherapy is often used in patients with locally advanced breast cancer. However, commonly used clinical and pathological parameters are poor predictors of response to this type of therapy. Recent studies have suggested that altered regulation of the cell cycle in cancer may be involved in resistance to chemotherapy. Over-expression of the ubiquitin ligase Skp2 results in loss of the cell cycle inhibitor p27Kip1 and is associated with poor prognosis in early breast cancer. The purpose of the present study was to examine the role of these proteins as predictors of clinical outcome and response to chemotherapy in locally advanced breast cancer. Methods The expression levels of Skp2 and p27Kip1 were determined by immunohistochemistry both before and after preoperative chemotherapy in 40 patients with locally advanced breast cancer. All patients were treated with cyclophosphamide/doxorubicin (adriamycin)/5-fluorouracil (CAF) and some patients received additional treatment with docetaxel. Expression data were compared with patients' clinical and pathological features, clinical outcome, and response to chemotherapy. Results Skp2 expression before preoperative chemotherapy was inversely related to p27Kip1 levels, tumor grade, and expression of estrogen and progesterone receptors. Both Skp2 and p27Kip1 were found to be accurate prognostic markers for disease-free and overall survival. High preoperative expression of Skp2 was associated with resistance to CAF therapy in 94% of patients (P < 0.0001) but not with resistance to docetaxel. Conclusion Skp2 expression may be a useful marker for predicting response to doxorubicin-based preoperative chemotherapy and clinical outcome in patients with locally advanced breast cancer. PMID:18644126

  14. Over-expression of neuron-derived orphan receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury.

    PubMed

    Rodríguez-Calvo, Ricardo; Guadall, Anna; Calvayrac, Olivier; Navarro, María A; Alonso, Judith; Ferrán, Beatriz; de Diego, Alicia; Muniesa, Pedro; Osada, Jesús; Rodríguez, Cristina; Martínez-González, José

    2013-05-15

    We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

  15. Over-expression of miR-196b-5p is significantly associated with the progression of myelodysplastic syndrome.

    PubMed

    Wen, Jing; Huang, Ying; Li, Hongying; Zhang, Xupai; Cheng, Peng; Deng, Donghong; Peng, Zhigang; Luo, Jun; Zhao, Weihua; Lai, Yongrong; Liu, Zhenfang

    2017-02-21

    Myelodysplastic syndrome (MDS) is a clonal stem cell disorder characterized by ineffective hematopoiesis with a high risk of transformation to acute myeloid leukemia (AML). miRNAs function as tumor suppressors and oncogenes in various cancers and regulate the differentiation potential of hematopoietic stem and progenitor cells (HSPCs). It has been suggested that miRNAs may play an important role in progression of MDS. We analyzed bone marrow samples collected from MDS patients according to different risk stratification indicated by the International Prognostic Scoring System (IPSS). We demonstrated that miR-196b-5p was up-regulated in intermediate II and higher groups, and in secondary AML (s-AML) patients in particular (P < 0.01) compared with healthy controls, suggesting that the higher expression levels are associated with increased risk of the development of MDS. We observed changes in proliferation and apoptosis in MDS-L cells following transfection with miR-196-5p mimics or inhibitors. After up-regulating the expression of miR-196b-5p, proliferation of MDS-L cells was up-regulated, whereas apoptosis was down-regulated (P < 0.05). In contrast, down-regulation of miR-196b-5p expression decreased cell proliferation and increased apoptosis (P < 0.05). We concluded that over-expression of miR-196b-5p may be closely associated with the risk of transformation to leukemia in MDS patients.

  16. 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

  17. Ubiquitous Over-Expression of Chromatin Remodeling Factor SRG3 Ameliorates the T Cell-Mediated Exacerbation of EAE by Modulating the Phenotypes of both Dendritic Cells and Macrophages.

    PubMed

    Lee, Sung Won; Park, Hyun Jung; Jeon, Sung Ho; Lee, Changjin; Seong, Rho Hyun; Park, Se-Ho; Hong, Seokmann

    2015-01-01

    Although SWI3-related gene (SRG3), a chromatin remodeling factor, is critical for various biological processes including early embryogenesis and thymocyte development, it is unclear whether SRG3 is involved in the differentiation of CD4+ T cells, the key mediator of adaptive immune responses. Because it is known that experimental autoimmune encephalomyelitis (EAE) development is determined by the activation of CD4+ T helper cells, here, we investigated the role of SRG3 in EAE development using SRG3 transgenic mouse models exhibiting two distinct SRG3 expression patterns: SRG3 expression driven by either the CD2 or β-actin promoter. We found that the outcome of EAE development was completely different depending on the expression pattern of SRG3. The specific over-expression of SRG3 using the CD2 promoter facilitated EAE via the induction of Th1 and Th17 cells, whereas the ubiquitous over-expression of SRG3 using the β-actin promoter inhibited EAE by promoting Th2 differentiation and suppressing Th1 and Th17 differentiation. In addition, the ubiquitous over-expression of SRG3 polarized CD4+ T cell differentiation towards the Th2 phenotype by converting dendritic cells (DCs) or macrophages to Th2 types. SRG3 over-expression not only reduced pro-inflammatory cytokine production by DCs but also shifted macrophages from the inducible nitric oxide synthase (iNOS)-expressing M1 phenotype to the arginase-1-expressing M2 phenotype during EAE. In addition, Th2 differentiation in β-actin-SRG3 Tg mice during EAE was associated with an increase in the basophil and mast cell populations and in IL4 production. Furthermore, the increased frequency of Treg cells in the spinal cord of β-actin-SRG3 Tg mice might induce the suppression of and accelerate the recovery from EAE symptoms. Taken together, our results provide the first evidence supporting the development of a new therapeutic strategy for EAE involving the modulation of SRG3 expression to induce M2 and Th2 polarization

  18. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide.

    PubMed

    Li, Shumin; Case, Adam J; Yang, Rui-Fang; Schultz, Harold D; Zimmerman, Matthew C

    2013-01-01

    Angiotensin II (AngII) is the main effector peptide of the renin-angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2 (•-)). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2 (•-). We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2 (•-) scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2 (•-). Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2 (•-) levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2 (•-), and inhibits AngII intra-neuronal signaling.

  19. 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

  20. 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.

  1. BAD induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL without loss of mitochondrial membrane potential.

    PubMed

    Schimmer, A D; Hedley, D W; Pham, N A; Chow, S; Minden, M D

    2001-07-01

    Inhibitors of Bcl-2 may be useful therapeutic agents for the treatment of a wide variety of malignancies including leukemia. A potential prototype of such a compound is the endogenous Bcl-2 and Bcl-xL binding protein BAD. Previous reports indicate that BAD can overcome the anti-apoptotic effect of Bcl-xL but not Bcl-2. If BAD cannot induce apoptosis in cells over-expressing Bcl-2, it would limit the application of molecules like BAD as novel anti-tumor agents. We report that transient transfection of BAD induced cell death in cells with and without over-expression of Bcl-2 or Bcl-xL. Forty-eight hours after transfection, BAD increased cell death in COS, COS Bcl-2, and COS Bcl-xL cells as demonstrated by decreased GFP expression, and an increase in the number of number of floating cells. In addition, BAD induced cell death in leukemic cell lines over-expressing Bcl-2 and Bcl-xL as determined by changes in luciferase activity. BAD-induced apoptosis was not accompanied by loss of mitochondrial membrane potential. Therefore, we conclude that transient transfection of BAD directly induces apoptosis in cells over-expressing Bcl-2 or Bcl-xL and validates the pursuit of molecules like BAD as novel therapeutic agents.

  2. Epigallocatechin-3-gallate suppressed the over-expression of HSP 70 and MDR1 induced by heat shock in SGC 7901.

    PubMed

    Tang, Xiao-Yan; Zhu, You-Qing

    2008-06-01

    This study investigated the effects of epigallocatechin-3-gallate (EGCG) on the expression of HSP 70 and MDR 1. SGC-7901 cells were cultured with RPMI 1640 medium. The single or combined effects of EGCG (0.1, 1, 10, 20, and 40 micromol/L) and heat shock were examined by MTT assay. The expression of HSP 70 and MDR 1 was semi-quantified by the reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry method (SP staining). EGCG suppressed cell proliferation at a time- and dose-dependent manner. The effects of combined treatment with EGCG and heat shock on the growth of SGC-7901 cells were stronger than single effects of EGCG. After using EGCG for 24 h, 48 h and 72 h, the IC50s were 112.5 micromol/l, 21.41 micromol/l and 5.24 micromol/l, respectively. Heat shock stimulated the over-expression of HSP 70, especially after heat shock for 8 h, as well as MDR1 after heat shock for 24 h. But EGCG suppressed the over-expression induced by heat shock. The authors conclude that EGCG inhibited the proliferation of SGC-7901, and EGCG combined with heat shock strengthened the effects. Heat shock weakened the over-expression of HSP 70 and MDR1; however, EGCG suppressed the over-expression of HSP 70 and MDR1 induced by heat shock. EGCG combined with heat shock may enhance the sensitivity of drugs to tumors.

  3. CPT1{alpha} over-expression increases long-chain fatty acid oxidation and reduces cell viability with incremental palmitic acid concentration in 293T cells

    SciTech Connect

    Jambor de Sousa, Ulrike L.; Koss, Michael D.; Fillies, Marion; Gahl, Anja; Scheeder, Martin R.L.; Cardoso, M. Cristina; Leonhardt, Heinrich; Geary, Nori; Langhans, Wolfgang; Leonhardt, Monika . E-mail: monika.leonhardt@inw.agrl.ethz.ch

    2005-12-16

    To test the cellular response to an increased fatty acid oxidation, we generated a vector for an inducible expression of the rate-limiting enzyme carnitine palmitoyl-transferase 1{alpha} (CPT1{alpha}). Human embryonic 293T kidney cells were transiently transfected and expression of the CPT1{alpha} transgene in the tet-on vector was activated with doxycycline. Fatty acid oxidation was measured by determining the conversion of supplemented, synthetic cis-10-heptadecenoic acid (C17:1n-7) to C15:ln-7. CPT1{alpha} over-expression increased mitochondrial long-chain fatty acid oxidation about 6-fold. Addition of palmitic acid (PA) decreased viability of CPT1{alpha} over-expressing cells in a concentration-dependent manner. Both, PA and CPT1{alpha} over-expression increased cell death. Interestingly, PA reduced total cell number only in cells over-expressing CPT1{alpha}, suggesting an effect on cell proliferation that requires PA translocation across the mitochondrial inner membrane. This inducible expression system should be well suited to study the roles of CPT1 and fatty acid oxidation in lipotoxicity and metabolism in vivo.

  4. Integrated genomic analyses identify frequent gene fusion events and VHL inactivation in gastrointestinal stromal tumors

    PubMed Central

    Sun, Choong-Hyun; Park, Inho; Lee, Seungmook; Kwon, Jekeun; Do, Ingu; Hong, Min Eui; Van Vrancken, Michael; Lee, Jeeyun; Park, Joon Oh; Cho, Jeonghee; Kim, Kyoung-Mee; Sohn, Tae Sung

    2016-01-01

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. We sequenced nine exomes and transcriptomes, and two genomes of GISTs for integrated analyses. We detected 306 somatic variants in nine GISTs and recurrent protein-altering mutations in 29 genes. Transcriptome sequencing revealed 328 gene fusions, and the most frequently involved fusion events were associated with IGF2 fused to several partner genes including CCND1, FUS, and LASP1. We additionally identified three recurrent read-through fusion transcripts: POLA2-CDC42EP2, C8orf42-FBXO25, and STX16-NPEPL1. Notably, we found intragenic deletions in one of three exons of the VHL gene and increased mRNAs of VEGF, PDGF-β, and IGF-1/2 in 56% of GISTs, suggesting a mechanistic link between VHL inactivation and overexpression of hypoxia-inducible factor target genes in the absence of hypoxia. We also identified copy number gain and increased mRNA expression of AMACR, CRIM1, SKP2, and CACNA1E. Mapping of copy number and gene expression results to the KEGG pathways revealed activation of the JAK-STAT pathway in small intestinal GISTs and the MAPK pathway in wild-type GISTs. These observations will allow us to determine the genetic basis of GISTs and will facilitate further investigation to develop new therapeutic options. PMID:25987131

  5. Integrated genomic analyses identify frequent gene fusion events and VHL inactivation in gastrointestinal stromal tumors.

    PubMed

    Kang, Guhyun; Yun, Hongseok; Sun, Choong-Hyun; Park, Inho; Lee, Seungmook; Kwon, Jekeun; Do, Ingu; Hong, Min Eui; Van Vrancken, Michael; Lee, Jeeyun; Park, Joon Oh; Cho, Jeonghee; Kim, Kyoung-Mee; Sohn, Tae Sung

    2016-02-09

    Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. We sequenced nine exomes and transcriptomes, and two genomes of GISTs for integrated analyses. We detected 306 somatic variants in nine GISTs and recurrent protein-altering mutations in 29 genes. Transcriptome sequencing revealed 328 gene fusions, and the most frequently involved fusion events were associated with IGF2 fused to several partner genes including CCND1, FUS, and LASP1. We additionally identified three recurrent read-through fusion transcripts: POLA2-CDC42EP2, C8orf42-FBXO25, and STX16-NPEPL1. Notably, we found intragenic deletions in one of three exons of the VHL gene and increased mRNAs of VEGF, PDGF-β, and IGF-1/2 in 56% of GISTs, suggesting a mechanistic link between VHL inactivation and overexpression of hypoxia-inducible factor target genes in the absence of hypoxia. We also identified copy number gain and increased mRNA expression of AMACR, CRIM1, SKP2, and CACNA1E. Mapping of copy number and gene expression results to the KEGG pathways revealed activation of the JAK-STAT pathway in small intestinal GISTs and the MAPK pathway in wild-type GISTs. These observations will allow us to determine the genetic basis of GISTs and will facilitate further investigation to develop new therapeutic options.

  6. Gene mutations and actionable genetic lesions in mantle cell lymphoma

    PubMed Central

    Ahmed, Makhdum; Zhang, Leo; Nomie, Krystle; Lam, Laura; Wang, Michael

    2016-01-01

    Mutations and epigenetic alterations are key events in transforming normal cells to cancer cells. Mantle cell lymphoma (MCL), a non-Hodgkin's lymphoma of the B-cell, is an aggressive malignancy with poor prognosis especially for those patients who are resistant to the frontline drugs. There is a great need to describe the molecular basis and mechanism of drug resistance in MCL to develop new strategies for treatment. We reviewed frequent somatic mutations and mutations involving the B-cell pathways in MCL and discussed clinical trials that attempted to disrupt these gene pathways and/or epigenetic events. Recurrent gene mutations were discussed in the light of prognostic and therapeutic opportunity and also the challenges of targeting these lesions. Mutations in the ATM, CCND1, TP53, MLL2, TRAF2 and NOTCH1 were most frequently encountered in mantle cell lymphoma. Translational models should be built that would assess mutations longitudinally to identify important compensatory, pro-survival and anti-apoptic pathways and actionable genetic targets. PMID:27449094

  7. 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.

  8. 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.

  9. Over-expression of ascorbate oxidase in the apoplast of transgenic tobacco results in altered ascorbate and glutathione redox states and increased sensitivity to ozone.

    PubMed

    Sanmartin, Maite; Drogoudi, Pavlina A M D; Lyons, Tom; Pateraki, Irene; Barnes, Jeremy; Kanellis, Angelos K

    2003-04-01

    Transgenic tobacco ( Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O(3)). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Over-expression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO over-expression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO over-expressing plants exposed to 100 nmol mol(-1) ozone for 7 h day(-1) exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO(2) assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO(2) assimilation rate when exposed to a brief ozone episode (300 nmol mol(-1) for 8 h). Stomatal conductance, hence O(3) uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.

  10. 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

  11. Over-expression of the Sirt3 sirtuin Protects neuronally differentiated PC12 Cells from degeneration induced by oxidative stress and trophic withdrawal.

    PubMed

    Shulyakova, Natalya; Sidorova-Darmos, Elena; Fong, Jamie; Zhang, Guangming; Mills, Linda R; Eubanks, James H

    2014-10-31

    Sirt3 is a mitochondrial sirtuin whose deacetylase activity regulates facets of oxidative metabolic efficiency, anti-oxidative capacity, and intra-mitochondrial signaling. In this study, we tested whether the over-expression of a human Sirt3-myc transgene in differentiated PC12 cells, a model of sympathetic catecholaminergic neurons, would affect the sensitivity of these cells to oxidative stress or trophic withdrawal insults. Expression analysis revealed the Sirt3-myc product was expressed as a 45kDa pro-form, which localized primarily within the cytosol, and a 30kDa processed form that localized predominantly within mitochondria. When subjected to acute glucose deprivation or acute oxygen-glucose deprivation, differentiated PC12 cells over-expressing Sirt3-myc displayed significantly lower levels of cytotoxicity, both at the end of the insult, and at different times following media reperfusion, than cells transfected with a control plasmid. Further, Sirt3-myc over-expression also protected differentiated PC12 cells from apoptosis induced by trophic withdrawal. Collectively, these data indicate that an elevation of Sirt3 is sufficient to protect neuronal PC12 cells from cytotoxic insults, and add to the growing evidence that Sirt3 could be targeted for neuroprotective intervention.

  12. Over-Expression of the Mycobacterial Trehalose-Phosphate Phosphatase OtsB2 Results in a Defect in Macrophage Phagocytosis Associated with Increased Mycobacterial-Macrophage Adhesion

    PubMed Central

    Li, Hao; Wu, Mei; Shi, Yan; Javid, Babak

    2016-01-01

    Trehalose-6-phosphate phosphatase (OtsB2) is involved in the OtsAB trehalose synthesis pathway to produce free trehalose and is strictly essential for mycobacterial growth. We wished to determine the effects of OtsB2 expression on mycobacterial phenotypes such as growth, phagocytosis and survival in macrophages. Mycobacterium bovis-bacillus calmette-guerin (BCG) over-expressing OtsB2 were able to better survive in stationary phase. Over-expression of OtsB2 led to a decrease in phagocytosis but not survival in THP-1 macrophage-like cells, and this was not due to a decrease in general macrophage phagocytic activity. Surprisingly, when we investigated macrophage–mycobacterial interactions by flow cytometry and atomic force microscopy, we discovered that BCG over-expressing OtsB2 have stronger binding to THP-1 cells than wild-type BCG. These results suggest that altering OtsB2 expression has implications for mycobacterial host–pathogen interactions. Macrophage–mycobacteria phagocytic interactions are complex and merit further study. PMID:27867377

  13. 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.

  14. Over-Expression of PDGFR-β Promotes PDGF-Induced Proliferation, Migration, and Angiogenesis of EPCs through PI3K/Akt Signaling Pathway

    PubMed Central

    Li, Wei; Zhao, Xiaohui; Yu, Yang; Zhu, Jinkun; Qin, Zhexue; Wang, Qiang; Wang, Kui; Lu, Wei; Liu, Jie; Huang, Lan

    2012-01-01

    The proliferation, migration, and angiogenesis of endothelial progenitor cells (EPCs) play critical roles in postnatal neovascularization and re-endothelialization following vascular injury. Here we evaluated whether the over-expression of platelet-derived growth factor receptor-β (PDGFR-β) can enhance the PDGF-BB-stimulated biological functions of EPCs through the PDGFR-β/phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. We first confirmed the expression of endogenous PDGFR-β and its plasma membrane localization in spleen-derived EPCs. We then demonstrated that the PDGFR-β over-expression in EPCs enhanced the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. Using AG1295 (a PDGFR kinase inhibitor), LY294002 (a PI3K inhibitor), and sc-221226 (an Akt inhibitor), we further showed that the PI3K/Akt signaling pathway participates in the PDGF-BB-induced proliferation, migration, and angiogenesis of EPCs. In addition, the PI3K/Akt signaling pathway is required for PDGFR-β over-expression to enhance these PDGF-BB-induced phenotypes. PMID:22355314

  15. BMP-6 over-expression in prostate cancer is associated with increased Id-1 protein and a more invasive phenotype.

    PubMed

    Darby, S; Cross, S S; Brown, N J; Hamdy, F C; Robson, C N

    2008-02-01

    Bone morphogenetic protein-6 (BMP-6) has been strongly implicated in prostate cancer development and bone metastasis. Our previous data showed that BMP-6 mRNA was absent in patients with benign prostatic hyperplasia, but evident in primary tumours with established secondary skeletal metastases. To examine the role of BMP-6 in prostate cancer progression, we have developed a BMP-6-regulatable, doxycycline-inducible gene expression system. BMP-6 induction by doxycycline addition led to increased levels of BMP-6 RNA and protein, associated with nuclear translocation of SMADs and activation of the downstream target gene Id-1. BMP-6 protein did not enhance the proliferation rate of PC3M cells but did significantly increase the rate of migration and invasion in both PC3M and DU145 cells. Increased metalloproteinase (MMP-1 and MMP-9) mRNA levels were also observed following BMP-6 induction. Luciferase reporter assays confirmed BMP-6-mediated activation of MMP-1 and MMP-9 promoters, indicating direct transcriptional activation of MMPs by BMP-6. BMP-6 stimulation also led to an increase in phosphorylation levels of MAPK proteins. We next examined the effects of BMP-6 on the downstream gene Id-1 in a cohort of prostate cancer patients. A tissue microarray (TMA) was constructed and samples stained for BMP-6 and Id-1 expression. We observed a significant increase in the intensity of staining of epithelial BMP-6 in the cancer cases compared to the benign cases (Mann-Whitney U test, p < 0.0005) and in the intensity of staining of epithelial Id-1 in the cancer cases compared to the benign cases (Mann-Whitney U test, p = 0.015). We further observed a significant positive correlation between epithelial staining for Id-1 and BMP-6 (p = 0.001) across all samples for both benign and cancer cases. These data demonstrate that BMP-6 promotes migration and invasion of prostate cancer cells, potentially through activation of Id-1 and MMP activation.

  16. Identification of new candidate therapeutic target genes in head and neck squamous cell carcinomas

    PubMed Central

    Klijanienko, Jerzy; Vacher, Sophie; Ouafi, Lamia; Chemlali, Walid; Caly, Martial; Sastre-Garau, Xavier; Lappartient, Emmanuelle; Mariani, Odette; Rodriguez, José; Jouffroy, Thomas; Girod, Angélique; Calugaru, Valentin; Hoffmann, Caroline; Lidereau, Rosette; Berger, Frédérique; Kamal, Maud; Bieche, Ivan; Le Tourneau, Christophe

    2016-01-01

    Background We aimed at identifying druggable molecular alterations at the RNA level from untreated HNSCC patients, and assessing their prognostic significance. Methods We retrieved 96 HNSCC patients who underwent primary surgery. Real-time quantitative RT-PCR was used to analyze a panel of 42 genes coding for major druggable proteins. Univariate and multivariate analyses were performed to assess the prognostic significance of overexpressed genes. Results Median age was 56 years [35–78]. Most of patients were men (80%) with a history of alcohol (70.4%) and/or tobacco consumption (72.5%). Twelve patients (12%) were HPV-positive. Most significantly overexpressed genes involved cell cycle regulation (CCND1 [27%], CDK6 [21%]), tyrosine kinase receptors (MET [18%], EGFR [14%]), angiogenesis (PGF [301%], VEGFA [14%]), and immune system (PDL1/CD274 [28%]). PIK3CA expression was an independent prognostic marker, associated with shorter disease-free survival. Conclusions We identified druggable overexpressed genes associated with a poor outcome that might be of interest for personalizing treatment of HNSCC patients. PMID:27329726

  17. Association of Thymidylate Synthase Gene Polymorphisms with Stavudine Triphosphate Intracellular Levels and Lipodystrophy▿

    PubMed Central

    Domingo, Pere; Cabeza, M. Carmen; Pruvost, Alain; Torres, Ferran; Salazar, Juliana; del Mar Gutierrez, M.; Mateo, M. Gracia; Fontanet, Angels; Fernandez, Irene; Domingo, Joan C.; Villarroya, Francesc; Vidal, Francesc; Baiget, Montserrat

    2011-01-01

    The antiviral activity and toxicity of stavudine (d4T) depend on its triphosphate metabolite, stavudine triphosphate (d4T-TP). Therefore, modifications in intracellular levels of d4T-TP may change the toxicity profile of stavudine. d4T-TP intracellular levels in peripheral blood mononuclear cells were determined with a prominence liquid chromatograph connected to a triple-quadruple mass spectrometer. Polymorphisms in the thymidylate synthase (TS), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), reduced folate carrier 1 (RFC1; SLC19A1), and cyclin D1 (CCND1) genes were determined by direct sequencing using an ABI Prism 3100 genetic analyzer or Fluidigm's Biomark system. The Mann-Whitney test, rank analysis of variance (with Bonferroni's adjusted post hoc comparisons), and logistic regression were used for the inferential analyses. Thirty-three stavudine-treated patients were enrolled in this cross-sectional study. d4T-TP intracellular levels were 11.50 fmol/106 cells (interquartile range [IQR] = 8.12 to 13.87 fmol/106 cells) in patients with a high-expression TS genotype (2/3G, 3C/3G, and 3G/3G), whereas in those with a low-expression TS genotype (2/2, 2/3C, and 3C/3C), they were 21.40 fmol/106 cells (IQR = 18.90 to 27.0 fmol/106 cells) (P < 0.0001). Polymorphisms in the MTHFR, DHFR, RFC1, and CCND1 genes did not influence the intracellular concentration of d4T-TP. d4T-TP levels were independently associated with the TS genotype (low versus high expression; odds ratio [OR] = 86.22; 95% confidence interval [CI] = 8.48 to nonestimable; P = 0.0023). The low-expression TS genotype was associated with the development of HIV/highly active antiretroviral therapy-associated lypodystrophy syndrome (HALS) (OR = 14.0; 95% CI = 2.09 to 108.0; P = 0.0032). Our preliminary data show that polymorphisms in the thymidylate synthase gene are strongly associated with d4T-TP intracellular levels and with development of HALS. PMID:21282454

  18. Selective Over Expression Of EAAT2 In Astrocytes Enhances Neuroprotection From Moderate But Not Severe Hypoxia-Ischemia

    PubMed Central

    Weller, Melodie L.; Stone, Ida M.; Goss, Amber; Rau, Thomas; Rova, Cherokee; Poulsen, David J.

    2008-01-01

    Attempts have been made to elevate EAAT2 expression in effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with β-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant Adeno-associated virus vector, AAV1-GFAP-EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral mediated gene delivery and β-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, the overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific-increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G. PMID:18620031

  19. 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.

  20. Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative high-resolution whole-genome profiling

    PubMed Central

    Beà, Sílvia; Salaverria, Itziar; Armengol, Lluís; Pinyol, Magda; Fernández, Verónica; Hartmann, Elena M.; Jares, Pedro; Amador, Virginia; Hernández, Luís; Navarro, Alba; Ott, German; Rosenwald, Andreas; Estivill, Xavier

    2009-01-01

    Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. However, only a limited number of target genes have been identified. We have studied 10 MCL cell lines and 28 primary tumors with a combination of a high-density single-nucleotide polymorphism array and gene expression profiling. We detected highly altered genomes in the majority of the samples with a high number of partial uniparental disomies (UPDs). The UPD at 17p was one of the most common, and it was associated with TP53 gene inactivation. Homozygous deletions targeted 4 known tumor suppressor genes (CDKN2C, BCL2L11, CDKN2A, and RB1) and 6 new genes (FAF1, MAP2, SP100, MOBKL2B, ZNF280A, and PRAME). Gene amplification coupled with overexpression was identified in 35 different regions. The most recurrent amplified regions were 11q13.3-q13.5, 13q31.3, and 18q21.33, which targeted CCND1, C13orf25, and BCL2, respectively. Interestingly, the breakpoints flanking all the genomic alterations, including UPDs, were significantly associated with genomic regions enriched in copy number variants and segmental duplications, suggesting that the recombination at these regions may play a role in the genomic instability of MCL. This integrative genomic analysis has revealed target genes that may be potentially relevant in MCL pathogenesis. PMID:18984860

  1. 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

  2. Over-expression of heat shock protein 70 protects mice against lung ischemia/reperfusion injury through SIRT1/AMPK/eNOS pathway

    PubMed Central

    Liu, Shumei; Xu, Junping; Fang, Chunfang; Shi, Chunjing; Zhang, Xin; Yu, Bo; Yin, Yantong

    2016-01-01

    Lung ischemia/reperfusion injury (LIRI) usually occurs during in lung transplantation and extracorporeal circulation operation and may develop into pulmonary infections, acute rejection and bronchiolitis obliterans syndrome. Recent studies have discovered the protective effect of heat shock protein 70 (HSP70) on various types of injuries. In the present study, we firstly explore the role of over-expressed HSP70 on the protection against LIRI. Lung Wet/Dry (W/D) ratio, biomarkers in the bronchoalveolar lavage fluid (BALF), lung histological changes and apoptosis markers, oxidative products and proinflammatory cytokines in the lung tissues were analyzed. Next, the expression of eNOS, SIRT1 and AMPK were measured. Finally, the changes of the lung W/D ratio and biomarkers in the BALF using the inhibitors of SIRT1/AMPK/eNOS pathway were evaluated. Mice exposed to LIRI procedure had significant increases in lung W/D ratio and biomarkers (protein level, LDH level, leukocytes and total cells) in BALF. LIRI also caused histological injury, demonstrated by hemorrhage, alveolar septal thickening and fibrin deposition. Apoptosis, oxidative products and proinflammatory cytokines in lung tissue were also induced by LIRI. The over-expression of HSP70 antagonized the impacts of LIRI by attenuating these parameters. It significantly increased the expression of eNOS, SIRT1 and AMPK, while the inhibition of SIRT1 and AMPK deactivated the eNOS expression. The lung W/D ratio and biomarkers in BALF were increased while mice were given inhibitors of eNOS, SIRT1 and AMPK. We concluded that over-expression of HSP70 had protective effect on LIRI and HSP70 might be involved in the protection through a SIRT1/AMPK/eNOS pathway. PMID:27830023

  3. Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

    PubMed Central

    Das, Sanchita; Shah, Priyanka; Baharia, Rajendra K.; Tandon, Rati; Khare, Prashant; Sundar, Shyam; Sahasrabuddhe, Amogh A.; Siddiqi, M. I.; Dube, Anuradha

    2013-01-01

    Background Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism. PMID:24340105

  4. The over-expression of FGFR4 could influence the features of gastric cancer cells and inhibit the efficacy of PD173074 and 5-fluorouracil towards gastric cancer.

    PubMed

    Li, Jingjing; Ye, Yanwei; Wang, Min; Lu, Lisha; Han, Chao; Zhou, Yubing; Zhang, Jingmin; Yu, Zujiang; Zhang, Xiefu; Zhao, Chunlin; Wen, Jianguo; Kan, Quancheng

    2016-05-01

    The aim was to investigate the function of fibroblast growth factor receptor 4 (FGFR4) in gastric cancer (GC) and explore the treatment value of agent targeted to FGFR4. Function assays in vitro and in vivo were performed to investigate the discrepancy of biological features among the GC cells with different expression of FGFR4. GC cells were treated with the single and combination of PD173074 (PD, an inhibitor of FGFR4) and 5-fluorouracil (5-Fu). The invasion ability were stronger, and the apoptosis rates were lower in MGC803 and BGC823 cells treated with FGFR4-LV5 (over-expression of FGFR4 protein) (P < 0.05). The proliferation ability of GC cells is reduced when treated by the single and combination of 5-Fu and PD while that of the FGFR4-LV5 group was less inhibited compared with control group (P < 0.05). The apoptosis rates are remarkably increased in GC cells treated with the single and combination of 5-Fu and PD (P < 0.05). However, the apoptosis rate obviously is reduced in GC cells treated with FGFR4-LV5 compared with control group (P < 0.05). The expression of PCNA and Bcl-XL is remarkably decreased, and the expression of Caspase-3 and cleaved Caspase-3 is obviously increased in GC cells treated with the single and combination of 5-Fu and PD. The tumor volumes of nude mice in FGFR4-LV5 group were much more increased (P < 0.05). The over-expression of FGFR4 enhanced the proliferation ability of GC in vitro and in vivo. The combination of 5-Fu and PD exerted synergetic effect in weakening the proliferation ability and promoting apoptosis in GC cells, while the over-expression of FGFR4 might inhibit the efficacy of two drugs.

  5. 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

  6. T-bet over-expression regulates aryl hydrocarbon receptor-mediated T helper type 17 differentiation through an interferon (IFN)γ-independent pathway.

    PubMed

    Yokosawa, M; Kondo, Y; Tahara, M; Iizuka-Koga, M; Segawa, S; Kaneko, S; Tsuboi, H; Yoh, K; Takahashi, S; Matsumoto, I; Sumida, T

    2017-04-01

    Various transcription factors are also known to enhance or suppress T helper type 17 (Th17) differentiation. We have shown previously that the development of collagen-induced arthritis was suppressed in T-bet transgenic (T-bet Tg) mice, and T-bet seemed to suppress Th17 differentiation through an interferon (IFN)-γ-independent pathway, although the precise mechanism remains to be clarified. The present study was designed to investigate further the mechanisms involved in the regulation of Th17 differentiation by T-bet over-expression, and we found the new relationship between T-bet and aryl hydrocarbon receptor (AHR). Both T-bet Tg mice and IFN-γ(-/-) -over-expressing T-bet (T-bet Tg/IFN-γ(-/-) ) mice showed inhibition of retinoic acid-related orphan receptor (ROR)γt expression and IL-17 production by CD4(+) T cells cultured under conditions that promote Th-17 differentiation, and decreased IL-6 receptor (IL-6R) expression and signal transducer and activator of transcription-3 (STAT-3) phosphorylation in CD4(+) T cells. The mRNA expression of ahr and rorc were suppressed in CD4(+) T cells cultured under Th-17 conditions from T-bet Tg mice and T-bet Tg/IFN-γ(-/-) mice. CD4(+) T cells of wild-type (WT) and IFN-γ(-/-) mice transduced with T-bet-expressing retrovirus also showed inhibition of IL-17 production, whereas T-bet transduction had no effect on IL-6R expression and STAT-3 phosphorylation. Interestingly, the mRNA expression of ahr and rorc were suppressed in CD4(+) T cells with T-bet transduction cultured under Th17 conditions. The enhancement of interleukin (IL)-17 production from CD4(+) T cells by the addition of AHR ligand with Th17 conditions was cancelled by T-bet over-expression. Our findings suggest that T-bet over-expression-induced suppression of Th17 differentiation is mediated through IFN-γ-independent AHR suppression.

  7. 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.

  8. 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

  9. Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies

    PubMed Central

    Soares, Janine; Neves, Manuel; Santos, Lúcio Lara; Ferreira, José Alexandre

    2016-01-01

    Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells. PMID:27835695

  10. Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.

    PubMed

    Lima, Luís; Gaiteiro, Cristiana; Peixoto, Andreia; Soares, Janine; Neves, Manuel; Santos, Lúcio Lara; Ferreira, José Alexandre

    2016-01-01

    Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells.

  11. 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.

  12. Over-expression of Id-1 induces cell proliferation in hepatocellular carcinoma through inactivation of p16INK4a/RB pathway.

    PubMed

    Lee, Terence Kin-Wah; Man, Kwan; Ling, Ming-Tat; Wang, Xiang-Hong; Wong, Yong-Chuan; Lo, Chung-Mau; Poon, Ronnie Tung-Ping; Ng, Irene Oi-Lin; Fan, Sheung-Tat

    2003-11-01

    Inhibitors of differentiation and DNA binding-1 (Id-1) have been demonstrated to oppose Ets-mediated activation of p16INK4a. As p16INK4a protein is inactivated in hepatocellular carcinoma (HCC), we aimed to investigate the role of Id-1 in regulating p16INK4a expression during the development of HCC in HCC patients and direct ectopic Id-1 introduction into the PLC/PRF/5 HCC cell line. Sixty-two HCC samples were recruited for evaluation of Id-1 and proliferating cell nuclear antigen (PCNA) protein expression. The messenger RNA (mRNA) expression of Id-1 and p16INK4a was detected by quantitative reverse transcription-polymerase chain reaction. For in vitro Id-1 transfection, five Id-1 transfected clones were isolated and the effect of ectopic Id-1 introduction was investigated by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, flow cytometry, immunostaining and western blot. Our results showed that Id-1 was over-expressed in HCC specimens both at mRNA and protein levels. Over-expression of Id-1 protein was correlated with PCNA (r = 0.334, P = 0.033). HCC samples showing low Id-1 protein expression had a lower Id-1 mRNA level (340.2 versus 1467%, P = 0.039) and higher p16INK4a expression (195 versus -78.6%, P = 0.039) than samples with high Id-1 protein expression. In the PLC/PRF/5 HCC cell line study, ectopic Id-1 expression resulted in proliferation of HCC cells and an increased percentage of S phase cells and PCNA expression. The results showed that over-expression of Id-1 induces cell proliferation in HCC through inactivation of p16INK4a/retinoblastoma pathway. In conclusion, the results provided an insight for the understanding of the role of Id-1 in functional inactivation of p16INK4a in HCC.

  13. 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

  14. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  15. Chromatographic purification of an insoluble histidine tag recombinant Ykt6p SNARE from Arabidopsis thaliana over-expressed in E. coli.

    PubMed

    Vincent, Patrick; Dieryck, Wilfrid; Maneta-Peyret, Lilly; Moreau, Patrick; Cassagne, Claude; Santarelli, Xavier

    2004-08-25

    In order to undertake in plant cell the study of the endoplasmic reticulum (ER)-Golgi apparatus (GA) protein and/or lipid vesicular transport pathway, expressed sequence tag (EST) coding for a homologue to the yeast soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) Ykt6p has been cloned in Arabidopsis thaliana by reverse transcription polymerase chain reaction (RT-PCR). The corresponding protein was over-expressed as a recombinant histidine-tag (his-tag) protein in E. coli. Starting from one litter of culture, an ultrasonic homogenization was performed for cell disruption and after centrifugation the Arabidopsis Ykt6p SNARE present in inclusion bodies in the pellet was solubilized. After centrifugation, the clarified feedstock obtained was injected onto an immobilized metal affinity chromatography (IMAC) in presence of 6 M guanidine and on-column refolding was performed. Folded and subsequently purified (94% purity) recombinant protein was obtained with 82% of recovery.

  16. Retraction: "Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells" by Bao et al.

    PubMed

    2016-08-01

    The above article, published online on April 18, 2011 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the second author that found Figures 1C and 4C to be inappropriately re-used and re-labeled. REFERENCE Bao B, Wang Z, Ali S, Kong D, Banerjee S, Ahmad A, Li Y, Azmi AS, Miele L, Sarkar FH. 2011. Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells. J Cell Biochem 112:2296-2306; doi: 10.1002/jcb.23150.

  17. Inhibition of intimal hyperplasia after stenting by over-expression of p15: a member of the INK4 family of cyclin-dependent kinase inhibitors.

    PubMed

    Segev, Amit; Nili, Nafiseh; Qiang, Beiping; Osherov, Azriel B; Giordano, Frank J; Jaffe, Ronen; Gauldie, Jack; Sparkes, John D; Fraser, Ashley R; Ladouceur-Wodzak, Michelle; Butany, Jagdish; Strauss, Bradley H

    2011-03-01

    We evaluated the role of p15(Ink4), a member of the INK4 family of CDK inhibitors on vascular smooth muscle cells (VSMCs) proliferation, cell cycle progression and intimal hyperplasia after stenting. Aortic VSMCs transduced with either adenovirus encoding for p15(Ink4) or β-galactosidase were assessed for DNA synthesis, cell cycle progression, and pRb phosphorylation. Rabbit carotid arteries were stented and treated with peri-adventitial delivery of saline or adenovirus encoding for p15(Ink4) or β-galactosidase. p15(Ink4) transgene and protein expression were evaluated at 24 h and 72 h, respectively. In-stent cell proliferation was evaluated by BrdU at day 7. Histomorphometric analysis of in-stent intimal hyperplasia was performed at 10 weeks. Human p15(Ink4) DNA was detected in transduced VSMCs at 24h. p15(Ink4) over-expression reduced VSMCs DNA synthesis by 60%. Cell cycle progression was inhibited, with a 30% increase in G1 population accompanied by inhibition of pRb phosphorylation. Human p15(Ink4) transgene was identified in transduced stented arteries but not in control arteries. p15(Ink4) immunostaining was increased and cell proliferation significantly reduced by 50% in p15(Ink4) transduced arteries. Intimal cross-sectional area (CSA) of p15(Ink4)-treated group was significantly lower than the β-gal treated and non-transduced groups (p=0.008). There were no differences in the intimal or medial inflammatory response between groups. p15(Ink4) over-expression blocks cell cycle progression leading to inhibition of VSMCs proliferation. Peri-adventitial delivery of p15(Ink4) significantly inhibits in-stent intimal hyperplasia.

  18. IKBKE is over-expressed in glioma and contributes to resistance of glioma cells to apoptosis via activating NF-κB.

    PubMed

    Guan, Hongyu; Zhang, Heng; Cai, Junchao; Wu, Jueheng; Yuan, Jie; Li, Jun; Huang, Zhengsong; Li, Mengfeng

    2011-02-01

    IκB kinase-ε (IKBKE), a member of the IκB kinase (IKK) family, has been identified as an oncogenic protein and found to be up-regulated in breast cancer, ovarian cancer and prostate cancer. Nonetheless, the expression status and functional significance of IKBKE in human glioma remain unexplored. For the first time, we have demonstrated that mRNA and protein levels of IKBKE were robustly up-regulated in glioma cell lines and human primary glioma tissues. Immunohistochemistry analysis revealed that 53.5% (38/71) paraffin-embedded archived glioma specimens exhibited high levels of IKBKE expression. Intriguingly, there was no significant difference in IKBKE expression among different grades of glioma. To understand the biological function of IKBKE in the development and progression of human glioma, glioma cells lines ectopically over-expressing IKBKE were established and tested for their responsiveness to apoptotic inducers. Our data showed that IKBKE over-expression inhibited cell apoptosis induced by UV irradiation or adriamycin and, in contrast, shRNAi-mediated suppression of IKBKE increased the sensitivity of glioma cells to the apoptotic inducers. Importantly, we found that up-regulated IKBKE could induce the expression of Bcl-2 through activating NF-κB signalling, and that, specifically, we identified IκB as a critical component for this signalling cascade. The current study suggests that up-regulation of IKBKE may represent an important molecular hallmark that is biologically and clinically relevant to the development and progression, as well as the chemo- and radio-resistance, of the disease.

  19. 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.

  20. Transgenic Over-expression of Plasminogen Activator Inhibitor-1 Results in Age-dependent and Gender-specific Increases in Bone Strength and Mineralization

    PubMed Central

    Nordstrom, S.M.; Carleton, S.M.; Carson, W.L.; Eren, M.; Phillips, C.L.; Vaughan, D.E.

    2014-01-01

    The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor- 1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-expresses a variant of human PAI-1 that exhibits enhanced functional stability. Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age (n=10). Femora were imaged via DEXA for BMD and µCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry (n=11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted. Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness was demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT. Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred

  1. Transgenic over-expression of plasminogen activator inhibitor-1 results in age-dependent and gender-specific increases in bone strength and mineralization.

    PubMed

    Nordstrom, S M; Carleton, S M; Carson, W L; Eren, M; Phillips, C L; Vaughan, D E

    2007-12-01

    The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor-1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-express a variant of human PAI-1 that exhibits enhanced functional stability. Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age (n=10). Femora were imaged via DEXA for BMD and muCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry (n=11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted. Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness were demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT. Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred normal

  2. 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.

  3. 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

  4. 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

  5. Over-expression of monoacylglycerol lipase (MGL) in small intestine alters endocannabinoid levels and whole body energy balance, resulting in obesity.

    PubMed

    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.

  6. 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.

  7. 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

  8. Calcitriol May Down-Regulate mRNA Over-Expression of Toll-Like Receptor-2 and -4, LL-37 and Proinflammatory Cytokines in Cultured Human Keratinocytes

    PubMed Central

    Jeong, Mi Sook; Kim, Ji-Yun; Lee, He In

    2014-01-01

    Background Although vitamin D analogs have been used in the topical treatment of psoriasis, their mechanisms of action are not well understand. Calcitriol, the hormonally active vitamin D3 metabolite, has been demonstrated to exert immunomodulatory effects in the skin by down-regulating the expression of Toll-like receptors (TLRs) and proinflammatory cytokines. Objective We investigated the effects of calcitriol on the expression of TLR2, TLR4, antimicrobial peptide LL-37, and proinflammatory cytokines in cultured human keratinocytes. Methods The mRNA expression levels of TLR2, TLR4, tumor necrosis factor α (TNF-α), interleukin (IL)-1β and LL-37 in cultured human keratinocytes were measured by real-time polymerase chain reaction (PCR) and reverse transcription (RT). Furthermore, we measured supernatant TNF-α levels by an enzyme-linked immunosorbent assay (ELISA) to confirm the effects of calcitriol on TLR2 and TLR4. Results As measured by RT-PCR and real-time PCR, calcitriol was found to suppress the lipopolysaccharide- and ultraviolet B radiation-mediated induction of expression of TLRs, LL-37 and proinflammatory cytokines such as TNF-α and IL-1β in normal human keratinocytes. The supernatant TNF-α levels measured by ELISA were also suppressed after treatment with calcitriol. Conclusion Calcitriol may down-regulate inflammatory stated over-expression of LL-37 and proinflammatory cytokines. PMID:24966627

  9. 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.

  10. MicroRNA 146a (miR-146a) Is Over-Expressed during Prion Disease and Modulates the Innate Immune Response and the Microglial Activation State

    PubMed Central

    Huzarewich, Rhiannon L. C. H.; Manguiat, Kathy; Medina, Sarah; Robertson, Catherine; Booth, Stephanie A.

    2012-01-01

    Increasing evidence supports the involvement of microRNAs (miRNAs) in inflammatory and immune processes in prion neuropathogenesis. MiRNAs are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes. We established miR-146a over-expression in prion-infected mouse brain tissues concurrent with the onset of prion deposition and appearance of activated microglia. Expression profiling of a variety of central nervous system derived cell-lines revealed that miR-146a is preferentially expressed in cells of microglial lineage. Prominent up-regulation of miR-146a was evident in the microglial cell lines BV-2 following TLR2 or TLR4 activation and also EOC 13.31 via TLR2 that reached a maximum 24–48 hours post-stimulation, concomitant with the return to basal levels of transcription of induced cytokines. Gain- and loss-of-function studies with miR-146a revealed a substantial deregulation of inflammatory response pathways in response to TLR2 stimulation. Significant transcriptional alterations in response to miR-146a perturbation included downstream mediators of the pro-inflammatory transcription factor, nuclear factor-kappa B (NF-κB) and the JAK-STAT signaling pathway. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in microglial activation states as well as phagocytic mediators of the oxidative burst such as CYBA and NOS3. Based on our results, we propose a role for miR-146a as a potent modulator of microglial function by regulating the activation state during prion induced neurodegeneration. PMID:22363497

  11. Intra-hippocampal transplantation of neural precursor cells with transgenic over-expression of IL-1 receptor antagonist rescues memory and neurogenesis impairments in an Alzheimer's disease model.

    PubMed

    Ben-Menachem-Zidon, Ofra; Ben Menachem-Zidon, Ofra; Ben-Menahem, Yair; Ben-Hur, Tamir; Yirmiya, Raz

    2014-01-01

    Ample evidence implicates neuroinflammatory processes in the etiology and progression of Alzheimer's disease (AD). To assess the specific role of the pro-inflammatory cytokine interleukin-1 (IL-1) in AD we examined the effects of intra-hippocampal transplantation of neural precursor cells (NPCs) with transgenic over-expression of IL-1 receptor antagonist (IL-1raTG) on memory functioning and neurogenesis in a murine model of AD (Tg2576 mice). WT NPCs- or sham-transplanted Tg2576 mice, as well as naive Tg2576 and WT mice served as controls. To assess the net effect of IL-1 blockade (not in the context of NPCs transplantation), we also examined the effects of chronic (4 weeks) intra-cerebroventricular (i.c.v.) administration of IL-1ra. We report that 12-month-old Tg2576 mice exhibited increased mRNA expression of hippocampal IL-1β, along with severe disturbances in hippocampal-dependent contextual and spatial memory as well as in neurogenesis. Transplantation of IL-1raTG NPCs 1 month before the neurobehavioral testing completely rescued these disturbances and significantly increased the number of endogenous hippocampal cells expressing the plasticity-related molecule BDNF. Similar, but less-robust effects were also produced by transplantation of WT NPCs and by i.c.v. IL-1ra administration. NPCs transplantation produced alterations in hippocampal plaque formation and microglial status, which were not clearly correlated with the cognitive effects of this procedure. The results indicate that elevated levels of hippocampal IL-1 are causally related to some AD-associated memory disturbances, and provide the first example for the potential use of genetically manipulated NPCs with anti-inflammatory properties in the treatment of AD.

  12. Interleukin-2 treatment reverses effects of cAMP-responsive element modulator α-over-expressing T cells in autoimmune-prone mice

    PubMed Central

    Ohl, K; Wiener, A; Schippers, A; Wagner, N; Tenbrock, K

    2015-01-01

    Systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), are often characterized by a failure of self-tolerance and result in an uncontrolled activation of B cells and effector T cells. Interleukin (IL)-2 critically maintains homeostasis of regulatory T cells (Treg) and effector T cells in the periphery. Previously, we identified the cAMP-responsive element modulator α (CREMα) as a major factor responsible for decreased IL-2 production in T cells from SLE patients. Additionally, using a transgenic mouse that specifically over-expresses CREMα in T cells (CD2CREMαtg), we provided in-vivo evidence that CREMα indeed suppresses IL-2 production. To analyse the effects of CREMα in an autoimmune prone mouse model we introduced a Fas mutation in the CD2CREMαtg mice (FVB/Fas–/–CD2CREMαtg). Overexpression of CREMα strongly accelerated the lymphadenopathy and splenomegaly in the FVB/Fas–/– mice. This was accompanied by a massive expansion of double-negative (DN) T cells, enhanced numbers of interferon (IFN)-γ-producing T cells and reduced percentages of Tregs. Treatment of FVB/Fas–/–CD2CREMαtg mice with IL-2 restored the percentage of Tregs and reversed increased IFN-γ production, but did not affect the number of DNTs. Our data indicate that CREMα contributes to the failure of tolerance in SLE by favouring effector T cells and decreasing regulatory T cells, partially mediated by repression of IL-2 in vivo. PMID:25817470

  13. 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.

  14. 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.

  15. Over-expression of mitochondrial ferritin affects the JAK2/STAT5 pathway in K562 cells and causes mitochondrial iron accumulation

    PubMed Central

    Santambrogio, Paolo; Erba, Benedetta Gaia; Campanella, Alessandro; Cozzi, Anna; Causarano, Vincenza; Cremonesi, Laura; Gallì, Anna; Della Porta, Matteo Giovanni; Invernizzi, Rosangela; Levi, Sonia

    2011-01-01

    Background Mitochondrial ferritin is a nuclear encoded iron-storage protein localized in mitochondria. It has anti-oxidant properties related to its ferroxidase activity, and it is able to sequester iron avidly into the organelle. The protein has a tissue-specific pattern of expression and is also highly expressed in sideroblasts of patients affected by hereditary sideroblastic anemia and by refractory anemia with ringed sideroblasts. The present study examined whether mitochondrial ferritin has a role in the pathogenesis of these diseases. Design and Methods We analyzed the effect of mitochondrial ferritin over-expression on the JAK2/STAT5 pathway, on iron metabolism and on heme synthesis in erythroleukemic cell lines. Furthermore its effect on apoptosis was evaluated on human erythroid progenitors. Results Data revealed that a high level of mitochondrial ferritin reduced reactive oxygen species and Stat5 phosphorylation while promoting mitochondrial iron loading and cytosolic iron starvation. The decline of Stat5 phosphorylation induced a decrease of the level of anti-apoptotic Bcl-xL transcript compared to that in control cells; however, transferrin receptor 1 transcript increased due to the activation of the iron responsive element/iron regulatory protein machinery. Also, high expression of mitochondrial ferritin increased apoptosis, limited heme synthesis and promoted the formation of Perls-positive granules, identified by electron microscopy as iron granules in mitochondria. Conclusions Our results provide evidence suggesting that Stat5-dependent transcriptional regulation is displaced by strong cytosolic iron starvation status induced by mitochondrial ferritin. The protein interferes with JAK2/STAT5 pathways and with the mechanism of mitochondrial iron accumulation. PMID:21712541

  16. 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.

  17. 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.

  18. Multifunctional silver nanocluster-hybrid oligonucleotide vehicle for cell imaging and microRNA-targeted gene silencing.

    PubMed

    Chen, Hau-Yun; Albert, Karunya; Wen, Cheng-Che; Hsieh, Pei-Ying; Chen, Sih-Yu; Huang, Nei-Chung; Lo, Shen-Chuan; Chen, Jen-Kun; Hsu, Hsin-Yun

    2017-04-01

    Novel therapeutics is urgently needed to prevent cancer-related deaths. MicroRNAs that act as tumor suppressors have been recognized as a next-generation tumor therapy, and the restoration of tumor-suppressive microRNAs using microRNA replacements or mimics may be a less toxic, more effective strategy due to fewer off-target effects. Here, we designed the novel multifunctional oligonucleotide nanocarrier complex composed of a tumor-targeting aptamer sequence specific to mucin 1 (MUC1), poly-cytosine region for fluorescent silver nanocluster (AgNC) synthesis, and complimentary sequence for microRNA miR-34a loading. MiR-34a was employed because of its therapeutic effect of inhibiting oncogene expression and inducing apoptosis in carcinomas. By monitoring the intrinsic fluorescence of AgNC, it was clearly shown that the constructed complex (MUC1-AgNCm-miR-34a) enters MCF-7 cells. To evaluate the efficacy of this nanocarrier for microRNA delivery, we investigated the gene and protein expression levels of downstream miR-34a targets (BCL-2, CDK6, and CCND1) by quantitative PCR and western blotting, respectively, and the results indicated their effective inhibition by miR-34a. This novel multifunctional AgNC-based nanocarrier can aid in improving the efficacy of breast cancer theranostics.

  19. An investigation of the role of gene copy number variations in sorafenib sensitivity in metastatic hepatocellular carcinoma patients

    PubMed Central

    Lee, Ji Yun; Hong, Mineui; Lee, Jeeyun; Lee, Sujin; Kim, Kyoung-Mee; Park, Cheolkeun; Lim, Ho Yeong

    2017-01-01

    Background: Metastatic hepatocellular carcinoma (HCC) is a highly aggressive tumor with limited treatment options. While sorafenib has recently been shown to provide a survival advantage in patients with advanced HCC, the overall outcomes such as time to progression (TTP) and overall survival (OS) ought to be further improved. To that end, several targeted agents aimed at amplified oncogenes such as HER2 and FGFR2 have recently been developed. In this study, we aimed to identify genetic markers in the form of copy number variations (CNVs) that influence clinical outcomes post-sorafenib treatment in advanced HCC patients. Methods: We surveyed 38 metastatic HCC patients who were treated with sorafenib for the presence of CNVs using the NanoString nCounter assay. Results: The median TTP and OS for all patients were 2.7 months (95% confidence interval [CI]: 2.0-3.3 months) and 13.4 months (95% CI: 8.4-18.4 months), respectively. Several genes previously implicated in liver cancer were amplified, including CCND1 (n = 4), CDKN1A (n = 2), KRAS (n = 2), MDM2 (n = 1), and ERBB2 (n = 1). However, we found no correlations between CNVs and survival in our sorafenib-treated patients. Conclusions: The clinical features and biomarkers that account for sensitivity to sorafenib in HCC are complicated and remain unclear. Further investigation to identify predictive biomarkers and therapeutic strategies, including combining sorafenib with other target agents, are warranted. PMID:28382134

  20. Cloning, large scale over-expression in E. coli and purification of the components of the human LAT 1 (SLC7A5) amino acid transporter.

    PubMed

    Galluccio, Michele; Pingitore, Piero; Scalise, Mariafrancesca; Indiveri, Cesare

    2013-08-01

    The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni(2+)-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.

  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. Over-expression of the special AT rich sequence binding protein 1 (SATB1) promotes the progression of nasopharyngeal carcinoma: association with EBV LMP-1 expression

    PubMed Central

    2013-01-01

    weakly expressed in NP-69 cells and CNE1 cells, and significant increasingly expressed in CNE-2Z and C666-1 cells. Targeted knockdown of SATB1 expression obviously attenuated the proliferation and migration of highly SATB1-expressing CNE2Z and C666-1 cells, but not NP-69 and CNE1 cells. Interestingly, forced LMP-1 expression in CNE1 cells led to a surprisingly increasing SATB1 expression and nuclear location, companying with an up-regulated PCNA expression. Conclusions Our results reveal that EBV LMP-1-mediated over-expression of SATB1 is associated with NPC progression, suggesting SATB1 may represent a promising biomarker and therapeutic target for NPC. PMID:24047082

  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. 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

  5. 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

  6. Genome-wide gene expression profiles of HPV-positive and HPV-negative oropharyngeal cancer: potential implications for treatment choices

    PubMed Central

    Lohavanichbutr, Pawadee; Houck, John; Fan, Wenhong; Yueh, Bevan; Mendez, Eduardo; Futran, Neal; Doody, David R.; Upton, Melissa P.; Farwell, D. Gregory; Schwartz, Stephen M.; Zhao, Lue Ping; Chen, Chu

    2009-01-01

    Objective To study the difference in gene expression between human papillomavirus (HPV)-positive and HPV-negative oral squamous cell carcinoma (OSCC). Design We used Affymetrix U133 plus 2.0 arrays to examine gene expression profiles of OSCC and normal oral tissue. HPV DNA was detected using PCR followed by the Roche Linear Array HPV Genotyping Test, and the differentially expressed genes were analyzed to examine their potential biological roles using the Ingenuity Pathway Analysis Software (IPA 5.0). Subjects Tumor tissue from 119 primary OSCC patients and normal oral tissue from 35 patients without cancer, all of whom were treated at three University of Washington-affiliated medical centers. Results HPV DNA was found in 41 of 119 (34.5%) tumors and 2 of 35 (5.7%) normal tissue samples, with 39 of 43 HPV being HPV type 16; there was a higher prevalence of HPV DNA in oropharyngeal cancer (23 of 31) than in oral cavity cancer (18 of 88). We found no significant difference in gene expression between HPV-positive and HPV-negative oral cavity cancer but found 446 probe sets (347 known genes) differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer. The most prominent functions of these genes are DNA replication, DNA repair, and cell cycle. Some genes differentially expressed between HPV-positive and HPV-negative oropharyngeal cancer (e.g., TYMS, STMN1, CCND1 and RBBP4) are involved in chemotherapy or radiation sensitivity. Conclusion These results suggest that differences in the biology of HPV-positive and HPV-negative oropharyngeal cancer may have implications for the management of patients with these different tumors. PMID:19221247

  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. Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL.

    PubMed Central

    Cao, Xiaobo X.; Mohuiddin, Imran; Chada, Sunil; Mhashilkar, Abner M.; Ozvaran, Mustafa K.; McConkey, David J.; Miller, Steven D.; Daniel, Jonathon C.; Smythe, W. Roy

    2002-01-01

    BACKGROUND: Malignant pleural mesothelioma (MPM) is unresponsive to conventional therapies. Forced expression of the novel tumor suppressor mda-7 gene in other cell types has resulted in decreased growth and apoptosis. We evaluated cell growth, apoptosis and tumor suppressor characteristics following forced expression of this gene in mesothelioma cell lines. METHODS: MDA-7 expression in human MPM cells at baseline, following pharmacologic differentiation and viral mda-7 transduction (Ad-mda7) were evaluated with Western blot. Cell viability was evaluated with a colorimetric (XTT) assay, and apoptosis with subG1 FACS and Hoescht. Caspase-3 expression was evaluated by functional assay. These parameters were also evaluated in a stable bcl-xl hyper-expressing MPM cell line. Bax mRNA levels were evaluated with real-time PCR. RESULTS: No baseline or differentiated MPM MDA7 expression was found, but was noted following Ad-mda7 exposure. More than 50% of MPM cells were killed at 5 days following Ad-mda7 exposure (p < 0.001). Apoptosis was accompanied by caspase-3 cleavage and increased BAX expression at both the protein (translational) and mRNA (transcriptional) level. These findings were reduced in a bcl-xl hyper-expressing cell line (P < 0.01). CONCLUSIONS: Although mda-7 does not appear to be a MPM suppressor gene, adenoviral-mediated expression in cell lines induces apoptotic cellular death related to BAX upregulation and caspase cleavage. This is supported by abrogation of effect in a bcl-xl hyper-expressing cell line. PMID:12606823

  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 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

  12. Over-Expression of GmGIa-Regulated Soybean miR172a Confers Early Flowering in Transgenic Arabidopsis thaliana.

    PubMed

    Wang, Tao; Sun, Ming-Yang; Wang, Xue-Song; Li, Wen-Bin; Li, Yong-Guang

    2016-04-29

    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.

  13. Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

    PubMed Central

    Tomita, Nao; Hattori, Takako; Itoh, Shinsuke; Aoyama, Eriko; Yao, Mayumi; Yamashiro, Takashi; Takigawa, Masaharu

    2013-01-01

    Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in

  14. Gene expression abnormalities and oligodendrocyte deficits in the internal capsule in schizophrenia.

    PubMed

    Kerns, David; Vong, Ghe S; Barley, Kevin; Dracheva, Stella; Katsel, Pavel; Casaccia, Patrizia; Haroutunian, Vahram; Byne, William

    2010-07-01

    Deficits in the expression of oligodendrocyte (Ol) and myelin genes have been described in numerous brain regions in schizophrenia (SZ) in association with abnormalities of cell cycle markers. We have previously reported a SZ-associated decrease in the expression of genes expressed after, but not prior to, the terminal differentiation of Ols in the posterior limb of the internal capsule (ICp). This pattern of deficits could reflect a failure of Ol precursors to exit the cell cycle and differentiate to meet the demands imposed by the high rate of apoptosis among myelinating Ols. Here we explore this hypothesis using quantitative real time PCR to examine the mRNA expression of additional genes in the ICp of the previously examined sample of 14 subjects with SZ and 15 normal controls (NCs). The genes examined in the present study were chosen because they are associated with particular phases of the cell cycle (CCND1, CCND2, p21(Cip1), p27(Kip1), and p57(Kip2)), with DNA replication and repair (PCNA), apoptosis (CASP3), or the Notch signaling pathway (JAG1, HES1, HES5, andDTX1). The Notch pathway influences whether Ol precursors continue to proliferate or exit the cell cycle. We also determined the densities of Ols in the ICp. Genes associated with maintenance of the cell cycle tended to exhibit increased expression levels in SZ relative to NCs and to be negatively correlated with the expression levels of the previously assessed mature Ol genes. In contrast, genes associated with cell cycle arrest tended to show the opposite pattern (decreased expression in SZ and positive correlations with mature Ol genes). CASP3 and PCNA expression levels were significantly decreased in SZ and positively correlated with mature Ol genes, suggesting that myelinating Ols may turnover more rapidly in normal controls than in subjects with SZ. JAG1 expression was significantly increased in SZ and exhibited positive correlations with mediators of the canonical Notch pathway but negative

  15. Reversible linkage of two distinct small molecule inhibitors of Myc generates a dimeric inhibitor with improved potency that is active in myc over-expressing cancer cell lines.

    PubMed

    Wanner, Jutta; Romashko, Darlene; Werner, Douglas S; May, Earl W; Peng, Yue; Schulz, Ryan; Foreman, Kenneth W; Russo, Suzanne; Arnold, Lee D; Pingle, Maneesh; Bergstrom, Donald E; Barany, Francis; Thomson, Stuart

    2015-01-01

    We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

  16. Reversible Linkage of Two Distinct Small Molecule Inhibitors of Myc Generates a Dimeric Inhibitor with Improved Potency That Is Active in Myc Over-Expressing Cancer Cell Lines

    PubMed Central

    Wanner, Jutta; Romashko, Darlene; Werner, Douglas S.; May, Earl W.; Peng, Yue; Schulz, Ryan; Foreman, Kenneth W.; Russo, Suzanne; Arnold, Lee D.; Pingle, Maneesh; Bergstrom, Donald E.; Barany, Francis; Thomson, Stuart

    2015-01-01

    We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes. PMID:25875098

  17. 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.

  18. Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology.

    PubMed

    Agrawal, Aditi; Rajamani, Vijayalakshmi; Reddy, Vanga Siva; Mukherjee, Sunil Kumar; Bhatnagar, Raj K

    2015-10-01

    The success of Bt transgenics in controlling predation of crops has been tempered by sporadic emergence of resistance in targeted insect larvae. Such emerging threats have prompted the search for novel insecticidal molecules that are specific and could be expressed through plants. We have resorted to small RNA-based technology for an investigative search and focused our attention to an insect-specific miRNA that interferes with the insect molting process resulting in the death of the larvae. In this study, we report the designing of a vector that produces artificial microRNA (amiR), namely amiR-24, which targets the chitinase gene of Helicoverpa armigera. This vector was used as transgene in tobacco. Northern blot and real-time analysis revealed the high level expression of amiR-24 in transgenic tobacco plants. Larvae feeding on the transgenic plants ceased to molt further and eventually died. Our results demonstrate that transgenic tobacco plants can express amiR-24 insectice specific to H. armigera.

  19. 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

  20. 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.

  1. 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.

  2. Rab1A over-expression prevents Golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of Parkinson's disease.

    PubMed

    Coune, P G; Bensadoun, J C; Aebischer, P; Schneider, B L

    2011-01-01

    Although the overabundance of human alpha-synuclein in nigral dopaminergic neurons is considered to play a pathogenic role in Parkinson's disease (PD), it remains unclear how alpha-synuclein leads to neuronal degeneration and motor symptoms. Here, we explored the effect of human alpha-synuclein in the rat substantia nigra following AAV-mediated gene delivery inducing a moderate loss of dopaminergic neurons together with motor impairments. A significant fraction of the surviving nigral neurons were found to express human αSyn and displayed a pathological fragmentation of the Golgi apparatus. This observation prompted further investigation on the role of the secretory pathway, in particular at the ER/Golgi level, in alpha-synuclein toxicity. To address this question, we co-expressed human alpha-synuclein with Rab1A, a regulator of ER-to-Golgi vesicular trafficking, and found a significant reduction of Golgi fragmentation. Rab1A did not protect the dopaminergic neurons from the alpha-synuclein-induced degeneration that occurred within several months following vector injection. However, we observed in animals co-expressing Rab1A an improvement of motor behavior that correlates with the rescue of normal Golgi morphology in alpha-synuclein-expressing dopaminergic neurons. The non-prenylable mutant Rab1A-DeltaCC did not produce any of the effects observed with the wild-type form of Rab1A, linking the protective role of Rab1A with its activity in ER-to-Golgi vesicular trafficking. In conclusion, Rab1A can rescue the Golgi fragmentation caused by the overabundance of alpha-synuclein in nigral dopaminergic neurons, improving the ability of the surviving neurons to control motor function in hemiparkinsonian animals.

  3. Inhibition of cyclin D1 expression by androgen receptor in breast cancer cells--identification of a novel androgen response element.

    PubMed

    Lanzino, Marilena; Sisci, Diego; Morelli, Catia; Garofalo, Cecilia; Catalano, Stefania; Casaburi, Ivan; Capparelli, Claudia; Giordano, Cinzia; Giordano, Francesca; Maggiolini, Marcello; Andò, Sebastiano

    2010-09-01

    Cyclin D1 gene (CCND1) is a critical mitogen-regulated cell-cycle control element whose transcriptional modulation plays a crucial role in breast cancer growth and progression. Here we demonstrate that the non-aromatizable androgen 5-α-dihydrotestosterone (DHT) inhibits endogenous cyclin D1 expression, as evidenced by reduction of cyclin D1 mRNA and protein levels, and decrease of CCND1-promoter activity, in MCF-7 cells. The DHT-dependent inhibition of CCND1 gene activity requires the involvement and the integrity of the androgen receptor (AR) DNA-binding domain. Site directed mutagenesis, DNA affinity precipitation assay, electrophoretic mobility shift assay and chromatin immunoprecipitation analyses indicate that this inhibitory effect is ligand dependent and it is mediated by direct binding of AR to an androgen response element (CCND1-ARE) located at -570 to -556-bp upstream of the transcription start site, in the cyclin D1 proximal promoter. Moreover, AR-mediated repression of the CCND1 involves the recruitment of the atypical orphan nuclear receptor DAX1 as a component of a multiprotein repressor complex also embracing the participation of Histone Deacetylase 1. In conclusion, identification of the CCND1-ARE allows defining cyclin D1 as a specific androgen target gene in breast and might contribute to explain the molecular basis of the inhibitory role of androgens on breast cancer cells proliferation.

  4. 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.

  5. Transdifferentiation of mesenchymal stem cells-derived adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and have a correlation with cell cycle arresting and driving genes.

    PubMed

    Ullah, Mujib; Stich, Stefan; Notter, Michael; Eucker, Jan; Sittinger, Michael; Ringe, Jochen

    2013-02-01

    It is generally accepted that after differentiation bone marrow mesenchymal stem cells (MSC) become lineage restricted and unipotent in an irreversible manner. However, current results imply that even terminally differentiated cells transdifferentiate across lineage boundaries and therefore act as a progenitor cells for other lineages. This leads to the questions that whether transdifferentiation occurs via direct cell-to-cell conversion or dedifferentiation to a progenitor cells and subsequent differentiation, and whether MSC potency decreases or increases during differentiation. To address these questions, MSC were differentiated into adipogenic lineage cells, followed by dedifferentiation. The process of dedifferentiation was also confirmed by single cell clonal analysis. Finally the dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated MSC, adipogenic-differentiated and dedifferentiated cells were performed. Interestingly, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes have an association with cell cycle arrest in adipogenic-differentiated cells and perhaps narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these genes have an association with cell cycle progression and maybe motivate dedifferentiation of adipogenic-differentiated cells. We found that dedifferentiated cells have a multilineage potency comparable to MSC, and also observed the associative role of proliferation genes with cell cycle arrest and progression. Concluded, our results indicate that transdifferentiation of adipogenic-differentiated cells into osteogenic- or chondrogenic-differentiated cells proceeds via dedifferentiation and correlates with cell cycle arresting and deriving genes. Regarding

  6. Vascular Endothelial Over-Expression of Human Soluble Epoxide Hydrolase (Tie2-sEH Tr) Attenuates Coronary Reactive Hyperemia in Mice: Role of Oxylipins and ω-Hydroxylases

    PubMed Central

    Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

    2017-01-01

    Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P < 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P < 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of

  7. 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

    Luo, Yue-Bei; Johnsen, Russell D; Griffiths, Lisa; Needham, Merrilee; Fabian, Victoria A; Fletcher, Sue; Wilton, Steve D; Mastaglia, Frank L

    2013-12-01

    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.

  8. Transcriptomic analysis of gene cascades involved in protein kinase A and C signalling in the KGN line of human ovarian granulosa tumour cells1.

    PubMed

    Tremblay, Patricia G; Sirard, Marc-André

    2017-04-05

    The developmental competence of an oocyte is its capacity to resume maturation, undergo successful fertilization and reach the blastocyst stage. This competence is acquired through interaction with somatic cells of the follicle. Cumulus and granulosa cells support oocyte development while the oocyte influences follicular cell growth and differentiation. Studies suggest that follicle-stimulating hormone and luteinizing hormone play an essential role in oocyte competence acquisition through signalling initiated by protein kinases A and C (PKA and PKC) in granulosa cells. Using a microarray and RT-qPCR, the transcriptome of human granulosa-like tumour cells (KGN) treated for 24 h with forskolin (FSK) or phorbol 12-myristate 13-acetate (PMA) was analyzed to determine the effects of PKA and PKC stimulation on gene expression. Protein-kinase-driven signalling appeared to involve five major upstream regulators, namely EGF, TGFB1, VEGF, FGF2 and HGF. Genes associations with seven major ovarian functions were identified: PTGS2, IL8 and IL6 with inflammation; STAR, CYP11A1 and CYP19A1 with steroidogenesis; VEGFC, VEGFA and CXCR4 with angiogenesis; AREG, EGFR and SPRY2 with differentiation, BAX, BCL2L12 and CASP1 with apoptosis, CCND1, CCNB1 and CCNB2 with division and MMP1, MMP9 and TIMP1 with ovulation. These results indicate overall that signalling via both PKA and PKC potentiates gene regulation of functions such as inflammation and apoptosis, while functions such as differentiation, ovulation and angiogenesis are partial to one kinase or the other. These results improve understanding of the pathways underlying the most important changes that occur in the follicle prior to ovulation.

  9. Study cell invasion by optical techniques

    NASA Astrophysics Data System (ADS)

    Yang, Ying; Hoban, Paul R.; Sule-Suso, Joseph; Holley, Sarah; El Haj, Alicia J.; Bahrami, Fariba; Wang, Ruikang K.

    2006-02-01

    Cancer is a world-wide health problem associated with an increasing death rate. The mechanisms of how normal cells transform into cancer cells are not fully understood. Intensive investigations have been undertaken to identify genes whose unregulated expression are involved in this process. In this study, we have grown, on collagen gel, adherent mouse embryo fibroblasts (MEFs) knocked out for Cyl-1 (MEF Cyl1-/-) which have been transfected with the human proto-oncogene cyclin D1 (CCND1) under the control of an inducible expression system. CCND1 expression can be regulated in the fibroblasts via the presence of an inducer, isopropyl β-D-Thiogalactopyranoside (IPTG). In the absence of IPTG, CCND1 expression is silenced. The migration ability of the resultant cells on the collagen gel has been monitored by complementary optical techniques: the conventional light microscopy; optical coherence tomography and Fourier Transform Infrared Microspcopic Spectroscopy (FTIR) using Synchrotron beam source. It is found that the cells expressing CCND1 exhibited cell invasion morphology and had different matrix compositions near the cell layer in comparison to the cells not expressing CCND1. The results from this study are consistent with published findings that expression of CCND1 has oncogenic potential and is involved in cell invasion in vitro. Application of complementary optical techniques proves to be an efficient way obtaining morphological and composition information of cell invasion.

  10. 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...

  11. 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

  12. 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

  13. Enhanced co-production of hydrogen and poly-(R)-3-hydroxybutyrate by recombinant PHB producing E. coli over-expressing hydrogenase 3 and acetyl-CoA synthetase.

    PubMed

    Wang, Rui-Yan; Shi, Zhen-Yu; Chen, Jin-Chun; Wu, Qiong; Chen, Guo-Qiang

    2012-09-01

    Recombinant Escherichia coli was constructed for co-production of hydrogen and polyhydroxybutyrate (PHB) due to its rapid growth and convenience of genetic manipulation. In particular, anaerobic metabolic pathways dedicated to co-production of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds such as formate and acetate to useful products. Here, recombinant E. coli expressing hydrogenase 3 and/or acetyl-CoA synthetase showed improved PHB and hydrogen production when grown with or without acetate as a carbon source. When hydrogenase 3 was over-expressed, hydrogen yield was increased from 14 to 153 mmol H(2)/mol glucose in a mineral salt (MS) medium with glucose as carbon source, accompanied by an increased PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

  14. The over expression of long non-coding RNA ANRIL promotes epithelial-mesenchymal transition by activating the ATM-E2F1 signaling pathway in pancreatic cancer: an in vivo and in vitro study.

    PubMed

    Chen, Shi; Zhang, Jia-Qiang; Chen, Jiang-Zhi; Chen, Hui-Xing; Qiu, Fu-Nan; Yan, Mao-Lin; Chen, Yan-Ling; Peng, Cheng-Hong; Tian, Yi-Feng; Wang, Yao-Dong

    2017-03-23

    This study aims to investigate the roles of lncRNA ANRIL in epithelial-mesenchymal transition (EMT) by regulating the ATM-E2F1 signaling pathway in pancreatic cancer (PC). PC rat models were established and ANRIL overexpression and interference plasmids were transfected. The expression of ANRIL, EMT markers (E-cadherin, N-cadherin and Vimentin) and ATM-E2F1 signaling pathway-related proteins (ATM, E2F1, INK4A, INK4B and ARF) were detected. Small molecule drugs were applied to activate and inhibit the ATM-E2F1 signaling pathway. Transwell assay and the scratch test were adopted to detect cell invasion and migration abilities. ANRIL expression in the PC cells was higher than in normal pancreatic duct epithelial cells. In the PC rat models and PC cells, ANRIL interference promoted the expressions of INK4B, INK4A, ARF and E-cadherin, while reduced N-cadherin and Vimentin expression. Over-expressed ANRIL decreased the expression of INK4B, INK4A, ARF and E-cadherin, but raised N-cadherin and Vimentin expressions. By inhibiting the ATM-E2F1 signaling pathway in PC cells, E-cadherin expression increased but N-cadherin and Vimentin expressions decreased. After ANRIL was silenced or the ATM-E2F1 signaling pathway inhibited, PC cell migration and invasion abilities were decreased. In conclusion, over-expression of lncRNA ANRIL can promote EMT of PC cells by activating the ATM-E2F1 signaling pathway.

  15. 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 ...

  16. 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.

  17. 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

  18. Over-expression of bacterial gamma-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula x Populus alba) dependent on the resulting gamma-glutamylcysteine and glutathione levels.

    PubMed

    Herschbach, Cornelia; Rizzini, Luca; Mult, Susanne; Hartmann, Tanja; Busch, Florian; Peuke, Andreas D; Kopriva, Stanislav; Ensminger, Ingo

    2010-07-01

    We compared three transgenic poplar lines over-expressing the bacterial gamma-glutamylcysteine synthetase (GSH1) targeted to plastids. Lines Lggs6 and Lggs12 have two copies, while line Lggs20 has three copies of the transgene. The three lines differ in their expression levels of the transgene and in the accumulation of gamma-glutamylcysteine (gamma-EC) and glutathione (GSH) in leaves, roots and phloem exudates. The lowest transgene expression level was observed in line Lggs6 which showed an increased growth, an enhanced rate of photosynthesis and a decreased excitation pressure (1-qP). The latter typically represents a lower reduction state of the plastoquinone pool, and thereby facilitates electron flow along the electron transport chain. Line Lggs12 showed the highest transgene expression level, highest gamma-EC accumulation in leaves and highest GSH enrichment in phloem exudates and roots. This line also exhibited a reduced growth, and after a prolonged growth of 4.5 months, symptoms of leaf injury. Decreased maximum quantum yield (F(v)/F(m)) indicated down-regulation of photosystem II reaction centre (PSII RC), which correlates with decreased PSII RC protein D1 (PsbA) and diminished light-harvesting complex (Lhcb1). Potential effects of changes in chloroplastic and cytosolic GSH contents on photosynthesis, growth and the whole-plant sulphur nutrition are discussed for each line.

  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. IL-17A but not IL-22 suppresses the replication of hepatitis B virus mediated by over-expression of MxA and OAS mRNA in the HepG2.2.15 cell line.

    PubMed

    Wang, Bing; Zhao, Xin-Ping; Fan, Yu-Chen; Zhang, Jian-Jun; Zhao, Jing; Wang, Kai

    2013-03-01

    Interleukin-17A (IL-17A) and interleukin-22 (IL-22), mainly secreted by interleukin-17-producing T help cells (Th17), are pleiotropic cytokines that regulate the biological responses of several target cells, including hepatocytes. Th17 frequency was reported to negatively correlate with plasma hepatitis B virus (HBV) DNA load in patients with HBV infection. Several studies have indicated that cytokines, such as IL-6 and IL-4, are involved in the noncytopathic suppression of HBV replication. We therefore hypothesized that IL-17A and IL-22 might have a potent suppressive effect on HBV replication. In our present study, we analyzed the suppressive effect of IL-17A and IL-22 on HBV replication in the hepatocellular carcinoma cell line HepG2.2.15. IL-17A did not inhibit the proliferation of HepG2.2.15 cells. It decreased the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) in culture medium and the levels of intracellular HBV DNA. By contrast, blockage of IL-17 receptor (IL-17R) increased the levels of HBsAg and extracellular HBV DNA in culture medium and the levels of intracellular HBV DNA. The expression of antiviral proteins, including myxovirus resistance A (MxA) and oligoadenylate synthetase (OAS), was enhanced by IL-17A. IL-22 and anti-human IL-22 receptor (IL-22R) antibody did not change any indexes. We demonstrated that IL-17A effectively suppressed HBV replication in a noncytopathic manner and the over-expression of MxA and OAS mRNA was involved in the suppression of HBV replication by IL-17A.

  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 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

  2. The occurrence of a deficit in contextual fear extinction in adult amyloid-over-expressing TASTPM mice is independent of the strength of conditioning but can be prevented by mild novel cage stress.

    PubMed

    Rattray, Ivan; Scullion, Gillian A; Soulby, Andrew; Kendall, David A; Pardon, Marie-Christine

    2009-06-08

    In the amyloid over-expressing TASTPM mouse model of Alzheimer's disease, impaired contextual fear memory occurs early, and is preceded, at 4 months of age, by a deficit in extinction of contextual fear that is resistant to improvement by repeated mild novel cage stress. The first aim of this study was thus to establish whether the extinction deficit could be prevented if the novel cage procedure was applied prior to its onset. The second aim was to establish whether the occurrence of the extinction deficit was dependent on the robustness of the conditioning protocol. We first compared 3-month-old wild-type and TASTPM mice for acquisition, retention and extinction of contextual fear and then, looked at the impact of 5 weeks of novel cage stress (4 x 1 h/week) applied from 3 months onwards, on age-related changes in these behaviours evaluated at 4.5 months of age. In another experiment, we compared 4-month-old TASTPM and wild-type mice for the impact of a 2 and 5-pairing conditioning procedure on the three phases of contextual fear conditioning. In 4.5-month-old TASTPM mice, the deficit in extinction was alleviated by repeated novel cage stress, applied from prior to its onset at 3 months. At 4 months of age, the occurrence of an extinction deficit was independent of the strength of the conditioning procedure, in TASTPM mice, which even showed an increase in aversive memory under the 2-pairing condition. The robust early impairment in the extinction of contextual fear seen in adult TASTPM mice suggests that a deficit in cognitive flexibility is the first sign of behavioural pathology in this model of Alzheimer's disease.

  3. Transgenic Mice Over-Expressing the C-99 Fragment of βPP with an α-Secretase Site Mutation Develop a Myopathy Similar to Human Inclusion Body Myositis

    PubMed Central

    Jin, Lee-Way; Hearn, Mark G.; Ogburn, Charles E.; Dang, Ngocthao; Nochlin, David; Ladiges, Warren C.; Martin, George M.

    1998-01-01

    Inclusion body myositis (IBM) is the most common muscle disease in the elderly. Amyloid-β protein (Aβ) has been shown to accumulate abnormally in the vacuolated fibers and to localize to amyloid-like fibrils in muscles from IBM patients. We studied the skeletal muscles from a line of transgenic mice over-expressing the carboxyl-terminal 99 amino acids (C99) of the β-amyloid precursor protein (βPP) with a substitution of lysine-612 to valine (K612V), intended to abolish α-secretase recognition and to preserve the Aβ domain of C99. The majority (87%) of the 24-month-old transgenic mice showed myopathic changes, and approximately one-third of them had degenerating fibers with sarcoplasmic vacuoles and thioflavin-S-positive deposits. Ultrastructurally, the inclusions were aggregates of short thin amyloid-like fibrils, 6 to 8 nm in diameter. These features are similar to those of human IBM. Immunocytochemistry using an antibody against Aβ showed membranous staining in most muscle fibers of transgenic mice, as well as granular or vacuolar cytoplasmic staining in the atrophic fibers. Western blots showed a high level of accumulation of carboxyl-terminal fragments of βPP in the muscles of the transgenic mice with the most severe IBM-like lesions. The expression of IBM-like lesions was age dependent. These transgenic mice provide a model for the study of IBM and for the peripheral expression of a key element in the pathogenesis of Alzheimer disease. PMID:9846957

  4. Identification of a therapeutic strategy targeting amplified FGF19 in liver cancer by Oncogenomic screening.

    PubMed

    Sawey, Eric T; Chanrion, Maia; Cai, Chunlin; Wu, Guanming; Zhang, Jianping; Zender, Lars; Zhao, Alice; Busuttil, Ronald W; Yee, Herman; Stein, Lincoln; French, Dorothy M; Finn, Richard S; Lowe, Scott W; Powers, Scott

    2011-03-08

    We screened 124 genes that are amplified in human hepatocellular carcinoma (HCC) using a mouse hepatoblast model and identified 18 tumor-promoting genes, including CCND1 and its neighbor on 11q13.3, FGF19. Although it is widely assumed that CCND1 is the main driving oncogene of this common amplicon (15% frequency in HCC), both forward-transformation assays and RNAi-mediated inhibition in human HCC cells established that FGF19 is an equally important driver gene in HCC. Furthermore, clonal growth and tumorigenicity of HCC cells harboring the 11q13.3 amplicon were selectively inhibited by RNAi-mediated knockdown of CCND1 or FGF19, as well as by an anti-FGF19 antibody. These results show that 11q13.3 amplification could be an effective biomarker for patients most likely to respond to anti-FGF19 therapy.

  5. The RUNX Genes as Conditional Oncogenes: Insights from Retroviral Targeting and Mouse Models.

    PubMed

    Neil, James C; Gilroy, Kathryn; Borland, Gillian; Hay, Jodie; Terry, Anne; Kilbey, Anna

    2017-01-01

    The observation that the Runx genes act as targets for transcriptional activation by retroviral insertion identified a new family of dominant oncogenes. However, it is now clear that Runx genes are 'conditional' oncogenes whose over-expression is growth inhibitory unless accompanied by another event such as concomitant over-expression of MYC or loss of p53 function. Remarkably, while the oncogenic activities of either MYC or RUNX over-expression are suppressed while p53 is intact, the combination of both neutralises p53 tumour suppression in vivo by as yet unknown mechanisms. Moreover, there is emerging evidence that endogenous, basal RUNX activity is important to maintain the viability and proliferation of MYC-driven lymphoma cells. There is also growing evidence that the human RUNX genes play a similar conditional oncogenic role and are selected for over-expression in end-stage cancers of multiple types. Paradoxically, reduced RUNX activity can also predispose to cell immortalisation and transformation, particularly by mutant Ras. These apparently conflicting observations may be reconciled in a stage-specific model of RUNX involvement in cancer. A question that has yet to be fully addressed is the extent to which the three Runx genes are functionally redundant in cancer promotion and suppression.

  6. Overexpression of several Arabidopsis histone genes increases Agrobacterium-medicated transformation and transgene expression in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Arabidopsis histone H2A-1 is important for Agrobacterium-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, in the rat5 mutant results in decreased T-(transferred) DNA integration into the plant genome, whereas over-expression of HTA1 increases transformation freq...

  7. Gene and protein expression in pituitary corticotroph adenomas: a systematic review of the literature.

    PubMed

    Seltzer, Justin; Ashton, Charles E; Scotton, Thomas C; Pangal, Dhiraj; Carmichael, John D; Zada, Gabriel

    2015-02-01

    , and gene underexpression in 58 genes and 15 proteins was reported. Immunohistochemistry was used in 39 of the studies, and reverse transcriptase polymerase chain reaction was used in 26 of the studies, primarily, and as validation for 4 others. Thirteen studies used both immunohistochemistry and reverse transcriptase polymerase chain reaction. Other methods used included microarray, in situ hybridization, Northern blot analysis, and Western blot analysis. Expression of prioritized genes emphasized in multiple studies were often validated on both the gene and protein levels. Genes/proteins found to be overexpressed in ACTH-PAs relative to the normal pituitary gland included hPTTG1/securin, NEUROD1/NeuroD1 (Beta2), HSD11B2/11β-hydroxysteroid dehydrogenase 2, AKT/Akt, protein kinase B, and CCND1/cyclin D1. Candidate genes/proteins found to be underexpressed in ACTH-PAs relative to the normal pituitary gland included CDKN1B/p27(Kip1), CDKN2A/p16, KISS1/kisspeptin, ACTHR/ACTH-R, and miR-493. CONCLUSIONS On the basis of the authors' systematic review, many significant gene and protein targets that may contribute to tumorigenesis, invasion, and hormone production/secretion of ACTH have been identified and validated in ACTH-PAs. Many of these potential targets have not been fully analyzed for their therapeutic and diagnostic potential but may represent candidate molecular targets for biomarker development and drug targeting. This review may help catalyze additional research efforts using modern profiling and sequencing techniques and alteration of gene expression.

  8. Cyclin D1 Gene G870A Polymorphism Predicts Response to Neoadjuvant Radiotherapy and Prognosis in Rectal Cancer

    SciTech Connect

    Ho-Pun-Cheung, Alexandre; Assenat, Eric; Thezenas, Simon; Bibeau, Frederic; Rouanet, Philippe; Azria, David; Cellier, Dominic; Grenier, Jean; Ychou, Marc; Senesse, Pierre; Lopez-Crapez, Evelyne . E-mail: ecrapez@valdorel.fnclcc.fr

    2007-07-15

    Purpose: To investigate whether CCND1 genetic variations associated with a constitutive nuclear protein may influence either the pathologic response to preoperative RT or the prognosis in a series of rectal cancer patients. Methods and Materials: Seventy rectal cancer patients treated by neoadjuvant radiotherapy were included in the study. CCND1 exon 5 mutations were screened, and the G870A polymorphism was assessed for correlation with clinical variables, tumor response, and patient outcome. Results: No exon 5 mutation was found. Concerning the G870A polymorphism, the A/A variant was significantly associated with radiosensitivity (p = 0.022). Moreover, patients harboring the A allele were correlated with a lower risk of local failure (p = 0.017). Also, combination of the G870A polymorphism with the post-therapeutic lymph node status allowed the elaboration of a prognostic index, which accurately distinguished subgroups of patients with predictable recurrence-free (p = 0.003) and overall (p = 0.044) survival. Conclusions: Although CCND1 exon 5 mutations are rare in rectal cancer, G870A polymorphism is a frequent variation that may predict radiosensitivity and prognosis.

  9. A transcriptional repressor of the ERF family confers drought tolerance to rice and regulates genes preferentially located on chromosome 11.

    PubMed

    Joo, Joungsu; Choi, Hae Jong; Lee, Youn Hab; Kim, Yeon-Ki; Song, Sang Ik

    2013-07-01

    Plant-specific ethylene response factors (ERFs) play important roles in abiotic and biotic stress responses in plants. Using a transgenic approach, we identified two rice ERF genes, OsERF4a and OsERF10a, which conferred drought stress tolerance. In particular, OsERF4a contains a conserved ERF-associated amphiphilic repression (EAR) motif in its C-terminal region that has been shown to function as a transcriptional repression domain. Expression profiling of transgenic rice plants over-expressing OsERF4a using either a constitutively active or an ABA-inducible promoter identified 45 down-regulated and 79 up-regulated genes in common. The increased stress tolerance by over-expression of the EAR domain-containing protein OsERF4a could result from suppression of a repressor of the defense response. Expression of the putative silent information regulator 2 (Sir2) repressor protein was repressed, and expression of several stress-response genes were induced by OsERF4a over-expression. The Sir2 and 7 out of 9 genes that were down-regulated by OsERF4a over-expression were induced by high salinity and drought treatments in non-transgenic control plants. Genes that were down- and up-regulated by OsERF4a over-expression were highly biased toward chromosome 11. Rice chromosome 11 has several large clusters of disease-resistance and defense-response genes. Taken together, our results suggest that OsERF4a is a positive regulator of shoot growth and water-stress tolerance in rice during early growth stages. We propose that OsERF4a could work by suppressing a repressor of the defense responses and/or by controlling the expression of a large number of genes located on chromosome 11.

  10. 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.

  11. CREPT/RPRD1B, a Recently Identified Novel Protein Highly Expressed in Tumors, Enhances the β-Catenin·TCF4 Transcriptional Activity in Response to Wnt Signaling*

    PubMed Central

    Zhang, Yanquan; Liu, Chunxiao; Duan, Xiaolin; Ren, Fangli; Li, Shan; Jin, Zhe; Wang, Yinyin; Feng, Yarui; Liu, Zewen; Chang, Zhijie

    2014-01-01

    CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domain-containing protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/β-catenin signaling activated genes through the β-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both β-catenin and TCF4, and enhances the association of β-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of β-catenin on TBS, and over-expression of CREPT enhances the activity of the β-catenin·TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the β-catenin·TCF4 complex in response to Wnt signaling. PMID:24982424

  12. Biofuel Potential of Plants Transformed Genetically with NAC Family Genes

    PubMed Central

    Singh, Sadhana; Grover, Atul; Nasim, M.

    2016-01-01

    NAC genes contribute to enhance survivability of plants under conditions of environmental stress and in secondary growth of the plants, thereby building biomass. Thus, genetic transformation of plants using NAC genes provides a possibility to tailor biofuel plants. Over-expression studies have indicated that NAC family genes can provide tolerance to various biotic and abiotic stresses, either by physiological or biochemical changes at the cellular level, or by affecting visible morphological and anatomical changes, for example, by development of lateral roots in a number of plants. Over-expression of these genes also work as triggers for development of secondary cell walls. In our laboratory, we have observed a NAC gene from Lepidium latifolium contributing to both enhanced biomass as well as cold stress tolerance of model plants tobacco. Thus, we have reviewed all the developments of genetic engineering using NAC genes which could enhance the traits required for biofuel plants, either by enhancing the stress tolerance or by enhancing the biomass of the plants. PMID:26858739

  13. FSHD muscular dystrophy region gene 1 binds Suv4-20h1 histone methyltransferase and impairs myogenesis.

    PubMed

    Neguembor, Maria Victoria; Xynos, Alexandros; Onorati, Maria Cristina; Caccia, Roberta; Bortolanza, Sergia; Godio, Cristina; Pistoni, Mariaelena; Corona, Davide F; Schotta, Gunnar; Gabellini, Davide

    2013-10-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.

  14. Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility.

    PubMed

    Preston, R S; Philp, A; Claessens, T; Gijezen, L; Dydensborg, A B; Dunlop, E A; Harper, K T; Brinkhuizen, T; Menko, F H; Davies, D M; Land, S C; Pause, A; Baar, K; van Steensel, M A M; Tee, A R

    2011-03-10

    Under conditions of reduced tissue oxygenation, hypoxia-inducible factor (HIF) controls many processes, including angiogenesis and cellular metabolism, and also influences cell proliferation and survival decisions. HIF is centrally involved in tumour growth in inherited diseases that give rise to renal cell carcinoma (RCC), such as Von Hippel-Lindau syndrome and tuberous sclerosis complex. In this study, we examined whether HIF is involved in tumour formation of RCC in Birt-Hogg-Dubé syndrome. For this, we analysed a Birt-Hogg-Dubé patient-derived renal tumour cell line (UOK257) that is devoid of the Birt-Hogg-Dubé protein (BHD) and observed high levels of HIF activity. Knockdown of BHD expression also caused a threefold activation of HIF, which was not as a consequence of more HIF1α or HIF2α protein. Transcription of HIF target genes VEGF, BNIP3 and CCND1 was also increased. We found nuclear localization of HIF1α and increased expression of VEGF, BNIP3 and GLUT1 in a chromophobe carcinoma from a Birt-Hogg-Dubé patient. Our data also reveal that UOK257 cells have high lactate dehydrogenase, pyruvate kinase and 3-hydroxyacyl-CoA dehydrogenase activity. We observed increased expression of pyruvate dehydrogenase kinase 1 (a HIF gene target), which in turn leads to increased phosphorylation and inhibition of pyruvate dehydrogenase. Together with increased protein levels of GLUT1, our data reveal that UOK257 cells favour glycolytic rather than lipid metabolism (a cancer phenomenon termed the 'Warburg effect'). UOK257 cells also possessed a higher expression level of the L-lactate influx monocarboxylate transporter 1 and consequently utilized L-lactate as a metabolic fuel. As a result of their higher dependency on glycolysis, we were able to selectively inhibit the growth of these UOK257 cells by treatment with 2-deoxyglucose. This work suggests that targeting glycolytic metabolism may be used therapeutically to treat Birt-Hogg-Dubé-associated renal lesions.

  15. Polytherapy with a combination of three repurposed drugs (PXT3003) down-regulates Pmp22 over-expression and improves myelination, axonal and functional parameters in models of CMT1A neuropathy.

    PubMed

    Chumakov, Ilya; Milet, Aude; Cholet, Nathalie; Primas, Gwenaël; Boucard, Aurélie; Pereira, Yannick; Graudens, Esther; Mandel, Jonas; Laffaire, Julien; Foucquier, Julie; Glibert, Fabrice; Bertrand, Viviane; Nave, Klaus-Armin; Sereda, Michael W; Vial, Emmanuel; Guedj, Mickaël; Hajj, Rodolphe; Nabirotchkin, Serguei; Cohen, Daniel

    2014-12-10

    Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited sensory and motor peripheral neuropathy. It is caused by PMP22 overexpression which leads to defects of peripheral myelination, loss of long axons, and progressive impairment then disability. There is no treatment available despite observations that monotherapeutic interventions slow progression in rodent models. We thus hypothesized that a polytherapeutic approach using several drugs, previously approved for other diseases, could be beneficial by simultaneously targeting PMP22 and pathways important for myelination and axonal integrity. A combination of drugs for CMT1A polytherapy was chosen from a group of authorised drugs for unrelated diseases using a systems biology approach, followed by pharmacological safety considerations. Testing and proof of synergism of these drugs were performed in a co-culture model of DRG neurons and Schwann cells derived from a Pmp22 transgenic rat model of CMT1A. Their ability to lower Pmp22 mRNA in Schwann cells relative to house-keeping genes or to a second myelin transcript (Mpz) was assessed in a clonal cell line expressing these genes. Finally in vivo efficacy of the combination was tested in two models: CMT1A transgenic rats, and mice that recover from a nerve crush injury, a model to assess neuroprotection and regeneration. Combination of (RS)-baclofen, naltrexone hydrochloride and D-sorbitol, termed PXT3003, improved myelination in the Pmp22 transgenic co-culture cellular model, and moderately down-regulated Pmp22 mRNA expression in Schwannoma cells. In both in vitro systems, the combination of drugs was revealed to possess synergistic effects, which provided the rationale for in vivo clinical testing of rodent models. In Pmp22 transgenic CMT1A rats, PXT3003 down-regulated the Pmp22 to Mpz mRNA ratio, improved myelination of small fibres, increased nerve conduction and ameliorated the clinical phenotype. PXT3003 also improved axonal regeneration and

  16. 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 ...

  17. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    PubMed

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  18. 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.

  19. Physical and spectral characterization of the human cyclin A gene and its interactions with anthracycline anticancer drugs

    NASA Astrophysics Data System (ADS)

    Bao, Huixin; Wang, Xiaohui; Yu, Haijia; Fu, Manliang; Qu, Xiaogang; Zheng, Yongchen; Ren, Jinsong

    2007-02-01

    Over expression of cyclin A in human tumors has been linked to cancer by various experimental lines of evidence. However, physical and spectral characterization of the human cyclin A gene and its interactions with anticancer drugs have not been reported. Our gene sequence analysis, singular value decomposition method and melting studies in the presence of antitumor agents, daunomycin, doxorubicin and Hoechst 33258 showed that cyclin A gene had both AT-rich and GC-rich domains. For a ligand with unknown DNA binding specificity, this gene sequence can be used to differentiate its DNA binding preference.

  20. 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.

  1. Gene expression profiling of precursor T-cell lymphoblastic leukemia/lymphoma identifies oncogenic pathways that are potential therapeutic targets

    PubMed Central

    Lin, Ying-Wei; Aplan, Peter D.

    2007-01-01

    We compared the gene expression pattern of thymic tumors from precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL) that arose in transgenic mice which over-expressed SCL, LMO1, or NUP98-HOXD13 (NHD13) with that of thymocytes from normal littermates. Only two genes, Ccl8 and Mrpl38, were consistently more than 4-fold over-expressed in pre-T LBL from all three genotypes analyzed, and a single gene, Prss16 was consistently under-expressed. However, we identified a number of genes, such as Cfl1, Tcra, Tcrb, Pbx3, Eif4a, Eif4b, and Cox8b that were over or under-expressed in pre-T LBL that arose in specific transgenic lines. Similar to the situation seen with human pre-T LBL, the SCL/LMO1 leukemias displayed an expression profile consistent with mature, late cortical thymocytes, whereas the NHD13 leukemias displayed an expression profile more consistent with immature thymocytes. We evaluated two of the most differentially regulated genes as potential therapeutic targets. Cfl1 was specifically over-expressed in SCL-LMO1 tumors; inactivation of Cfl1 using Okadaic acid resulted in suppression of leukemic cell growth. Overexpression of Ccl8 was a consistent finding in all 3 transgenic lines, and an antagonist for the Ccl8 receptor induced death of leukemic cell lines, suggesting a novel therapeutic approach. PMID:17429429

  2. 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.

  3. 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

  4. 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.

  5. Noggin Along with a Self-Assembling Peptide Nanofiber Containing Long Motif of Laminin Induces Tyrosine Hydroxylase Gene Expression.

    PubMed

    Tavakol, Shima; Musavi, Sayed Mostafa Modaress; Tavakol, Behnaz; Hoveizi, Elham; Ai, Jafar; Rezayat, Seyed Mahdi

    2016-07-08

    Tyrosine hydroxylase (TH), a rate-limiting step in catecholamine synthesis in which its activity influences Alzheimer disease, Parkinson disease, and IQ of schizophrenia patients, has been studied for a long time. In the meantime, the present investigation assessed the effect of noggin and type of self-assembling nanofibers in TH gene over-expression by neuron-like cells derived from human endometrial-derived stromal cells (hEnSCs). Neuroblastoma cells and hEnSCs encapsulated into nanofibers including Matrigel, (RADA)4, laminin, and BMHP-1 motif bounded to (RADA)4 and their cell viability were studied for 48 h and 18 days in basal and neurogenic media, respectively, in noggin-rich media. Then, expression of neural genes and proteins has been investigated by immunocytochemistry (ICC) and real-time PCR methods, respectively. The results indicated that neuroblastoma cell and hEnSC viability is in good agreement with the level of Bcl2 and β-tubulin III gene expression; however, -BMHP-1 and -laminin nanofibers exhibited significantly higher cell viability eventually through Wnt/β-catenin signaling pathway as compared to others, respectively. The gene expression analysis of nanofibers showed that none of them induced gamma-aminobutyric acid (GABA) gene expression while glial fibrillary acidic protein (GFAP) gene just over-expressed in cells encapsulated into Matrigel with a low level of Bcl2 gene expression. However, the TH gene just had been over-expressed in cells encapsulated into -laminin nanofiber and 2D cell culture. In the absence of noggin with -laminin nanofibers, TH gene expression was suppressed. It might be concluded that although noggin through anti-BMP pathways resulted in GFAP decrement and TH gene increment, the type of scaffold that defined the final fate of cells and -laminin accompaniment might be useful for the recovery of Alzheimer and Parkinson disease patients.

  6. 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

  7. Gene Therapy

    MedlinePlus

    ... cells in an effort to treat or stop disease. Genes contain your DNA — the code that controls much of your body's form and function, from making you grow taller to regulating your body systems. Genes that don't work properly can cause disease. Gene therapy replaces a faulty gene or adds ...

  8. Roles of plant hormones and anti-apoptosis genes during drought stress in rice (Oryza sativa L.).

    PubMed

    Ubaidillah, Mohammad; Safitri, Fika Ayu; Jo, Jun-Hyeon; Lee, Sang-Kyu; Hussain, Adil; Mun, Bong-Gyu; Chung, Il Kyung; Yun, Byung-Wook; Kim, Kyung-Min

    2016-12-01

    We previously identified the rice (Oryza sativa) senescence-associated gene OsSAP which encodes a highly conserved protein involved in anti-apoptotic activity. This novel Bax suppressor-related gene regulates tolerance to multiple stresses in yeast. Here, we show the effects of drought stress on leaf and root tissues of plants over-expressing OsSAP in relation to the levels of phytohormones, abscisic acid (ABA), jasmonic acid (JA), indole-3-carboxylic acid (ICA), gibberellic acid (GA3), and zeatin. Results showed that rice plants over-expressing SAP were tolerant to drought stress compared to wild type and the plants over-expressing AtBI-1, which is a homolog of the human Bax inhibitor-1 in Arabidopsis. ABA and JA levels in OsSAP and AtBI-1 transgenic plants consistently increased up to at least 3 days after drought treatment, whereas lower GA3 levels were recorded during early drought period. Comparison between control and transgenic plants overexpressing anti-apoptosis genes OsSAP and AtBI-1 resulted in different patterns of hormone levels, indicating that these genes are involved in the plant responses to drought stress and present an opportunity for further study on drought stress tolerance in rice and other plant species.

  9. MicroRNA-193b Represses Cell Proliferation and Regulates Cyclin D1 in Melanoma

    PubMed Central

    Chen, Jiamin; Feilotter, Harriet E.; Paré, Geneviève C.; Zhang, Xiao; Pemberton, Joshua G.W.; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A.

    2010-01-01

    Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by ≥50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3′untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development. PMID:20304954

  10. Immunologic applications of conditional gene modification technology in the mouse.

    PubMed

    Sharma, Suveena; Zhu, Jinfang

    2014-04-02

    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.

  11. 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

  12. 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

  13. Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain.

    PubMed

    Pridgeon, J W; Yildirim-Aksoy, M; Klesius, P H; Kojima, K; Mobley, J A; Srivastava, K K; Reddy, P G

    2013-10-25

    A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.

  14. [Packaging of lentivirus carrying gene hβc and overexpression of gene hβc in NB4 cells].

    PubMed

    Yang, Jing-Hui; Wu, Yong; Zi, You-Mei; Li, Xian-Fang; Liao, Xiao-Ying; Chen, Yuan-Zhong

    2011-06-01

    This study was aimed to overexpress gene hβc in NB4 cells via the method of lentivirus-mediated gene transfer, to observe the differentiation behaviour change of hβc over-expressing NB4 cells treated with IL-3 or GM-CSF, to explore the relationship between hβc gene and the differentiation behaviour of NB4 cells. The targeted hβc gene was amplified by PCR from the cloned vector carrying ORF of hβc. The PCR product containing PmeI and BstBI site introduced by primer was digested, and then cloned into lentivirus vector pRRLSIN.cPPT.PGK/IRES/GFP.WPRE to construct a lentiviral vector carrying hβc, named pLV-hβc. And the pLV-hβc plasmid was confirmed by restriction and sequencing. The recombinant lentivirus was produced by co-transfecting three plasmids into 293T packing cells. After transfection, the lentiviral supernatant was collected to transfect NB4 cells. GFP expression was examined by fluorescent microscope and the expression of hβc gene was detected by Western blot. Then, the NB4 cells over-expressing hβc were treated with IL-3 (10 ng/ml), GM-CSF (10 ng/ml), ATRA (1 µmol/L) respectively, and the CD11b expression, morphology and differentiation behaviour changes of every groups were observed by flow cytometry and microscopy, while NB4 cells transfected with blank lentivirus (NB4-blank cells) were used as controls. The results showed that the recombinant lentivirus vector carrying hβc gene could efficiently transfect NB4 cells and made NB4 cells to stably over-express hβc gene. The expression of CD11b was up-regulated in NB4-hβc cells treated with of IL-3 or GM-CSF, but it was not as obvious as the effect of ATRA, and no morphological change was observed in NB4 hβc cells treated with the IL-3 or GM-CSF. It is concluded that IL-3 or GM-CSF can induce NB4 cells over-expressing hβc to differentiate to neutrophils, but can not make them fully matured.

  15. Gene Positioning

    PubMed Central

    Ferrai, Carmelo; de Castro, Inês Jesus; Lavitas, Liron; Chotalia, Mita; Pombo, Ana

    2010-01-01

    Eukaryotic gene expression is an intricate multistep process, regulated within the cell nucleus through the activation or repression of RNA synthesis, processing, cytoplasmic export, and translation into protein. The major regulators of gene expression are chromatin remodeling and transcription machineries that are locally recruited to genes. However, enzymatic activities that act on genes are not ubiquitously distributed throughout the nucleoplasm, but limited to specific and spatially defined foci that promote preferred higher-order chromatin arrangements. The positioning of genes within the nuclear landscape relative to specific functional landmarks plays an important role in gene regulation and disease. PMID:20484389

  16. Hepatic Xenobiotic Metabolizing Enzyme Gene Expression ...

    EPA Pesticide Factsheets

    BACKGROUND: Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species. RESULTS: Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs w

  17. 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.

  18. Gene doping.

    PubMed

    Azzazy, Hassan M E

    2010-01-01

    Gene doping abuses the legitimate approach of gene therapy. While gene therapy aims to correct genetic disorders by introducing a foreign gene to replace an existing faulty one or by manipulating existing gene(s) to achieve a therapeutic benefit, gene doping employs the same concepts to bestow performance advantages on athletes over their competitors. Recent developments in genetic engineering have contributed significantly to the progress of gene therapy research and currently numerous clinical trials are underway. Some athletes and their staff are probably watching this progress closely. Any gene that plays a role in muscle development, oxygen delivery to tissues, neuromuscular coordination, or even pain control is considered a candidate for gene dopers. Unfortunately, detecting gene doping is technically very difficult because the transgenic proteins expressed by the introduced genes are similar to their endogenous counterparts. Researchers today are racing the clock because assuring the continued integrity of sports competition depends on their ability to develop effective detection strategies in preparation for the 2012 Olympics, which may mark the appearance of genetically modified athletes.

  19. Gene therapy.

    PubMed

    Williamson, B

    1982-07-29

    Gene therapy is not yet possible, but may become feasible soon, particularly for well understood gene defects. Although treatment of a patient raises no ethical problems once it can be done well, changing the genes of an early embryo is more difficult, controversial and unlikely to be required clinically.

  20. A three component latent class model for robust semiparametric gene discovery.

    PubMed

    Alfo', Marco; Farcomeni, Alessio; Tardella, Luca

    2011-01-01

    We propose a robust model for discovering differentially expressed genes which directly incorporates biological significance, i.e., effect dimension. Using the so-called c-fold rule, we transform the expressions into a nominal observed random variable with three categories: below a fixed lower threshold, above a fixed upper threshold or within the two thresholds. Gene expression data is then transformed into a nominal variable with three levels possibly originated by three different distributions corresponding to under expressed, not differential, and over expressed genes. This leads to a statistical model for a 3-component mixture of trinomial distributions with suitable constraints on the parameter space. In order to obtain the MLE estimates, we show how to implement a constrained EM algorithm with a latent label for the corresponding component of each gene. Different strategies for a statistically significant gene discovery are discussed and compared. We illustrate the method on a little simulation study and a real dataset on multiple sclerosis.

  1. Expression of the yeast cpd1 gene in tobacco confers resistance to the fungal toxin cercosporin.

    PubMed

    Panagiotis, Madesis; Kritonas, Kalantidis; Irini, Nianiou Obeidat; Kiriaki, Chatzidimitriou; Nicolaos, Panopoulos; Athanasios, Tsaftaris

    2007-06-01

    Many phytopathogenic species of the fungus Cercospora produce cercosporin, a photoactivated perylenequinone toxin that belongs to a family of photosensitizers, which absorb light energy and produce extremely cytotoxic, reactive oxygen species. The cpd1 (cercosporin photosensitizer detoxification) gene of yeast (Saccharomyces cerevisiae), which encodes for a novel protein with significant similarity to the FAD-dependent pyridine nucleotide reductases, confers resistance to cercosporin when over-expressed in yeast. The aim of this work was to investigate the potential ability of cpd1 gene to confer resistance to cercosporin when expressed in tobacco plants (Nicotiana tabacum). Transgenic tobacco plants were produced using Agrobacterium tumefaciens, with cpd1 integrated as the gene of interest. We report here that expression of cpd1 gene in tobacco can mediate resistance to cercosporin. The involvement of cpd1 gene in the detoxification of the cercosporin reinforces previous observations, which suggested that resistance to cercosporin is mediated by a mechanism involving toxin reduction.

  2. Gene expression profile in monocyte during in vitro mineral fiber degradation.

    PubMed

    Dika Nguea, Hermine; de Reydellet, Aymon; Lehuédé, Patrice; De Meringo, Alain; Le Faou, Alain; Marcocci, Lucia; Rihn, Bertrand H

    2008-06-01

    A human monocytes cell line, U-937, incubated in the presence of filtered medium from Escherichia coli culture (FS) has been previously reported to degrade man made mineral fiber and it has been indicated as a good paradigm of in vivo fiber biopersistence evaluation (manuscript accepted for publication). In the present paper, a study is reported aimed to define the molecular modification occurring in the U-937 monocytes during in vitro fiber degradation. The induction of gene expression was investigated in U-937 exposed to rock wool fibers (HDN) in the presence of FS by transcriptome analysis using 20 K DNA microarrays and quantitative RT-PCR. The over-expression of genes related to mobility and cellular adhesion, oxidative stress, immune system stimulation, enzymes, and ions transport protein systems were identified. Among them NCF1 gene, the gene encoding a subunit of NADPH oxidase, over-expression was detected. As the product of this gene allows the formation of superoxide anion that could lead to oxidative stress, HDN fibers were exposed to hydrogen peroxide. Fiber degradation similar to those observed upon incubation with U-937 in the presence of FS was obtained thus suggesting that reactive oxygen species production may be responsible for fiber degradation by U-937 monocytes.

  3. Transcriptional regulation of the Drosophila ANT gene by the DRE/DREF system.

    PubMed

    Kim, Young Shin; Shin, Meong Joo; Yang, Dong Jin; Yamaguchi, Masamitsu; Park, So Young; Yoo, Mi Ae

    2007-05-01

    Adenine nucleotide translocase (ANT) is a crucial component in the maintenance of cellular energy homeostasis, as well as in the formation of the mitochondrial permeability transition pores. However, the molecular mechanisms regulating the expression of the ANT gene are poorly understood. In this study, we have identified three DNA replication-related elements (DRE; 5'-TATCGATA) in the 5'-flanking region of the Drosophila ANT (dANT) gene. Gel-mobility shift analyses revealed that all three of the DREs were recognized by the DRE-binding factor (DREF). The site-directed mutagenesis of these DRE sites induces a considerable reduction in the activity of the dANT gene promoter in vitro. Analyses with transgenic flies harboring a dANT-lacZ fusion gene bearing the wild-type or mutant DRE sites showed that the DRE sites were required for the expression of dANT in vivo. We determined that the over-expression or knockdown of DREF exerts a regulatory effect on the activity of the dANT promoter. In addition, we observed the collapse of mitochondrial membrane potential in the eye imaginal discs in which DREF was over-expressed. These results show that DRE/DREF is a crucial regulator of dANT gene expression, and also suggest the possibility that cross-talk may occur between the DRE/DREF system and mitochondrial functioning.

  4. Discovery of a novel tumour metastasis-promoting gene, NVM-1.

    PubMed

    Thiele, Wilko; Novac, Natalia; Mink, Sigrun; Schreiber, Caroline; Plaumann, Diana; Fritzmann, Johannes; Cremers, Natascha; Rothley, Melanie; Schwager, Christian; Regiert, Thomas; Huber, Peter E; Stein, Ulrike; Schlag, Peter; Moll, Jürgen; Abdollahi, Amir; Sleeman, Jonathan P

    2011-09-01

    We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.

  5. Search for neuro-endocrine markers (chromogranin A, synaptophysin and VGF) in breast cancers. An integrated approach using immunohistochemistry and gene expression profiling.

    PubMed

    Annaratone, Laura; Medico, Enzo; Rangel, Nelson; Ca