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Sample records for neuroplasticity genes overexpressed

  1. Ependymin, a gene involved in regeneration and neuroplasticity in vertebrates, is overexpressed during regeneration in the echinoderm Holothuria glaberrima.

    PubMed

    Suárez-Castillo, Edna C; Medina-Ortíz, Wanda E; Roig-López, José L; García-Arrarás, José E

    2004-06-01

    We report the characterization of an ependymin-related gene (EpenHg) from a regenerating intestine cDNA library of the sea cucumber Holothuria glaberrima. This finding is remarkable because no ependymin sequence has ever been reported from invertebrates. Database comparisons of the conceptual translation of the EpenHg gene reveal 63% similarity (47% identity) with mammalian ependymin-related proteins (MERPs) and close relationship with the frog and piscine ependymins. We also report the partial sequences of ependymin representatives from another species of sea cucumber and from a sea urchin species. Conventional and real-time reverse transcriptase polymerase chain reaction (RT-PCRs) show that the gene is expressed in several echinoderm tissues, including esophagus, mesenteries, gonads, respiratory trees, hemal system, tentacles and body wall. Moreover, the ependymin product in the intestine is overexpressed during sea cucumber intestinal regeneration. The discovery of ependymins in echinoderms, a group well known for their regenerative capacities, can give us an insight on the evolution and roles of ependymin molecules. PMID:15256263

  2. ALTERED EXPRESSION OF NEUROPLASTICITY-RELATED GENES IN THE BRAIN OF DEPRESSED SUICIDES

    PubMed Central

    FUCHSOVA, B.; ALVAREZ JULIÁ, A.; RIZAVI, H. S.; FRASCH, A. C.; PANDEY, G. N.

    2015-01-01

    Background Expression of the neuronal membrane glycoprotein M6a (GPM6A), the proteolipid protein (PLP/DM20) family member, is downregulated in the hippocampus of chronically stressed animals. Its neuroplastic function involves a role in neurite formation, filopodium outgrowth and synaptogenesis through an unknown mechanism. Disruptions in neuroplasticity mechanisms have been shown to play a significant part in the etiology of depression. Thus, the current investigation examined whether GPM6A expression is also altered in human depressed brain. Methods Expression levels and coexpression patterns of GPM6A, GPM6B, and PLP1 (two other members of PLP/DM20 family) as well as of the neuroplasticity-related genes identified to associate with GPM6A were determined using quantitative polymerase chain reaction (qPCR) in postmortem samples from the hippocampus (n =18) and the prefrontal cortex (PFC) (n= 25) of depressed suicide victims and compared with control subjects (hippocampus n= 18; PFC n =25). Neuroplasticity-related proteins that form complexes with GPM6A were identified by coimmunoprecipitation technique followed by mass spectrometry. Results Results indicated transcriptional downregulation of GPM6A and GPM6B in the hippocampus of depressed suicides. The expression level of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) and coronin1A (CORO1A) was also significantly decreased. Subsequent analysis of coexpression patterns demonstrated coordinated gene expression in the hippocampus and in the PFC indicating that the function of these genes might be coregulated in the human brain. However, in the brain of depressed suicides this coordinated response was disrupted. Conclusions Disruption of coordinated gene expression as well as abnormalities in GPM6A and GPM6B expression and expression of the components of GPM6A complexes were detected in the brain of depressed suicides. PMID:25934039

  3. Invited address: "The times they are a-changin'" gene expression, neuroplasticity, and developmental research.

    PubMed

    Simons, Ronald L; Klopack, Eric T

    2015-03-01

    For good reason, social scientists have a long history of being suspicious of biological explanations of human behavior. Importantly, however, recent paradigmatic shifts in the life sciences have largely obviated these longstanding concerns. We highlight the changes that have occurred in genetics with its movement away from genetic determinism to an emphasis on epigenetics and in neuroscience with its switch from a fixed to a neuroplastic view of the brain. We describe these new developments noting the way they recognize, indeed place a premium upon, the role of the environment. The remainder of the paper focuses upon the challenges and opportunities for social scientists, especially those involved in developmental work, proffered by these paradigmatic shifts. The evidence clearly shows that nature and nurture are inextricably interlinked. Importantly, however, it also indicates that they are connected in a manner that honors the priority that the social sciences place upon the environment. We contend that incorporating biological processes into our developmental work will sharpen our theories and enhance their significance. We argue that such biologically integrated models will provide a clearer and more comprehensive understanding of how nurture influences behavior across the life course. PMID:25550091

  4. Social Isolation Stress Induces Anxious-Depressive-Like Behavior and Alterations of Neuroplasticity-Related Genes in Adult Male Mice

    PubMed Central

    Ieraci, Alessandro; Mallei, Alessandra; Popoli, Maurizio

    2016-01-01

    Stress is a major risk factor in the onset of several neuropsychiatric disorders including anxiety and depression. Although several studies have shown that social isolation stress during postweaning period induces behavioral and brain molecular changes, the effects of social isolation on behavior during adulthood have been less characterized. Aim of this work was to investigate the relationship between the behavioral alterations and brain molecular changes induced by chronic social isolation stress in adult male mice. Plasma corticosterone levels and adrenal glands weight were also analyzed. Socially isolated (SI) mice showed higher locomotor activity, spent less time in the open field center, and displayed higher immobility time in the tail suspension test compared to group-housed (GH) mice. SI mice exhibited reduced plasma corticosterone levels and reduced difference between right and left adrenal glands. SI showed lower mRNA levels of the BDNF-7 splice variant, c-Fos, Arc, and Egr-1 in both hippocampus and prefrontal cortex compared to GH mice. Finally, SI mice exhibited selectively reduced mGluR1 and mGluR2 levels in the prefrontal cortex. Altogether, these results suggest that anxious- and depressive-like behavior induced by social isolation stress correlates with reduction of several neuroplasticity-related genes in the hippocampus and prefrontal cortex of adult male mice. PMID:26881124

  5. Changes in gene expression associated with FTO overexpression in mice.

    PubMed

    Merkestein, Myrte; McTaggart, James S; Lee, Sheena; Kramer, Holger B; McMurray, Fiona; Lafond, Mathilde; Boutens, Lily; Cox, Roger; Ashcroft, Frances M

    2014-01-01

    Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected. PMID:24842286

  6. Changes in Gene Expression Associated with FTO Overexpression in Mice

    PubMed Central

    Kramer, Holger B.; McMurray, Fiona; Lafond, Mathilde; Boutens, Lily; Cox, Roger; Ashcroft, Frances M.

    2014-01-01

    Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected. PMID:24842286

  7. [Overexpression of Penicillium expansum lipase gene in Pichia pastoris].

    PubMed

    Yuan, Cai; Lin, Lin; Shi, Qiao-Qin; Wu, Song-Gang

    2003-03-01

    The alkaline lipase gene of Penicillium expansum (PEL) was coloned into the yeast integrative plasmid pPIC3.5K, which was then transformed into His4 mutant yeast GS115. Recombinant Pichia strains were obtained by minimal olive oil-methanol plates screening and confirmed by PCR. The expression producus of PEL gene was analysis by SDS-PAGE and olive oil plate, the result indicated that PEL gene was functionally overexpressed in Pichia pastoris and up to 95% of the secreted protein. Recombinant lipase had a molecular mass of 28kD, showing a range similar to that of PEL, could hydrolyze olive oil and formed clear halos in the olive oil plates. Four different strategies (different media, pH, glycerol and methanol concentration) were applied to optimize the cultivation conditions, the activity of lipase was up to 260 u/mL under the optimal cultivation conditions. It is pointed out that the absence of the expensive biotin and yeast nitrogen base in the medium increased the lipase production. The possible reason of this result is absence of yeast nitrogen base increased the medium pH during cultivation, and PEL shows a higher stability at this condition. The lipase activity of the supernatant from the culture grown at pH 7 was higher than the one from the culture in the same medium at pH 6.0 is due to the pH stability of PEL too. The results also showed that the methanol and glycerol concentration had a marked effect on the production of lipase. PMID:15966328

  8. Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer.

    PubMed

    Duffy, Supipi; Fam, Hok Khim; Wang, Yi Kan; Styles, Erin B; Kim, Jung-Hyun; Ang, J Sidney; Singh, Tejomayee; Larionov, Vladimir; Shah, Sohrab P; Andrews, Brenda; Boerkoel, Cornelius F; Hieter, Philip

    2016-09-01

    Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1 Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors. PMID:27551064

  9. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes.

    PubMed

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  10. Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes

    PubMed Central

    Shaposhnikov, Mikhail; Proshkina, Ekaterina; Shilova, Lyubov; Zhavoronkov, Alex; Moskalev, Alexey

    2015-01-01

    DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster. PMID:26477511

  11. Consciousness, brain, neuroplasticity

    PubMed Central

    Askenasy, Jean; Lehmann, Joseph

    2013-01-01

    Subjectivity, intentionality, self-awareness and will are major components of consciousness in human beings. Changes in consciousness and its content following different brain processes and malfunction have long been studied. Cognitive sciences assume that brain activities have an infrastructure, but there is also evidence that consciousness itself may change this infrastructure. The two-way influence between brain and consciousness has been at the center of philosophy and less so, of science. This so-called bottom-up and top-down interrelationship is controversial and is the subject of our article. We would like to ask: how does it happen that consciousness may provoke structural changes in the brain? The living brain means continuous changes at the synaptic level with every new experience, with every new process of learning, memorizing or mastering new and existing skills. Synapses are generated and dissolved, while others are preserved, in an ever-changing process of so-called neuroplasticity. Ongoing processes of synaptic reinforcements and decay occur during wakefulness when consciousness is present, but also during sleep when it is mostly absent. We suggest that consciousness influences brain neuroplasticity both during wakefulness as well as sleep in a top-down way. This means that consciousness really activates synaptic flow and changes brain structures and functional organization. The dynamic impact of consciousness on brain never stops despite the relative stationary structure of the brain. Such a process can be a target for medical intervention, e.g., by cognitive training. PMID:23847580

  12. Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Jacinto, Pauline L; Buensalido, Joseph Adrian L; Seo, Susan M; Kaatz, Glenn W

    2015-05-01

    Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus. Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S. aureus (MSSA), in clinical isolates are suggestive of clonal dissemination. It is also possible that related strains are prone to mutations resulting in overexpression of specific MDR-EP genes. Exposure of non-MDR-EP-overexpressing clinical isolates to biocides and dyes can select for MDR-EP-overexpressing mutants. spa types t002 and t008 isolates are predominated by multilocus sequencing typing sequence types (STs) 5 and 8, respectively. In this study, non-MDR-EP gene-overexpressing clinical isolates (MRSA and MSSA) representing ST5 and ST8 were subjected to single exposures of ethidium bromide (EtBr) to select for EtBr-resistant mutants. Measurements of active EtBr transport among mutants were used to demonstrate an efflux-proficient phenotype. Using quantitative reverse-transcription PCR, it was found that EtBr-resistant mutants of ST5 and ST8 parental strains predominantly overexpressed mepA (100%) and mdeA (83%), respectively, regardless of meticillin sensitivity. Associations between clonal lineage and MDR-EP gene overexpression differed from those previously observed and suggest the latter is due to clonal spread of efflux-proficient strains. The predilection of in vitro-selected mutants of related strains to overexpress the same MDR-EP gene indicates the presence of a consistent mutational process. PMID:25548027

  13. Human cancers overexpress genes that are specific to a variety of normal human tissues

    PubMed Central

    Lotem, Joseph; Netanely, Dvir; Domany, Eytan; Sachs, Leo

    2005-01-01

    We have analyzed gene expression data from three different kinds of samples: normal human tissues, human cancer cell lines, and leukemic cells from lymphoid and myeloid leukemia pediatric patients. We have searched for genes that are overexpressed in human cancer and also show specific patterns of tissue-dependent expression in normal tissues. Using the expression data of the normal tissues, we identified 4,346 genes with a high variability of expression and clustered these genes according to their relative expression level. Of 91 stable clusters obtained, 24 clusters included genes preferentially expressed either only in hematopoietic tissues or in hematopoietic and one to two other tissues; 28 clusters included genes preferentially expressed in various nonhematopoietic tissues such as neuronal, testis, liver, kidney, muscle, lung, pancreas, and placenta. Analysis of the expression levels of these two groups of genes in the human cancer cell lines and leukemias identified genes that were highly expressed in cancer cells but not in their normal counterparts and, thus, were overexpressed in the cancers. The different cancer cell lines and leukemias varied in the number and identity of these overexpressed genes. The results indicate that many genes that are overexpressed in human cancer cells are specific to a variety of normal tissues, including normal tissues other than those from which the cancer originated. It is suggested that this general property of cancer cells plays a major role in determining the behavior of the cancers, including their metastatic potential. PMID:16339305

  14. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes.

    PubMed

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2015-01-01

    Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a "fine-tune" regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression. PMID:26483828

  15. Transcriptomic and field evaluation of apple trees overexpressing a peach CBF gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of CBF genes in cold response and acclimation has been well documented in both herbaceous and woody plants. Our initial research demonstrated that overexpression of a peach CBF gene (PpCBF1) in ‘M.26’ apple increases freezing tolerance of non-acclimated plants and unexpectedly also results...

  16. Heterologous Overexpression of Poplar SnRK2 Genes Enhanced Salt Stress Tolerance in Arabidopsis thaliana

    PubMed Central

    Song, Xueqing; Yu, Xiang; Hori, Chiaki; Demura, Taku; Ohtani, Misato; Zhuge, Qiang

    2016-01-01

    Subfamily 2 of SNF1-related protein kinase (SnRK2) plays important roles in plant abiotic stress responses as a global positive regulator of abscisic acid signaling. In the genome of the model tree Populus trichocarpa, 12 SnRK2 genes have been identified, and some are upregulated by abiotic stresses. In this study, we heterologously overexpressed the PtSnRK2 genes in Arabidopsis thaliana and found that overexpression of PtSnRK2.5 and PtSnRK2.7 genes enhanced stress tolerance. In the PtSnRK2.5 and PtSnRK2.7 overexpressors, chlorophyll content, and root elongation were maintained under salt stress conditions, leading to higher survival rates under salt stress compared with those in the wild type. Transcriptomic analysis revealed that PtSnRK2.7 overexpression affected stress-related metabolic genes, including lipid metabolism and flavonoid metabolism, even under normal growth conditions. However, the stress response genes reported to be upregulated in Arabidopsis SRK2C/SnRK2.6 and wheat SnRK2.8 overexpressors were not changed by PtSnRK2.7 overexpression. Furthermore, PtSnRK2.7 overexpression widely and largely influenced the transcriptome in response to salt stress; genes related to transport activity, including anion transport-related genes, were characteristically upregulated, and a variety of metabolic genes were specifically downregulated. We also found that the salt stress response genes were greatly upregulated in the PtSnRK2.7 overexpressor. Taken together, poplar subclass 2 PtSnRK2 genes can modulate salt stress tolerance in Arabidopsis, through the activation of cellular signaling pathways in a different manner from that by herbal subclass 2 SnRK2 genes. PMID:27242819

  17. Harnessing neuroplasticity for clinical applications

    PubMed Central

    Sur, Mriganka; Dobkin, Bruce H.; O'Brien, Charles; Sanger, Terence D.; Trojanowski, John Q.; Rumsey, Judith M.; Hicks, Ramona; Cameron, Judy; Chen, Daofen; Chen, Wen G.; Cohen, Leonardo G.; deCharms, Christopher; Duffy, Charles J.; Eden, Guinevere F.; Fetz, Eberhard E.; Filart, Rosemarie; Freund, Michelle; Grant, Steven J.; Haber, Suzanne; Kalivas, Peter W.; Kolb, Bryan; Kramer, Arthur F.; Lynch, Minda; Mayberg, Helen S.; McQuillen, Patrick S.; Nitkin, Ralph; Pascual-Leone, Alvaro; Reuter-Lorenz, Patricia; Schiff, Nicholas; Sharma, Anu; Shekim, Lana; Stryker, Michael; Sullivan, Edith V.; Vinogradov, Sophia

    2011-01-01

    Neuroplasticity can be defined as the ability of the nervous system to respond to intrinsic or extrinsic stimuli by reorganizing its structure, function and connections. Major advances in the understanding of neuroplasticity have to date yielded few established interventions. To advance the translation of neuroplasticity research towards clinical applications, the National Institutes of Health Blueprint for Neuroscience Research sponsored a workshop in 2009. Basic and clinical researchers in disciplines from central nervous system injury/stroke, mental/addictive disorders, paediatric/developmental disorders and neurodegeneration/ageing identified cardinal examples of neuroplasticity, underlying mechanisms, therapeutic implications and common denominators. Promising therapies that may enhance training-induced cognitive and motor learning, such as brain stimulation and neuropharmacological interventions, were identified, along with questions of how best to use this body of information to reduce human disability. Improved understanding of adaptive mechanisms at every level, from molecules to synapses, to networks, to behaviour, can be gained from iterative collaborations between basic and clinical researchers. Lessons can be gleaned from studying fields related to plasticity, such as development, critical periods, learning and response to disease. Improved means of assessing neuroplasticity in humans, including biomarkers for predicting and monitoring treatment response, are needed. Neuroplasticity occurs with many variations, in many forms, and in many contexts. However, common themes in plasticity that emerge across diverse central nervous system conditions include experience dependence, time sensitivity and the importance of motivation and attention. Integration of information across disciplines should enhance opportunities for the translation of neuroplasticity and circuit retraining research into effective clinical therapies. PMID:21482550

  18. Functional overexpression and characterization of lipogenesis-related genes in the oleaginous yeast Yarrowia lipolytica.

    PubMed

    Silverman, Andrew M; Qiao, Kangjian; Xu, Peng; Stephanopoulos, Gregory

    2016-04-01

    Single cell oil (SCO) is an attractive energy source due to scalability, utilization of low-cost renewable feedstocks, and type of product(s) made. Engineering strains capable of producing high lipid titers and yields is crucial to the economic viability of these processes. However, lipid synthesis in cells is a complex phenomenon subject to multiple layers of regulation, making gene target identification a challenging task. In this study, we aimed to identify genes in the oleaginous yeast Yarrowia lipolytica whose overexpression enhances lipid production by this organism. To this end, we examined the effect of the overexpression of a set of 44 native genes on lipid production in Y. lipolytica, including those involved in glycerolipid synthesis, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport and characterized each resulting strain's ability to produce lipids growing on both glucose and acetate as a sole carbon source. Our results suggest that a diverse subset of genes was effective at individually influencing lipid production in Y. lipolytica, sometimes in a substrate-dependent manner. The most productive strain on glucose overexpressed the diacylglycerol acyltransferase DGA2 gene, increasing lipid titer, cellular content, and yield by 236, 165, and 246 %, respectively, over our control strain. On acetate, our most productive strain overexpressed the acylglycerol-phosphate acyltransferase SLC1 gene, with a lipid titer, cellular content, and yield increase of 99, 91, and 151 %, respectively, over the control strain. Aside from genes encoding enzymes that directly catalyze the reactions of lipid synthesis, other ways by which lipogenesis was increased in these cells include overexpressing the glycerol-3-phosphate dehydrogenase (GPD1) gene to increase production of glycerol head groups and overexpressing the 6-phosphogluconolactonase (SOL3) gene from the oxidative pentose phosphate pathway to increase NADPH

  19. Lactobacillus plantarum ldhL gene: overexpression and deletion.

    PubMed Central

    Ferain, T; Garmyn, D; Bernard, N; Hols, P; Delcour, J

    1994-01-01

    Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged. PMID:8300514

  20. Increase in pectin deposition by overexpression of an ERF gene in cultured cells of Arabidopsis thaliana.

    PubMed

    Nakano, Toshitsugu; Naito, Yuki; Kakegawa, Koich; Ohtsuki, Namie; Tsujimoto-Inui, Yayoi; Shinshi, Hideaki; Suzuki, Kaoru

    2012-04-01

    Ethylene-responsive transcription factor (ERF) family genes, which are involved in regulation of metabolic pathways and/or are useful for metabolic engineering, were investigated in the cultured cells of Arabidopsis thaliana. The pectin content in the gelatinous precipitates after the ethanol precipitation of extracts derived from calli of a transgenic cell line, A17, overexpressing an ERF gene (At1g44830), increased in comparison with the control. Expression of genes involved in pectin biosynthesis was up-regulated in the A17 calli. Overexpression of the ERF gene coordinately activates the pectin biosynthetic pathway genes and increases the content of pectin. These results therefore will be useful as a genetic resource for engineering pectin biosynthesis in plants. PMID:22160296

  1. Permethrin induces overexpression of multiple genes in Aedes aegypti.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using the PCR-select subtractive cDNA hybridization technique, 18 different genes were isolated from a permethrin-treated vs acetone-treated Aedes aegypti subtractive library. QPCR results revealed that eight of the 18 gene’s transcriptional levels in permethrin-treated Ae. aegypti were at least 2- ...

  2. Increased drought tolerance through the suppression of ESKMO1 gene and overexpression of CBF-related genes in Arabidopsis.

    PubMed

    Xu, Fuhui; Liu, Zhixue; Xie, Hongyan; Zhu, Jian; Zhang, Juren; Kraus, Josef; Blaschnig, Tasja; Nehls, Reinhard; Wang, Hong

    2014-01-01

    Improved drought tolerance is always a highly desired trait for agricultural plants. Significantly increased drought tolerance in Arabidopsis thaliana (Columbia-0) has been achieved in our work through the suppression of ESKMO1 (ESK1) gene expression with small-interfering RNA (siRNA) and overexpression of CBF genes with constitutive gene expression. ESK1 has been identified as a gene linked to normal development of the plant vascular system, which is assumed directly related to plant drought response. By using siRNA that specifically targets ESK1, the gene expression has been reduced and drought tolerance of the plant has been enhanced dramatically in the work. However, the plant response to external abscisic acid application has not been changed. ICE1, CBF1, and CBF3 are genes involved in a well-characterized plant stress response pathway, overexpression of them in the plant has demonstrated capable to increase drought tolerance. By overexpression of these genes combining together with suppression of ESK1 gene, the significant increase of plant drought tolerance has been achieved in comparison to single gene manipulation, although the effect is not in an additive way. Accompanying the increase of drought tolerance via suppression of ESK1 gene expression, the negative effect has been observed in seeds yield of transgenic plants in normal watering conditions comparing with wide type plant. PMID:25184213

  3. 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

  4. Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression.

    PubMed

    Xu, Jian-zhong; Zhang, Wei-guo

    2016-02-01

    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

  5. Transcript Profile of Flowering Regulatory Genes in VcFT-Overexpressing Blueberry Plants.

    PubMed

    Walworth, Aaron E; Chai, Benli; Song, Guo-Qing

    2016-01-01

    In order to identify genetic components in flowering pathways of highbush blueberry (Vaccinium corymbosum L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora ('VcFT-Aurora'), which overexpresses a blueberry FLOWERING LOCUS T-like gene (VcFT). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in 'VcFT-Aurora'. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing VcFT. A blueberry CONSTANS-LIKE 5-like (VcCOL5) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, i.e., a MADS-AFFECTING FLOWERING 2-like gene (VcMAF2), a MADS-AFFECTING FLOWERING 5-like gene (VcMAF5), and a VERNALIZATION1-like gene (VcVRN1), may function as integrators in place of FLOWERING LOCUS C (FLC) in the vernalization pathway. Because no CONSTAN1-like or FLOWERING LOCUS C-like genes were found in blueberry, VcCOL5 and VcMAF2/VcMAF5 or VRN1 might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of VcFT, i.e., SUPPRESSOR of Overexpression of Constans 1-like (VcSOC1), LEAFY-like (VcLFY), APETALA1-like (VcAP1), CAULIFLOWER 1-like (VcCAL1), and FRUITFULL-like (VcFUL) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of VcFT promoted expression of all of these

  6. Transcript Profile of Flowering Regulatory Genes in VcFT-Overexpressing Blueberry Plants

    PubMed Central

    Walworth, Aaron E.; Chai, Benli; Song, Guo-qing

    2016-01-01

    In order to identify genetic components in flowering pathways of highbush blueberry (Vaccinium corymbosum L.), a transcriptome reference composed of 254,396 transcripts and 179,853 gene contigs was developed by assembly of 72.7 million reads using Trinity. Using this transcriptome reference and a query of flowering pathway genes of herbaceous plants, we identified potential flowering pathway genes/transcripts of blueberry. Transcriptome analysis of flowering pathway genes was then conducted on leaf tissue samples of transgenic blueberry cv. Aurora (‘VcFT-Aurora’), which overexpresses a blueberry FLOWERING LOCUS T-like gene (VcFT). Sixty-one blueberry transcripts of 40 genes showed high similarities to 33 known flowering-related genes of herbaceous plants, of which 17 down-regulated and 16 up-regulated genes were identified in ‘VcFT-Aurora’. All down-regulated genes encoded transcription factors/enzymes upstream in the signaling pathway containing VcFT. A blueberry CONSTANS-LIKE 5-like (VcCOL5) gene was down-regulated and associated with five other differentially expressed (DE) genes in the photoperiod-mediated flowering pathway. Three down-regulated genes, i.e., a MADS-AFFECTING FLOWERING 2-like gene (VcMAF2), a MADS-AFFECTING FLOWERING 5-like gene (VcMAF5), and a VERNALIZATION1-like gene (VcVRN1), may function as integrators in place of FLOWERING LOCUS C (FLC) in the vernalization pathway. Because no CONSTAN1-like or FLOWERING LOCUS C-like genes were found in blueberry, VcCOL5 and VcMAF2/VcMAF5 or VRN1 might be the major integrator(s) in the photoperiod- and vernalization-mediated flowering pathway, respectively. The major down-stream genes of VcFT, i.e., SUPPRESSOR of Overexpression of Constans 1-like (VcSOC1), LEAFY-like (VcLFY), APETALA1-like (VcAP1), CAULIFLOWER 1-like (VcCAL1), and FRUITFULL-like (VcFUL) genes were present and showed high similarity to their orthologues in herbaceous plants. Moreover, overexpression of VcFT promoted expression of all

  7. Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

    PubMed

    Mahajan, Ameya S; Kondhare, Kirtikumar R; Rajabhoj, Mohit P; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K

    2016-07-01

    Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

  8. Overexpression of a single Leishmania major gene enhances parasite infectivity in vivo and in vitro

    PubMed Central

    Reiling, Linda; Chrobak, Mareike; Schmetz, Christel; Clos, Joachim

    2010-01-01

    We identified a Leishmania major-specific gene that can partly compensate for the loss of virulence observed for L. major HSP100 null mutants. The gene, encoding a 46 kD protein of unknown function and lineage, also enhances the virulence of wild type L. major upon overexpression. Surprisingly, the approximately sixfold overexpression of this protein also extends the host range of L. major to normally resistant C57BL/6 mice, causing persisting lesions in this strain, even while eliciting a strong cellular immune response. This enhanced virulence in vivo is mirrored in vitro by increased parasite burden inside bone marrow-derived macrophages. The localization of the protein in the macrophage cytoplasm suggests that it may modulate the macrophage effector mechanisms. In summary, our data show that even minor changes of gene expression in L. major may alter the outcome of an infection, regardless of the host's genetic predisposition. PMID:20345655

  9. Overexpression of Actinidia deliciosa pyruvate decarboxylase 1 gene enhances waterlogging stress in transgenic Arabidopsis thaliana.

    PubMed

    Zhang, Ji-Yu; Huang, Sheng-Nan; Wang, Gang; Xuan, Ji-Ping; Guo, Zhong-Ren

    2016-09-01

    Ethanolic fermentation is classically associated with waterlogging tolerance when plant cells switch from respiration to anaerobic fermentation. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we cloned a full-length PDC cDNA sequence from kiwifruit, named AdPDC1. We determined the expression of the AdPDC1 gene in kiwifruit under different environmental stresses using qRT-PCR, and the results showed that the increase of AdPDC1 expression during waterlogging stress was much higher than that during salt, cold, heat and drought stresses. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis enhanced the resistance to waterlogging stress but could not enhance resistance to cold stress at five weeks old seedlings. Overexpression of kiwifruit AdPDC1 in transgenic Arabidopsis could not enhance resistance to NaCl and mannitol stresses at the stage of seed germination and in early seedlings. These results suggested that the kiwifruit AdPDC1 gene is required during waterlogging but might not be required during other environmental stresses. Expression of the AdPDC1 gene was down-regulated by abscisic acid (ABA) in kiwifruit, and overexpression of the AdPDC1 gene in Arabidopsis inhibited seed germination and root length under ABA treatment, indicating that ABA might negatively regulate the AdPDC1 gene under waterlogging stress. PMID:27191596

  10. Overexpression and lack of copy number variation in the BMI-1 gene in human glioma

    PubMed Central

    MADATHAN KANDY, SIBIN; ISHWARA BHAT, DHANANJAYA; CHOPPAVARAPU, LAVANYA; SUVATHA, ARATI; GHATI KASTURIRANGAN, CHETAN

    2015-01-01

    Malignant gliomas are neoplasms of the brain that are associated with a poor prognosis. The B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) gene is one of the major cancer stem cell factors responsible for treatment failure in glioma. In the present study, the DNA-RNA-protein alterations in the BMI-1 gene were assessed in 50 glioma samples. Copy number variations in the BMI-1 gene were analyzed using SYBR® Green quantitative polymerase chain reaction. Gene expression analysis was performed using a Taqman assay and protein quantitation was performed using western blotting. A comparative Ct analysis showed the absence of copy number variations in all glioma samples. BMI-1 mRNA expression was found to be overexpressed in 36 out of 50 samples (72.0%), and 37 out of 50 samples showed overexpression (74.0%) of BMI-1 protein; this was statistically significant when compared with non-glioma tissues. It was observed that the protein and RNA expression in glioma were concordant. In this study on the BMI-1 gene, transcription and translation in glioma were observed and BMI-1 overexpression was found to be a common phenomenon. PMID:26722333

  11. MYEOV gene overexpression in primary plasma cell leukemia with t(11;14)(q13;q32)

    PubMed Central

    Coccaro, Nicoletta; Tota, Giuseppina; Anelli, Luisa; Zagaria, Antonella; Casieri, Paola; Cellamare, Angelo; Minervini, Crescenzio Francesco; Minervini, Angela; Cumbo, Cosimo; Impera, Luciana; Brunetti, Claudia; Orsini, Paola; Parciante, Elisa; Mestice, Anna; Specchia, Giorgina; Albano, Francesco

    2016-01-01

    Primary plasma cell leukemia (pPCL) is an uncommon form of plasma cell dyscrasia, and the most aggressive of the human monoclonal gammopathies. The t(11;14)(q13;q32) rearrangement is the most common alteration in pPCL, promoting cyclin D1 (CCND1) gene overexpression caused by its juxtaposition with the immunoglobulin heavy locus chromosome region. The myeloma overexpressed (MYEOV) gene maps very close to the CCND1 gene on chromosome 11, but its overexpression is rarely observed in multiple myeloma. The present study describes a case of pPCL with t(11;14) characterized by a breakpoint on der(11), unlike the one usually observed. Droplet digital polymerase chain reaction analysis revealed overexpression of CCND1 and MYEOV. To the best of our knowledge, MYEOV gene overexpression has never been previously described in pPCL. PMID:27446453

  12. Interference in transcription of overexpressed genes by promoter-proximal downstream sequences.

    PubMed

    Turchinovich, A; Surowy, H M; Tonevitsky, A G; Burwinkel, B

    2016-01-01

    Despite a high sequence homology among four human RNAi-effectors Argonaute proteins and their coding sequences, the efficiency of ectopic overexpression of AGO3 and AGO4 coding sequences in human cells is greatly reduced as compared to AGO1 and AGO2. While investigating this phenomenon, we documented the existence of previously uncharacterized mechanism of gene expression regulation, which is manifested in greatly varying basal transcription levels from the RNApolII promoters depending on the promoter-proximal downstream sequences. Specifically, we show that distinct overexpression of Argonaute coding sequences cannot be explained by mRNA degradation in the cytoplasm or nucleus, and exhibits on transcriptional level. Furthermore, the first 1000-2000 nt located immediately downstream the promoter had the most critical influence on ectopic gene overexpression. The transcription inhibiting effect, associated with those downstream sequences, subsided with increasing distance to the promoter and positively correlated with promoter strength. We hypothesize that the same mechanism, which we named promoter proximal inhibition (PPI), could generally contribute to basal transcription levels of genes, and could be mainly responsible for the essence of difficult-to-express recombinant proteins. Finally, our data reveal that expression of recombinant proteins in human cells can be greatly enhanced by using more permissive promoter adjacent downstream sequences. PMID:27485701

  13. Interference in transcription of overexpressed genes by promoter-proximal downstream sequences

    PubMed Central

    Turchinovich, A.; Surowy, H. M.; Tonevitsky, A. G.; Burwinkel, B.

    2016-01-01

    Despite a high sequence homology among four human RNAi-effectors Argonaute proteins and their coding sequences, the efficiency of ectopic overexpression of AGO3 and AGO4 coding sequences in human cells is greatly reduced as compared to AGO1 and AGO2. While investigating this phenomenon, we documented the existence of previously uncharacterized mechanism of gene expression regulation, which is manifested in greatly varying basal transcription levels from the RNApolII promoters depending on the promoter-proximal downstream sequences. Specifically, we show that distinct overexpression of Argonaute coding sequences cannot be explained by mRNA degradation in the cytoplasm or nucleus, and exhibits on transcriptional level. Furthermore, the first 1000–2000 nt located immediately downstream the promoter had the most critical influence on ectopic gene overexpression. The transcription inhibiting effect, associated with those downstream sequences, subsided with increasing distance to the promoter and positively correlated with promoter strength. We hypothesize that the same mechanism, which we named promoter proximal inhibition (PPI), could generally contribute to basal transcription levels of genes, and could be mainly responsible for the essence of difficult-to-express recombinant proteins. Finally, our data reveal that expression of recombinant proteins in human cells can be greatly enhanced by using more permissive promoter adjacent downstream sequences. PMID:27485701

  14. Overexpression of a Triticum aestivum Calreticulin gene (TaCRT1) Improves Salinity Tolerance in Tobacco

    PubMed Central

    Song, Min; Wang, Yun; Xu, Wenqi; Wu, Lintao; Wang, Hancheng; Ma, Zhengqiang

    2015-01-01

    Calreticulin (CRT) is a highly conserved and abundant multifunctional protein that is encoded by a small gene family and is often associated with abiotic/biotic stress responses in plants. However, the roles played by this protein in salt stress responses in wheat (Triticum aestivum) remain obscure. In this study, three TaCRT genes were identified in wheat and named TaCRT1, TaCRT2 and TaCRT3-1 based on their sequence characteristics and their high homology to other known CRT genes. Quantitative real-time PCR expression data revealed that these three genes exhibit different expression patterns in different tissues and are strongly induced under salt stress in wheat. The calcium-binding properties of the purified recombinant TaCRT1 protein were determined using a PIPES/Arsenazo III analysis. TaCRT1 gene overexpression in Nicotiana tabacum decreased salt stress damage in transgenic tobacco plants. Physiological measurements indicated that transgenic tobacco plants showed higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than non-transgenic tobacco under normal growth conditions. Interestingly, overexpression of the entire TaCRT1 gene or of partial TaCRT1 segments resulted in significantly higher tolerance to salt stress in transgenic plants compared with their WT counterparts, thus revealing the essential role of the C-domain of TaCRT1 in countering salt stress in plants. PMID:26469859

  15. Field evaluation of apple overexpressing a peach CBF gene confirms its effect on cold hardiness, dormancy, and growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In recent years, the scientific literature has become replete with examples of the improvement of abiotic stress tolerance by overexpression of specific genes. Few studies, however, have evaluated transgenic plants under field conditions or the impact of overexpression on non-target traits. We pre...

  16. Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

    PubMed

    Sato, Yuko; Hliscs, Marion; Dunst, Josefine; Goosmann, Christian; Brinkmann, Volker; Montagna, Georgina N; Matuschewski, Kai

    2016-07-15

    Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping. PMID:27226484

  17. Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene.

    PubMed

    Folta, Adam; Bargsten, Joachim W; Bisseling, Ton; Nap, Jan-Peter; Mlynarova, Ludmila

    2016-02-01

    Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value. PMID:25974127

  18. Overexpression of UDP-glucose pyrophosphorylase gene could increase cellulose content in Jute (Corchorus capsularis L.).

    PubMed

    Zhang, Gaoyang; Qi, Jianmin; Xu, Jiantang; Niu, Xiaoping; Zhang, Yujia; Tao, Aifen; Zhang, Liwu; Fang, Pingping; Lin, Lihui

    2013-12-13

    In this study, the full-length cDNA of the UDP-glucose pyrophosphorylase gene was isolated from jute by homologous cloning (primers were designed according to the sequence of UGPase gene of other plants) and modified RACE techniques; the cloned gene was designated CcUGPase. Using bioinformatic analysis, the gene was identified as a member of the UGPase gene family. Real-time PCR analysis revealed differential spatial and temporal expression of the CcUGPase gene, with the highest expression levels at 40 and 120d. PCR and Southern hybridization results indicate that the gene was integrated into the jute genome. Overexpression of CcUGPase gene in jute revealed increased height and cellulose content compared with control lines, although the lignin content remained unchanged. The results indicate that the jute UGPase gene participates in cellulose biosynthesis. These data provide an important basis for the application of the CcUGPase gene in the improvement of jute fiber quality. PMID:24269810

  19. Overexpression of a Chimeric Gene, OsDST-SRDX, Improved Salt Tolerance of Perennial Ryegrass

    PubMed Central

    Cen, Huifang; Ye, Wenxing; Liu, Yanrong; Li, Dayong; Wang, Kexin; Zhang, Wanjun

    2016-01-01

    The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement. PMID:27251327

  20. Overexpression of a Chimeric Gene, OsDST-SRDX, Improved Salt Tolerance of Perennial Ryegrass.

    PubMed

    Cen, Huifang; Ye, Wenxing; Liu, Yanrong; Li, Dayong; Wang, Kexin; Zhang, Wanjun

    2016-01-01

    The Drought and Salt Tolerance gene (DST) encodes a C2H2 zinc finger transcription factor, which negatively regulates salt tolerance in rice (Oryza sativa). Phylogenetic analysis of six homologues of DST genes in different plant species revealed that DST genes were conserved evolutionarily. Here, the rice DST gene was linked to an SRDX domain for gene expression repression based on the Chimeric REpressor gene-Silencing Technology (CRES-T) to make a chimeric gene (OsDST-SRDX) construct and introduced into perennial ryegrass by Agrobacterium-mediated transformation. Integration and expression of the OsDST-SRDX in transgenic plants were tested by PCR and RT-PCR, respectively. Transgenic lines overexpressing the OsDST-SRDX fusion gene showed obvious phenotypic differences and clear resistance to salt-shock and to continuous salt stresses compared to non-transgenic plants. Physiological analyses including relative leaf water content, electrolyte leakage, proline content, malondialdehyde (MDA) content, H2O2 content and sodium and potassium accumulation indicated that the OsDST-SRDX fusion gene enhanced salt tolerance in transgenic perennial ryegrass by altering a wide range of physiological responses. To our best knowledge this study is the first report of utilizing Chimeric Repressor gene-Silencing Technology (CRES-T) in turfgrass and forage species for salt-tolerance improvement. PMID:27251327

  1. MMP-9 overexpression is associated with intragenic hypermethylation of MMP9 gene in melanoma

    PubMed Central

    Falzone, Luca; Salemi, Rossella; Travali, Salvatore; Scalisi, Aurora; McCubrey, James A.; Candido, Saverio; Libra, Massimo

    2016-01-01

    Tumor spreading is associated with the degradation of extracellular matrix proteins, mediated by the overexpression of matrix metalloproteinase 9 (MMP-9). Although, such overexpression was linked to epigenetic promoter methylation, the role of intragenic methylation was not clarified yet. Melanoma was used as tumor model to investigate the relationship between the DNA intragenic methylation of MMP9 gene and MMP-9 overexpression at transcriptional and protein levels. Computational analysis revealed DNA hypermethylation within the intragenic CpG-2 region of MMP9 gene in melanoma samples with high MMP-9 transcript levels. In vitro validation showed that CpG-2 hotspot region was hypermethylated in the A375 melanoma cell line with highest mRNA and protein levels of MMP-9, while low methylation levels were observed in the MEWO cell line where MMP-9 was undetectable. Concordant results were demonstrated in both A2058 and M14 cell lines. This correlation may give further insights on the role of MMP-9 upregulation in melanoma. PMID:27115178

  2. Overexpression of β-expansin gene GmEXPB2 improves phosphorus efficiency in soybean.

    PubMed

    Zhou, Jia; Xie, Jianna; Liao, Hong; Wang, Xiurong

    2014-02-01

    Soybean (Glycine max) is an important oil crop in agricultural production, but low phosphorus (P) availability limits soybean growth and production. Expansin is a family of plant cell wall proteins and involved in a variety of physiological processes, including cell division and enlargement, root growth and leaf development. To test the potential effects of expansins on crop production, we have developed soybean transgenic plants overexpressing a soybean β-expansin gene GmEXPB2, which was significantly induced by phosphate (Pi) starvation. The results indicated that constitutive overexpression of GmEXPB2 promoted leaf expansion, sequentially stimulated root growth and consequently resulted in improved P efficiency in the transgenic plants under P-limited conditions in hydroponics. In particular, when tested in calcareous (CS) and acid soils (AS), the two GmEXPB2 transgenic soybean lines showed above 25 and 40% increases in plant dry weight and P content, respectively to wild-type plants in low-P CS, but not in AS. To our knowledge, this is the first report in which improvement of P efficiency could be achieved through constitutive overexpression of an endogenous EXPB gene in soybean. These findings suggest that genetic modification of root and leaf traits might be a suitable strategy for improving crop production in low-P soils. PMID:23773128

  3. Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

    PubMed

    Toyokawa, Chihana; Shobu, Misaki; Tsukamoto, Rie; Okamura, Saki; Honda, Yoichi; Kamitsuji, Hisatoshi; Izumitsu, Kousuke; Suzuki, Kazumi; Irie, Toshikazu

    2016-09-01

    We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a β-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus. PMID:26979984

  4. Overexpression of the CmACS-3 gene in melon causes abnormal pollen development.

    PubMed

    Zhang, H; Luan, F

    2015-01-01

    Sexual diversity expressed by the Curcurbitaceae family is a primary example of developmental plasticity in plants. Most melon genotypes are andromonoecious, where an initial phase of male flowers is followed by a mixture of bisexual and male flowers. Over-expression of the CmACS-3 gene in melon plants showed an increased number of flower buds, and increased femaleness as demonstrated by a larger number bisexual buds. Transformation of CmACS-3 in melons showed earlier development of and an increased number of bisexual buds that matured to anthesis but also increased the rate of development of the bisexual buds to maturity. Field studies showed that CmACS-3-overexpressing melons had earlier mature bisexual flowers, earlier fruit set, and an increased number of fruits set on closely spaced nodes on the main stem. PMID:26400274

  5. Enhancing cellulase production by overexpression of xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain.

    PubMed

    Okuda, Naoyuki; Fujii, Tatsuya; Inoue, Hiroyuki; Ishikawa, Kazuhiko; Hoshino, Tamotsu

    2016-10-01

    We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression. PMID:27309759

  6. Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression.

    PubMed

    Zhang, Jie-Yu; Liu, Jin-Hui; Liu, Fa-Di; Xia, Yan-Hua; Wang, Jing; Liu, Xi; Zhang, Zhi-Qin; Zhu, Na; Yan-Yan; Ying, Ying; Huang, Xiao-Tian

    2014-10-01

    The increasing incidence of vulvovaginal candidiasis (VVC) and the emergence of fluconazole resistance are an indisputable fact. However, little information is available regarding the correlation between fluconazole resistance in vaginal Candida albicans and the expression of drug efflux pump genes. In this study, we investigated the species distribution, fluconazole susceptibility profiles and the mechanisms of fluconazole resistance in Candida strains. In total, 785 clinical Candida isolates were collected from patients with VVC. C. albicans was the most frequently isolated species(n = 529) followed by C. glabrata (n = 164) and C. krusei (n = 57). Of all Candida isolates, 4.7% were resistant to fluconazole. We randomly selected 18 fluconazole resistant isolates of C. albicans to evaluate the expression of CDR1, CDR2, MDR1 and FLU1 genes. Compared with fluconazole-susceptible C. albicans isolates, CDR1 gene expression displayed 3.16-fold relative increase, which was statistically significant. CDR2, MDR1 and FLU1 overexpression was observed in several fluconazole-resistant C. albicans isolates, but statistical significance was not achieved. These results demonstrate a high frequency of non-albicans species (32.6%); however, C. albicans is the most common Candida species implicated in vaginitis, and this strain displays considerable fluconazole resistance. Meanwhile, our study further indicates that fluconazole resistance in C. albicans may correlate with CDR1 gene overexpression. PMID:24962255

  7. Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells

    PubMed Central

    Mann, K; Kullberg, M

    2016-01-01

    We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150–250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core. PMID:27199219

  8. Overexpression of wheat ubiquitin gene, Ta-Ub2, improves abiotic stress tolerance of Brachypodium distachyon.

    PubMed

    Kang, Hanhan; Zhang, Meng; Zhou, Shumei; Guo, Qifang; Chen, Fengjuan; Wu, Jiajie; Wang, Wei

    2016-07-01

    Ubiquitination plays an important role in regulating plant's development and adaptability to abiotic stress. To investigate the possible functions of a wheat monoubiquitin gene Ta-Ub2 in abiotic stress in monocot and compare it with that in dicot, we generated transgenic Brachypodium plants overexpressing Ta-Ub2 under the control of CaMV35s and stress-inducible RD29A promoters. The constitutive expression of Ta-Ub2 displayed slight growth inhibition in the growth of transgenic Brachypodium distachyon under the control conditions. However, this inhibition was minimized by expression of Ta-Ub2 under the control of stress-inducible RD29A promoter. Compared with WT, the transgenic plants preserved more water and showed higher enzymatic antioxidants under drought stress, which might be related to the change in the expression of some antioxidant genes. The expression of C-repeat binding factors transcription factor genes in the transgenic B. distachyon lines were upregulated under water stress. Salt and cold tolerances of transgenic B. distachyon were also improved. Although the phenotypic changes in the transgenic plants were different, overexpression of Ta-Ub2 improved the abiotic stress tolerance in both dicot and monocot plants. The improvement in Ta-Ub2 transgenic plants in abiotic stress tolerance might be, at least partly, through regulating the gene expression and increasing the enzymatic antioxidants. PMID:27181952

  9. Trastuzumab-targeted gene delivery to Her2-overexpressing breast cancer cells.

    PubMed

    Mann, K; Kullberg, M

    2016-07-01

    We describe a novel gene delivery system that specifically targets human epidermal growth factor receptor 2 (Her2)-overexpressing breast cancer cells. The targeting complexes consist of a PEGylated polylysine core that is bound to DNA molecules coding for either green fluorescent protein or shrimp luciferase. The complex is disulfide linked to the monoclonal antibody trastuzumab and to a pore-forming protein, Listeriolysin O (LLO). Trastuzumab is responsible for specific targeting of Her2 receptors and uptake of the gene delivery complex into endosomes of recipient cells, whereas LLO ensures that the DNA molecules are capable of transit from the endosomes into the cytoplasm. Omission of either trastuzumab or LLO from the nanocomplexes results in minimal gene product in targeted cells. Treatment of isogeneic MCF7 and MCF7/Her18 cell lines, differing only in number of Her2 receptors, with the complete gene delivery system results in a 30-fold greater expression of luciferase activity in the Her2-overexpressing MCF7/Her18 cells. Our nanocomplexes are small (150-250 nm), stable to storage, nontoxic and generic in make-up such that any plasmid DNA or antibody specific for cell-surface receptors can be coupled to the PEGylated polylysine core. PMID:27199219

  10. Overexpression of a rice TIFY gene increases grain size through enhanced accumulation of carbohydrates in the stem.

    PubMed

    Hakata, Makoto; Kuroda, Masaharu; Ohsumi, Akihiro; Hirose, Tatsuro; Nakamura, Hidemitsu; Muramatsu, Masayuki; Ichikawa, Hiroaki; Yamakawa, Hiromoto

    2012-01-01

    Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene. PMID:23132589

  11. Overexpression of p53 mRNA in colorectal cancer and its relationship to p53 gene mutation.

    PubMed Central

    el-Mahdani, N.; Vaillant, J. C.; Guiguet, M.; Prévot, S.; Bertrand, V.; Bernard, C.; Parc, R.; Béréziat, G.; Hermelin, B.

    1997-01-01

    We analysed the frequency of p53 mRNA overexpression in a series of 109 primary colorectal carcinomas and its association with p53 gene mutation, which has been correlated with short survival. Sixty-nine of the 109 cases (63%) demonstrated p53 mRNA overexpression, without any correlation with stage or site of disease. Comparison with p53 gene mutation indicated that, besides cases in which p53 gene mutation and p53 mRNA overexpression were either both present (40 cases) or both absent (36 cases), there were also cases in which p53 mRNA was overexpressed in the absence of any mutation (29 cases) and those with a mutant gene in which the mRNA was not overexpressed (four cases). Moreover, the mutant p53 tumours exhibited an increase of p53 mRNA expression, which was significantly higher in tumours expressing the mutated allele alone than in tumours expressing both wild- and mutated-type alleles. These data (1) show that p53 mRNA overexpression is a frequent event in colorectal tumours and is not predictive of the status of the gene, i.e. whether or not a mutation is present; (2) provide further evidence that p53 protein overexpression does not only result from an increase in the half-life of mutated p53 and suggest that inactivation of the p53 function in colorectal cancers involves at least two distinct mechanisms, including p53 overexpression and/or mutation; and (3) suggest that p53 mRNA overexpression is an early event, since it is not correlated with Dukes stage. PMID:9052405

  12. The Novel Gene CRNDE Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

    PubMed Central

    Szafron, Lukasz Michal; Balcerak, Anna; Grzybowska, Ewa Anna; Pienkowska-Grela, Barbara; Felisiak-Golabek, Anna; Podgorska, Agnieszka; Kulesza, Magdalena; Nowak, Natalia; Pomorski, Pawel; Wysocki, Juliusz; Rubel, Tymon; Dansonka-Mieszkowska, Agnieszka; Konopka, Bozena; Lukasik, Martyna; Kupryjanczyk, Jolanta

    2015-01-01

    CRNDE, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the CRNDE gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two CRNDE transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of CRNDE. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our in silico results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the CRNDE gene. PMID:25978564

  13. Gene Amplification-Associated Overexpression of the RNA Editing Enzyme ADAR1 Enhances Human Lung Tumorigenesis

    PubMed Central

    Anadón, Carmen; Guil, Sonia; Simó-Riudalbas, Laia; Moutinho, Catia; Setien, Fernando; Martínez-Cardús, Anna; Moran, Sebastian; Villanueva, Alberto; Calaf, Monica; Vidal, August; Lazo, Pedro A.; Zondervan, Ilse; Savola, Suvi; Kohno, Takashi; Yokota, Jun; Ribas de Pouplana, Lluís; Esteller, Manel

    2015-01-01

    The introduction of new therapies against particular genetic mutations in non-small cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  14. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

    PubMed

    Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; de Pouplana, L R; Esteller, M

    2016-08-18

    The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  15. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    PubMed

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators. PMID:26552797

  16. Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker.

    PubMed

    Zhou, Shang-Hui; Yang, Wen-Jun; Liu, Sheng-Wen; Li, Jiang; Zhang, Chun-Ye; Zhu, Yun; Zhang, Chen-Ping

    2014-01-01

    Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to normal bone tissues, including 1,200 upregulated genes and 681 downregulated genes. Pathway analysis indicated that obviously activated pathways are Ribosome and ECM-receptor interaction pathways; downregulated pathways are "Hepatitis C" and "cancer" signaling pathways. We further validated the expression of ADAMTS2, one of most differentiated expressed genes, by Immunohistochemistry (IHC) in 40 of FD cases. Results showed that ADAMTS2 was significantly overexpressed in FD tissues, but rarely expressed in normal bone tissues, suggesting that ADAMTS2 could be a potential biomarker for FD. Thus, this study uncovered differentially expressed candidate genes in FD, which provides pilot data for understanding FD pathogenesis, and developing novel biomarkers for diagnosis and targeting of FD. PMID:25674217

  17. Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker

    PubMed Central

    Zhou, Shang-Hui; Yang, Wen-Jun; Liu, Sheng-Wen; Li, Jiang; Zhang, Chun-Ye; Zhu, Yun; Zhang, Chen-Ping

    2014-01-01

    Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to normal bone tissues, including 1,200 upregulated genes and 681 downregulated genes. Pathway analysis indicated that obviously activated pathways are Ribosome and ECM-receptor interaction pathways; downregulated pathways are “Hepatitis C” and “cancer” signaling pathways. We further validated the expression of ADAMTS2, one of most differentiated expressed genes, by Immunohistochemistry (IHC) in 40 of FD cases. Results showed that ADAMTS2 was significantly overexpressed in FD tissues, but rarely expressed in normal bone tissues, suggesting that ADAMTS2 could be a potential biomarker for FD. Thus, this study uncovered differentially expressed candidate genes in FD, which provides pilot data for understanding FD pathogenesis, and developing novel biomarkers for diagnosis and targeting of FD. PMID:25674217

  18. FHL1 Reduces Dystrophy in Transgenic Mice Overexpressing FSHD Muscular Dystrophy Region Gene 1 (FRG1)

    PubMed Central

    Feeney, Sandra J.; McGrath, Meagan J.; Sriratana, Absorn; Gehrig, Stefan M.; Lynch, Gordon S.; D’Arcy, Colleen E.; Price, John T.; McLean, Catriona A.; Tupler, Rossella; Mitchell, Christina A.

    2015-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1. PMID:25695429

  19. Overexpression of OsHMA3 enhances Cd tolerance and expression of Zn transporter genes in rice

    PubMed Central

    Sasaki, Akimasa; Yamaji, Naoki; Ma, Jian Feng

    2014-01-01

    As a member of the heavy metal ATPase (HMA) family, OsHMA3 is a tonoplast-localized transporter for Cd in the roots of rice (Oryza sativa). Overexpression of OsHMA3 selectively reduces Cd accumulation in the grain. Further characterization in the present study revealed that overexpression of OsHMA3 also enhances the tolerance to toxic Cd. The growth of both the roots and shoots was similar in the absence of Cd between an OsHMA3-overexpressed line and vector control, but the Cd-inhibited growth was significantly alleviated in the OsHMA3-overexpressed line. The overexpressed line showed higher Cd concentration in the roots, but lower Cd concentration in the shoots compared with the wild-type rice and vector control line, indicating that overexpression of OsHMA3 enhanced vacuolar sequestration of Cd in the roots. The Zn concentration in the roots of the OsHMA3-overexpressed line was constantly higher than that of vector control, but the Zn concentration in the shoots was similar between the overexpressed line and vector control. Five transporter genes belonging to the ZIP family were constitutively up-regulated in the OsHMA3-overexpressed line. These results suggest that shoot Zn level was maintained by up-regulating these genes involved in the Zn uptake/translocation. Taken together, overexpression of OsHMA3 is an efficient way to reduce Cd accumulation in the grain and to enhance Cd tolerance in rice. PMID:25151617

  20. Increase in ascorbate content of transgenic tobacco plants overexpressing the acerola (Malpighia glabra) phosphomannomutase gene.

    PubMed

    Badejo, Adebanjo A; Eltelib, Hani A; Fukunaga, Kazunari; Fujikawa, Yukichi; Esaka, Muneharu

    2009-02-01

    Phosphomannomutase (PMM; EC 5.4.2.8) catalyzes the interconversion of mannose-6-phosphate to mannose-1-phosphate in the Smirnoff-Wheeler pathway for the biosynthesis of l-ascorbic acid (AsA). We have cloned the PMM cDNA from acerola (Malpighia glabra), a plant containing an enormous amount of AsA. The AsA contents correlate with the PMM gene expression of the ripening fruits and leaves. The PMM activities in the leaves of acerola, tomato and Arabidopsis correlate with their respective AsA contents. Transgenic tobacco plants overexpressing the acerola PMM gene showed about a 2-fold increase in AsA contents compared with the wild type, with a corresponding correlation with the PMM transcript levels and activities. PMID:19122187

  1. Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli.

    PubMed

    Kirubakaran, S Isaac; Sakthivel, N

    2007-03-01

    Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover. PMID:17029984

  2. Human wings apart-like gene is specifically overexpressed in cervical cancer

    PubMed Central

    LU, XIAOQIN; CUI, JINQUAN; FU, MEIZHOU; WANG, WULIANG

    2016-01-01

    Cervical cancer is the third most commonly diagnosed cancer in women. The human wings apart-like (hWAPL) gene, which is 30,793 bp long and located on 10q23.2., is a human homologue of the WAPL gene in Drosophila melanogaster. hWAPL has the characteristics of an oncogene in uterine cervical cancer. The present study investigated the expression of the hWAPL gene in tissues, including 9 common cancers, consisting of cervical, gastric and lung cancers, liver, bladder, esophageal, endometrial, renal and rectal carcinomas, cervical intraepithelial neoplasia (CIN) and benign squamous epithelia. The immunohistochemical analysis was conducted using paraffin-embedded tissues obtained from 413 patients, consisting of 27 benign squamous epithelial tissue samples, and 47 cervical cancer, 30 cervical intraepithelial neoplasia (CIN)I, 33 CINII, 38 CINIII, 29 gastric cancer, 28 liver carcinoma, 26 bladder carcinoma, 35 esophageal carcinoma, 25 endometrial, 26 renal carcinoma, 36 rectal carcinoma and 33 lung cancer tissues. The expression of hWAPL mRNA was evaluated by reverse transcription-quantitative polymerase chain reaction in 8 benign squamous epithelia and 11 cervical cancer tissues. Compared to benign squamous epithelia and the 8 other cancers, hWAPL protein was significantly increased in cervical cancer (P<0.001). The expression of the hWAPL protein in cervical cancer and CINIII tissues was markedly increased compared to the expression in CINI and CINII tissues (P<0.001). Despite the significant difference in the staining scores (P<0.001), no significant difference was observed in the percentage of tissues expressing hWAPL (P=0.102) between cervical cancer and CINIII. The hWAPL gene may therefore be specifically overexpressed in cervical cancer. The overexpression of hWAPL may play an important role in occurrence and development of cervical cancer. PMID:27347120

  3. The over-expression of cell migratory genes in alveolar rhabdomyosarcoma could contribute to metastatic spread.

    PubMed

    Rapa, Elizabeth; Hill, Sophie K; Morten, Karl J; Potter, Michelle; Mitchell, Chris

    2012-06-01

    Alveolar (ARMS) and Embryonal (ERMS) rhabdomyosarcoma differ in their response to current treatments. The ARMS subtype has a less favourable prognosis and often presents with widespread metastases, while the less metastatic ERMS has a 5 year survival rate of more than 80 %. In this study we investigate gene expression differences that could contribute to the high frequency of metastasis in ARMS. Microarray analysis identified significant differences in DNA repair, cell cycle and cell migration between the two RMS subtypes. Two genes up regulated in ARMS and involved in cell migration; the engulfment and cell motility gene 1 (ELMO1) and NEL-like 1 gene (NELL1) were selected for further investigation. Over-expression of ELMO1 significantly increased cell invasion from 24.70 ± 7% to 93 ± 5.4% in primary myoblasts and from 29.43 ± 2.1% to 87.33 ± 4.1% in the ERMS cell line RD. siRNA knockout of ELMO1 in the ARMS cell line RH30 significantly reduced cell invasion from 88.2 ± 3.8% to 35.2 ± 2.5%. Over-expression of NELL1 significantly increased myoblast invasion from 23.6 ± 6.9% to 100 ± 0.1%, but had no effect on invasion of the ERMS cell line RD. These findings suggest that ELMO1 may play a key role in ARMS metastasis. NELL1 increased invasion in primary myoblasts, but other factors required for it to enhance motility were not present in the RD ERMS cell line. Impairing ELMO1 function by pharmacological or siRNA knockdown could be a highly effective approach to reduce the metastatic spread of RMS. PMID:22415709

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

    PubMed

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

    2016-07-01

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

  5. Tau Overexpression Impacts a Neuroinflammation Gene Expression Network Perturbed in Alzheimer’s Disease

    PubMed Central

    Wes, Paul D.; Easton, Amy; Corradi, John; Barten, Donna M.; Devidze, Nino; DeCarr, Lynn B.; Truong, Amy; He, Aiqing; Barrezueta, Nestor X.; Polson, Craig; Bourin, Clotilde; Flynn, Marianne E.; Keenan, Stefanie; Lidge, Regina; Meredith, Jere; Natale, Joanne; Sankaranarayanan, Sethu; Cadelina, Greg W.; Albright, Charlie F.; Cacace, Angela M.

    2014-01-01

    Filamentous inclusions of the microtubule-associated protein, tau, define a variety of neurodegenerative diseases known as tauopathies, including Alzheimer’s disease (AD). To better understand the role of tau-mediated effects on pathophysiology and global central nervous system function, we extensively characterized gene expression, pathology and behavior of the rTg4510 mouse model, which overexpresses a mutant form of human tau that causes Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). We found that the most predominantly altered gene expression pathways in rTg4510 mice were in inflammatory processes. These results closely matched the causal immune function and microglial gene-regulatory network recently identified in AD. We identified additional gene expression changes by laser microdissecting specific regions of the hippocampus, which highlighted alterations in neuronal network activity. Expression of inflammatory genes and markers of neuronal activity changed as a function of age in rTg4510 mice and coincided with behavioral deficits. Inflammatory changes were tau-dependent, as they were reversed by suppression of the tau transgene. Our results suggest that the alterations in microglial phenotypes that appear to contribute to the pathogenesis of Alzheimer’s disease may be driven by tau dysfunction, in addition to the direct effects of beta-amyloid. PMID:25153994

  6. Neuroplasticity changes during space flight.

    PubMed

    Slenzka, K

    2003-01-01

    Neuroplasticity refers to the ability of neurons to alter some functional property in response to alterations in input. Most of the inputs received by the brain and thus the neurons are coming from the overall sensory system. The lack of gravity during space flight or even the reduction of gravity during the planned Mars missions are and will change these inputs. The often observed "loop swimming" of some aquatic species is under discussion to be based on sensory input changes as well as the observed motion sickness of astronauts and cosmonauts. Several reports are published regarding these changes being based on alterations of general neurophysiological parameters. In this paper a summing-up of recent results obtained in the last years during space flight missions will be presented. Beside data obtained from astronauts and cosmonauts, main focus of this paper will be on animal model system data. PMID:12971415

  7. Glutamatergic neuroplasticity in cocaine addiction.

    PubMed

    Uys, Joachim D; Reissner, Kathryn J

    2011-01-01

    Neuroadaptations among glutamatergic projections within the mesocorticolimbic circuits engaged by drugs of abuse have been described since the 1990s. There is now substantial evidence that drugs of abuse lead to long-term changes in glutamatergic signaling and encompass multiple levels of analysis. For example, cocaine induces changes in extracellular glutamate concentrations and in synaptic glutamatergic transmission. In addition, glutamate receptors are required for the expression of cocaine-related behaviors, and long-term changes have been reported in the expression of proteins at glutamatergic synapses, in glutamate-related redox regulation of neurons, and in glutamatergic synaptic and structural plasticity following chronic exposure to cocaine. In this chapter, we will describe the neurocircuitry involved, and will summarize evidence for adaptations in glutamatergic neuroplasticity as a mechanism for cocaine addiction. Finally, we will discuss progress in the development of glutamate-mediated pharmacotherapies for the treatment of cocaine dependence. PMID:21199777

  8. Identification of Novel Genes Responsible for Overexpression of ampC in Pseudomonas aeruginosa PAO1

    PubMed Central

    Tsutsumi, Yuko; Tomita, Haruyoshi

    2013-01-01

    The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to β-lactams. The expression of genes reported to be involved in β-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR. PMID:24041903

  9. Overexpression of an F-box protein gene disrupts cotyledon vein patterning in Arabidopsis.

    PubMed

    Cui, Xianghuan; Xu, Xiaofeng; He, Yangyang; Du, Xiling; Zhu, Jian

    2016-05-01

    Plant vascular patterning is complex. However, the detailed molecular mechanism of vascular patterning is still unknown. In this study, FBXL, an Arabidopsis F-box motif gene, was isolated by using 3' rapid amplification of cDNA ends (RACE) technique. The gene contained a coding sequence of 1407 nucleotides coding 468 amino acid residues. Amino acid sequence analysis revealed that the gene encoded a protein harboring an F-box motif at the N terminus, an LRRs motif in the middle, and an FBD motif at the C terminus. FBXL promoter-β-glucuronidase (GUS) and 35S promoter-FBXL vectors were constructed and transformed into Arabidopsis thaliana to understand the function of the FBXL gene. GUS expression analysis indicated that FBXL was specifically expressed in the vascular tissues of the root, stem, leaf, and inflorescence. FBXL overexpression in Arabidopsis displayed an abnormal venation pattern in cotyledons. Furthermore, FBXL expression was not induced by exogenous auxin and its transcript accumulation did not overlap with the distribution of endogenous auxin. These results suggested that FBXL may be involved in cotyledon vein patterning via auxin-independent pathway. PMID:26901782

  10. Ibuprofen reverts antifungal resistance on Candida albicans showing overexpression of CDR genes.

    PubMed

    Ricardo, Elisabete; Costa-de-Oliveira, Sofia; Dias, Ana Silva; Guerra, José; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália

    2009-06-01

    Several mechanisms may be associated with Candida albicans resistance to azoles. Ibuprofen was described as being able to revert resistance related to efflux activity in Candida. The aim of this study was to uncover the molecular base of antifungal resistance in C. albicans clinical strains that could be reverted by ibuprofen. Sixty-two clinical isolates and five control strains of C. albicans were studied: the azole susceptibility phenotype was determined according to the Clinical Laboratory for Standards Institute, M27-A2 protocol and minimal inhibitory concentration values were recalculated with ibuprofen (100 microg mL(-1)); synergistic studies between fluconazole and FK506, a Cdr1p inhibitor, were performed using an agar disk diffusion assay and were compared with ibuprofen results. Gene expression was quantified by real-time PCR, with and without ibuprofen, regarding CDR1, CDR2, MDR1, encoding for efflux pumps, and ERG11, encoding for azole target protein. A correlation between susceptibility phenotype and resistance gene expression profiles was determined. Ibuprofen and FK506 showed a clear synergistic effect when combined with fluconazole. Resistant isolates reverting to susceptible after incubation with ibuprofen showed CDR1 and CDR2 overexpression especially of the latter. Conversely, strains that did not revert displayed a remarkable increase in ERG11 expression along with CDR genes. Ibuprofen did not alter resistance gene expression significantly (P>0.05), probably acting as a Cdrp blocker. PMID:19416368

  11. Diverse gene sequences are overexpressed in werner syndrome fibroblasts undergoing premature replicative senescence.

    PubMed Central

    Murano, S; Thweatt, R; Shmookler Reis, R J; Jones, R A; Moerman, E J; Goldstein, S

    1991-01-01

    Genes that play a role in the senescent arrest of cellular replication are likely to be overexpressed in human diploid fibroblasts (HDF) derived from subjects with Werner syndrome (WS) because these cells have a severely curtailed replicative life span. To identify some of these genes, a cDNA library was constructed from WS HDF after they had been serum depleted and repleted (5 days in medium containing 1% serum followed by 24 h in medium containing 20% serum). Differential screening of 7,500 colonies revealed 102 clones that hybridized preferentially with [32P]cDNA derived from RNA of WS cells compared with [32P]cDNA derived from normal HDF. Cross-hybridization and partial DNA sequence determination identified 18 independent gene sequences, 9 of them known and 9 unknown. The known genes included alpha 1(I) procollagen, alpha 2(I) procollagen, fibronectin, ferritin heavy chain, insulinlike growth factor-binding protein-3 (IGFBP-3), osteonectin, human tissue plasminogen activator inhibitor type I, thrombospondin, and alpha B-crystallin. The nine unknown clones included two novel gene sequences and seven additional sequences that contained both novel segments and the Alu class of repetitive short interspersed nuclear elements; five of these seven Alu+ clones also contained the long interpersed nuclear element I (KpnI) family of repetitive elements. Northern (RNA) analysis, using the 18 sequences as probes, showed higher levels of these mRNAs in WS HDF than in normal HDF. Five selected mRNAs studied in greater detail [alpha 1(I) procollagen, fibronectin, insulinlike growth factor-binding protein-3, WS3-10, and WS9-14] showed higher mRNA levels in both WS and late-passage normal HDF than in early-passage normal HDF at various intervals following serum depletion/repletion and after subculture and growth from sparse to high-density confluent arrest. These results indicate that senescence of both WS and normal HDF is accompanied by overexpression of similar sets of

  12. Improvement of exopolysaccharide production in Lactobacillus casei LC2W by overexpression of NADH oxidase gene.

    PubMed

    Li, Nan; Wang, Yuanlong; Zhu, Ping; Liu, Zhenmin; Guo, Benheng; Ren, Jing

    2015-02-01

    Lactobacillus casei LC2W is an exopolysaccharide (EPS)-producing strain with probiotic effects. To investigate the regulation mechanism of EPS biosynthesis and to improve EPS production through cofactor engineering, a H₂O-forming NADH oxidase gene was cloned from Streptococcus mutans and overexpressed in L. casei LC2W under the control of constitutive promoter P₂₃. The recombinant strain LC-nox exhibited 0.854 U/mL of NADH oxidase activity, which was elevated by almost 20-fold in comparison with that of wild-type strain. As a result, overexpression of NADH oxidase resulted in a reduction in growth rate. In addition, lactate production was decreased by 22% in recombinant strain. It was proposed that more carbon source was saved and used for the biosynthesis of EPS, the production of which was reached at 219.4 mg/L, increased by 46% compared to that of wild-type strain. This work provided a novel and convenient genetic approach to manipulate metabolic flux and to increase EPS production. To the best of our knowledge, this is the first report which correlates cofactor engineering with EPS production. PMID:25644955

  13. Overexpression of a Metarhizium robertsii HSP25 gene increases thermotolerance and survival in soil.

    PubMed

    Liao, Xinggang; Lu, Hsiao-Ling; Fang, Weiguo; St Leger, Raymond J

    2014-01-01

    Temperature extremes are an important adverse factor limiting the effectiveness of microbial pest control agents. They reduce virulence and persistence in the plant root-colonizing insect pathogen Metarhizium robertsii. Small heat shock proteins have been shown to confer thermotolerance in many organisms. In this study, we report on the cloning and characterization of a small heat shock protein gene hsp25 from M. robertsii. hsp25 expression was upregulated when the fungus was grown at extreme temperatures (4, 35, and 42 °C) or in the presence of oxidative or osmotic agents. Expression of hsp25 in Escherichia coli increased bacterial thermotolerance confirming that hsp25 encodes a functional heat shock protein. Overexpressing hsp25 in M. robertsii increased fungal growth under heat stress either in nutrient-rich medium or on locust wings and enhanced the tolerance of heat shock-treated conidia to osmotic stress. In addition, overexpression of hsp25 increased the persistence of M. robertsii in rhizospheric soils in outdoor microcosms, though it did not affect survival in bulk soil, indicating that M. robertsii's survival in soil is dependent on interactions with plant roots. PMID:24265026

  14. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato.

    PubMed

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K; Arias, Cintia L; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E; González, María de la Cruz; Hernández, Jose A; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-02-01

    Drought stress conditions modify source-sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of the

  15. Ectopic overexpression of the cell wall invertase gene CIN1 leads to dehydration avoidance in tomato

    PubMed Central

    Albacete, Alfonso; Cantero-Navarro, Elena; Großkinsky, Dominik K.; Arias, Cintia L.; Balibrea, María Encarnación; Bru, Roque; Fragner, Lena; Ghanem, Michel E.; González, María de la Cruz; Hernández, Jose A.; Martínez-Andújar, Cristina; van der Graaff, Eric; Weckwerth, Wolfram; Zellnig, Günther; Pérez-Alfocea, Francisco; Roitsch, Thomas

    2015-01-01

    Drought stress conditions modify source–sink relations, thereby influencing plant growth, adaptive responses, and consequently crop yield. Invertases are key metabolic enzymes regulating sink activity through the hydrolytic cleavage of sucrose into hexose monomers, thus playing a crucial role in plant growth and development. However, the physiological role of invertases during adaptation to abiotic stress conditions is not yet fully understood. Here it is shown that plant adaptation to drought stress can be markedly improved in tomato (Solanum lycopersicum L.) by overexpression of the cell wall invertase (cwInv) gene CIN1 from Chenopodium rubrum. CIN1 overexpression limited stomatal conductance under normal watering regimes, leading to reduced water consumption during the drought period, while photosynthetic activity was maintained. This caused a strong increase in water use efficiency (up to 50%), markedly improving water stress adaptation through an efficient physiological strategy of dehydration avoidance. Drought stress strongly reduced cwInv activity and induced its proteinaceous inhibitor in the leaves of the wild-type plants. However, the CIN1-overexpressing plants registered 3- to 6-fold higher cwInv activity in all analysed conditions. Surprisingly, the enhanced invertase activity did not result in increased hexose concentrations due to the activation of the metabolic carbohydrate fluxes, as reflected by the maintenance of the activity of key enzymes of primary metabolism and increased levels of sugar-phosphate intermediates under water deprivation. The induced sink metabolism in the leaves explained the maintenance of photosynthetic activity, delayed senescence, and increased source activity under drought stress. Moreover, CIN1 plants also presented a better control of production of reactive oxygen species and sustained membrane protection. Those metabolic changes conferred by CIN1 overexpression were accompanied by increases in the concentrations of

  16. Muscle cell atrophy induced by HSP gene silencing was counteracted by HSP overexpression

    NASA Astrophysics Data System (ADS)

    Choi, Inho; Lee, Joo-Hee; Nikawa, Takeshi; Gwag, Taesik; Park, Kyoungsook; Park, Junsoo

    Heat shock proteins (HSP), as molecular chaperones, are known to assist protein quality control under various stresses. Although overexpression of HSP70 was found to contribute to muscle size retention under an unloading condition, it remains largely unclarified whether muscle atrophy is induced by active suppression of HSP expression. In this study, we pre-treated Hsp70 siRNA to rat L6 cells for the HSP gene silencing, and determined myotube diameter, HSP72 expression and anabolic and catabolic signaling activities in the absence or presence of triterpene celastrol (CEL), the HSP70 inducer. Relative to a negative control (NC), muscle cell diameter was reduced 0.89-fold in the siRNA-treated group, increased 1.2-fold in the CEL-treated group and retained at the size of NC in the siRNA+CEL group. HSP72 expression was decreased 0.35-fold by siRNA whereas the level was increased 6- to 8-fold in the CEL and siRNA+CEL groups. Expression of FoxO3 and atrogin-1 was increased 1.8- to 4.8-fold by siRNA, which was abolished by CEL treatment. Finally, phosphorylation of Akt1, S6K and ERK1/2 was not affected by siRNA, but was elevated 2- to 6-fold in the CEL and siRNA+CEL groups. Taken together, HSP downregulation by Hsp gene silencing led to muscle cell atrophy principally via increases in catabolic activities and that such anti-atrophic effect was counteracted by HSP overexpression.

  17. Improved Salinity Tolerance in Carrizo Citrange Rootstock through Overexpression of Glyoxalase System Genes

    PubMed Central

    Alvarez-Gerding, Ximena; Cortés-Bullemore, Rowena; Medina, Consuelo; Romero-Romero, Jesús L.; Inostroza-Blancheteau, Claudio; Aquea, Felipe; Arce-Johnson, Patricio

    2015-01-01

    Citrus plants are widely cultivated around the world and, however, are one of the most salt stress sensitive crops. To improve salinity tolerance, transgenic Carrizo citrange rootstocks that overexpress glyoxalase I and glyoxalase II genes were obtained and their salt stress tolerance was evaluated. Molecular analysis showed high expression for both glyoxalase genes (BjGlyI and PgGlyII) in 5H03 and 5H04 lines. Under control conditions, transgenic and wild type plants presented normal morphology. In salinity treatments, the transgenic plants showed less yellowing, marginal burn in lower leaves and showed less than 40% of leaf damage compared with wild type plants. The transgenic plants showed a significant increase in the dry weight of shoot but there are no differences in the root and complete plant dry weight. In addition, a higher accumulation of chlorine is observed in the roots in transgenic line 5H03 but in shoot it was lower. Also, the wild type plant accumulated around 20% more chlorine in the shoot compared to roots. These results suggest that heterologous expression of glyoxalase system genes could enhance salt stress tolerance in Carrizo citrange rootstock and could be a good biotechnological approach to improve the abiotic stress tolerance in woody plant species. PMID:26236739

  18. Production of Dwarf Lettuce by Overexpressing a Pumpkin Gibberellin 20-Oxidase Gene

    PubMed Central

    Niki, Tomoya; Nishijima, Takaaki; Nakayama, Masayoshi; Hisamatsu, Tamotsu; Oyama-Okubo, Naomi; Yamazaki, Hiroko; Hedden, Peter; Lange, Theo; Mander, Lewis N.; Koshioka, Masaji

    2001-01-01

    We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2–35S-Ω). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T2 generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T2 generation, indicating that the transgene was stable and dominant. The endogenous levels of GA1 and GA4 were reduced in the dwarfs, whereas large amounts of GA17 and GA25, which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

  19. Production of dwarf lettuce by overexpressing a pumpkin gibberellin 20-oxidase gene.

    PubMed

    Niki, T; Nishijima, T; Nakayama, M; Hisamatsu, T; Oyama-Okubo, N; Yamazaki, H; Hedden, P; Lange, T; Mander, L N; Koshioka, M

    2001-07-01

    We investigated the effect of overexpressing a pumpkin gibberellin (GA) 20-oxidase gene encoding an enzyme that forms predominantly biologically inactive products on GA biosynthesis and plant morphology in transgenic lettuce (Lactuca sativa cv Vanguard) plants. Lettuce was transformed with the pumpkin GA 20-oxidase gene downstream of a strong constitutive promoter cassette (El2-35S-Omega). The transgenic plants in which the pumpkin gene was detected by polymerase chain reaction were dwarfed in the T(2) generation, whereas transformants with a normal growth phenotype did not contain the transgene. The result of Southern-blot analysis showed that the transgene was integrated as a single copy; the plants segregated three dwarfs to one normal in the T(2) generation, indicating that the transgene was stable and dominant. The endogenous levels of GA(1) and GA(4) were reduced in the dwarfs, whereas large amounts of GA(17) and GA(25), which are inactive products of the pumpkin GA 20-oxidase, accumulated in these lines. These results indicate that a functional pumpkin GA 20-oxidase is expressed in the transgenic lettuce, resulting in a diversion of the normal pathway of GA biosynthesis to inactive products. Furthermore, this technique may be useful for controlling plant stature in other agricultural and horticultural species. PMID:11457947

  20. Overexpression of ubiquitin-like LpHUB1 gene confers drought tolerance in perennial ryegrass.

    PubMed

    Patel, Minesh; Milla-Lewis, Susana; Zhang, Wanjun; Templeton, Kerry; Reynolds, William C; Richardson, Kim; Biswas, Margaret; Zuleta, Maria C; Dewey, Ralph E; Qu, Rongda; Sathish, Puthigae

    2015-06-01

    HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses. PMID:25487628

  1. Overexpression of FABP7 in Down syndrome fetal brains is associated with PKNOX1 gene-dosage imbalance.

    PubMed

    Sánchez-Font, Ma Francisca; Bosch-Comas, Anna; Gonzàlez-Duarte, Roser; Marfany, Gemma

    2003-06-01

    Suppression subtractive hybridization performed on Down syndrome (DS) fetal brains revealed a differentially expressed gene, FABP7, mapped to 6q22-23. FABP7 overexpression in DS brains was verified by real-time PCR (1.63-fold). To elucidate the molecular basis of FABP7 overexpression and establish the relationship with chromosome 21 trisomy, the FABP7 promoter was cloned by genomic inverse PCR. Comparison to the mouse ortholog revealed conservation of reported regulatory elements, among them a Pbx/POU binding site, known to be the target of PBX heteromeric complexes. PBX partners include homeobox-containing proteins, such as PKNOX1 (PREP1), a transcription factor mapping at 21q22.3. We report here: (i) overexpression of PKNOX1 in DS fetal brains; (ii) in vitro specific binding of PKNOX1 to the Pbx/POU site of the FABP7 promoter; (iii) in vivo FABP7 promoter trans-activation in cultured neuroblastoma cells caused by PKNOX1 overexpression. To our knowledge this is the first report of a direct relation between dosage imbalance of a chromosome 21 gene and altered expression of a downstream gene mapping on another chromosome. Given the role of FABP7 in the establishment, development and maintenance of the CNS, we suggest that the overexpression of FABP7 could contribute to DS-associated neurological disorders. PMID:12771203

  2. Simulating pancreatic neuroplasticity: in vitro dual-neuron plasticity assay.

    PubMed

    Demir, Ihsan Ekin; Tieftrunk, Elke; Schäfer, Karl-Herbert; Friess, Helmut; Ceyhan, Güralp O

    2014-01-01

    Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ. PMID:24797813

  3. rre37 Overexpression Alters Gene Expression Related to the Tricarboxylic Acid Cycle and Pyruvate Metabolism in Synechocystis sp. PCC 6803

    PubMed Central

    Iijima, Hiroko; Watanabe, Atsuko; Takanobu, Junko; Hirai, Masami Yokota; Osanai, Takashi

    2014-01-01

    The tricarboxylic acid (TCA) cycle and pyruvate metabolism of cyanobacteria are unique and important from the perspectives of biology and biotechnology research. Rre37, a response regulator induced by nitrogen depletion, activates gene expression related to sugar catabolism. Our previous microarray analysis has suggested that Rre37 controls the transcription of genes involved in sugar catabolism, pyruvate metabolism, and the TCA cycle. In this study, quantitative real-time PCR was used to measure the transcript levels of 12 TCA cycle genes and 13 pyruvate metabolism genes. The transcripts of 6 genes (acnB, icd, ppc, pyk1, me, and pta) increased after 4 h of nitrogen depletion in the wild-type GT strain but the induction was abolished by rre37 overexpression. The repression of gene expression of fumC, ddh, and ackA caused by nitrogen depletion was abolished by rre37 overexpression. The expression of me was differently affected by rre37 overexpression, compared to the other 24 genes. These results indicate that Rre37 differently controls the genes of the TCA cycle and pyruvate metabolism, implying the key reaction of the primary in this unicellular cyanobacterium. PMID:25614900

  4. Overexpression of c-myc in diabetic mice restores altered expression of the transcription factor genes that regulate liver metabolism.

    PubMed Central

    Riu, Efren; Ferre, Tura; Mas, Alex; Hidalgo, Antonio; Franckhauser, Sylvie; Bosch, Fatima

    2002-01-01

    Overexpression of the c-Myc transcription factor in liver induces glucose uptake and utilization. Here we examined the effects of c- myc overexpression on the expression of hepatocyte-specific transcription factor genes which regulate the expression of genes controlling hepatic metabolism. At 4 months after streptozotocin (STZ) treatment, most diabetic control mice were highly hyperglycaemic and died, whereas in STZ-treated transgenic mice hyperglycaemia was markedly lower, the serum levels of beta-hydroxybutyrate, triacylglycerols and non-esterified fatty acids were normal, and they had greater viability in the absence of insulin. Furthermore, long-term STZ-treated transgenic mice showed similar glucose utilization and storage to healthy controls. This was consistent with the expression of glycolytic genes becoming normalized. In addition, restoration of gene expression of the transcription factor, sterol receptor element binding protein 1c, was observed in the livers of these transgenic mice. Further, in STZ-treated transgenic mice the expression of genes involved in the control of gluconeogenesis (phosphoenolpyruvate carbokykinase), ketogenesis (3-hydroxy-3-methylglutaryl-CoA synthase) and energy metabolism (uncoupling protein 2) had returned to normal. These findings were correlated with decreased expression of genes encoding the transcription factors hepatocyte nuclear factor 3gamma, peroxisome proliferator-activated receptor alpha and retinoid X receptor. These results indicate that c- myc overexpression may counteract diabetic changes by controlling hepatic glucose metabolism, both directly by altering the expression of metabolic genes and through the expression of key transcription factor genes. PMID:12230428

  5. Synonymous mutation gene design to overexpress ACCase in creeping bentgrass to obtain resistance to ACCase-inhibiting herbicides.

    PubMed

    Heckart, Douglas L; Schwartz, Brian M; Raymer, Paul L; Parrott, Wayne A

    2016-08-01

    Overexpression of a native gene can cause expression of both introduced and native genes to be silenced by posttranscriptional gene silencing (PTGS) mechanisms. PTGS mechanisms rely on sequence identity between the transgene and native genes; therefore, designing genes with mutations that do not cause amino acid changes, known as synonymous mutations, may avoid PTGS. For proof of concept, the sequence of acetyl-coA carboxylase (ACCase) from creeping bentgrass (Agrostis stolonifera L.) was altered with synonymous mutations. A native bentgrass ACCase was cloned and used as a template for the modified gene. Wild-type (WT) and modified genes were further modified with a non-synonymous mutation, coding for an isoleucine to leucine substitution at position 1781, known to confer resistance to ACCase-inhibiting herbicides. Five-hundred calli of creeping bentgrass 'Penn A-4' were inoculated with Agrobacterium containing either the WT or modified genes, with or without the herbicide-resistance mutation. Six herbicide-resistant-transgenic events containing the modified gene with the 1781 mutation were obtained. Transcription of the modified ACCase was confirmed in transgenic plants, showing that gene-silencing mechanisms were avoided. Transgenic plants were confirmed to be resistant to the ACCase-inhibiting herbicide, sethoxydim, providing evidence that the modified gene was functional. The result is a novel herbicide-resistance trait and shows that overexpression of a native enzyme with a gene designed with synonymous mutations is possible. PMID:27116460

  6. Overexpression of acetylcholinesterase gene in rice results in enhancement of shoot gravitropism.

    PubMed

    Yamamoto, Kosuke; Shida, Satoshi; Honda, Yoshihiro; Shono, Mariko; Miyake, Hiroshi; Oguri, Suguru; Sakamoto, Hikaru; Momonoki, Yoshie S

    2015-09-25

    Acetylcholine (ACh), a known neurotransmitter in animals and acetylcholinesterase (AChE) exists widely in plants, although its role in plant signal transduction is unclear. We previously reported AChE in Zea mays L. might be related to gravitropism based on pharmacological study using an AChE inhibitor. Here we clearly demonstrate plant AChE play an important role as a positive regulator in the gravity response of plants based on a genetic study. First, the gene encoding a second component of the ACh-mediated signal transduction system, AChE was cloned from rice, Oryza sativa L. ssp. Japonica cv. Nipponbare. The rice AChE shared high homology with maize, siratro and Salicornia AChEs. Similar to animal and other plant AChEs, the rice AChE hydrolyzed acetylthiocholine and propionylthiocholine, but not butyrylthiocholine. Thus, the rice AChE might be characterized as an AChE (E.C.3.1.1.7). Similar to maize and siratro AChEs, the rice AChE exhibited low sensitivity to the AChE inhibitor, neostigmine bromide, compared with the electric eel AChE. Next, the functionality of rice AChE was proved by overexpression in rice plants. The rice AChE was localized in extracellular spaces of rice plants. Further, the rice AChE mRNA and its activity were mainly detected during early developmental stages (2 d-10 d after sowing). Finally, by comparing AChE up-regulated plants with wild-type, we found that AChE overexpression causes an enhanced gravitropic response. This result clearly suggests that the function of the rice AChE relate to positive regulation of gravitropic response in rice seedlings. PMID:26277389

  7. Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato.

    PubMed

    Yu, Chao; Wang, Hua-Sen; Yang, Sha; Tang, Xian-Feng; Duan, Ming; Meng, Qing-Wei

    2009-01-01

    An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 degrees C), but inhibited by high temperature (40 degrees C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F(v)/F(m)) and the O(2) evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress. PMID:19648018

  8. Overexpression of bacterial mtlD gene in peanut improves drought tolerance through accumulation of mannitol.

    PubMed

    Bhauso, Tengale Dipak; Radhakrishnan, Thankappan; Kumar, Abhay; Mishra, Gyan Prakash; Dobaria, Jentilal Ramjibhai; Patel, Kirankumar; Rajam, Manchikatla Venkat

    2014-01-01

    In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated. PMID:25436223

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

    PubMed

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

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. PMID:23384757

  10. Overexpression of Bacterial mtlD Gene in Peanut Improves Drought Tolerance through Accumulation of Mannitol

    PubMed Central

    Bhauso, Tengale Dipak; Radhakrishnan, Thankappan; Kumar, Abhay; Mishra, Gyan Prakash; Dobaria, Jentilal Ramjibhai; Patel, Kirankumar; Rajam, Manchikatla Venkat

    2014-01-01

    In the changing global environmental scenarios, water scarcity and recurrent drought impose huge reductions to the peanut (Arachis hypogaea L.) crop yield. In plants, osmotic adjustments associated with efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms. Mannitol, a compatible solute, is known to scavenge hydroxyl radicals generated during various abiotic stresses, thereby conferring tolerance to water-deficit stress in many plant species. However, peanut plant is not known to synthesize mannitol. Therefore, bacterial mtlD gene coding for mannitol 1-phosphate dehydrogenase under the control of constitutive promoter CaMV35S was introduced and overexpressed in the peanut cv. GG 20 using Agrobacterium tumefaciens-mediated transformation. A total of eight independent transgenic events were confirmed at molecular level by PCR, Southern blotting, and RT-PCR. Transgenic lines had increased amount of mannitol and exhibited enhanced tolerance in response to water-deficit stress. Improved performance of the mtlD transgenics was indicated by excised-leaf water loss assay and relative water content under water-deficit stress. Better performance of transgenics was due to the ability of the plants to synthesize mannitol. However, regulation of mtlD gene expression in transgenic plants remains to be elucidated. PMID:25436223

  11. Overexpression of PaFT gene in the wild orchid Phalaenopsis amabilis (L.) Blume

    NASA Astrophysics Data System (ADS)

    Semiarti, Endang; Mercuriani, Ixora S.; Rizal, Rinaldi; Slamet, Agus; Utami, Bekti S.; Bestari, Ida A.; Aziz-Purwantoro, Moeljopawiro, S.; Jang, Soenghoe; Machida, Y.; Machida, C.

    2015-09-01

    To shorten vegetative stage and induce transition from vegetative to reproductive stage in orchids, we overexpressed Phalaenopsis amabilis Flowering LocusT (PaFT) gene under the control of Ubiquitin promoter into protocorm of Indonesian Wild Orchid Phalaenopsis amabilis (L.) Blume. The dynamic expression of vegetative gene Phalaenopsis Homeobox1 (POH1) and flowering time gene PaFT has been analyzed. Accumulation of mRNA was detected in shoot and leaves of both transgenic and non transgenic plants by using Reverse transcriptase-PCR (RT-PCR) with specific gene primers for POH1 and PaFT in 24 months old plants. To analyze the POH1 and PaFT genes, three pairs of degenerate primers PaFT degF1R1, F2R2 and F3R3 that amplified 531 bp PaFT cDNA were used. We detected 700 bp PaFTcDNA from leaves and shoots of transgenic plants, but not in NT plants. POH1 mRNA was detected in plants. PaFT protein consists of Phospatidyl Ethanolamine-Binding Protein (PEBP) in interval base 73-483 and CETS family protein at base 7-519, which are important motif for transmembrane protein. We inserted Ubipro::PaFT/pGAS101 into P. amabilis protocorm using Agrobacterium. Analysis of transgenic plants showed that PaFTmRNA was accumulated in leaves of 12 months after sowing, although it is not detected in non transgeic plants. Compare to the wild type (NT plants), ectopic expression of PaFT shows alter phenotype as follows: 31% normal, 19% with short-wavy leaves, 5% form rosette leaves and 45% produced multishoots. Analysis of protein profiles of trasgenic plants showed that a putative PaFT protein (MW 19,7 kDa) was produced in 1eaves and shoots.This means that at 12 months, POH1 gene expression gradually decreased/negatively regulated, the expression of PaFT gene was activated, although there is no flower initiation yet. Some environmental factors might play a role to induce inflorescens. This experiment is in progress.

  12. Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

    SciTech Connect

    Kaisho, Yoshihiko . E-mail: Kaisho_Yoshihiko@takeda.co.jp; Watanabe, Takuya; Nakata, Mitsugu; Yano, Takashi; Yasuhara, Yoshitaka; Shimakawa, Kozo; Mori, Ikuo; Sakura, Yasufumi; Terao, Yasuko; Matsui, Hideki; Taketomi, Shigehisa

    2005-05-13

    The human MrgX3 gene, belonging to the mrgs/SNSRs (mass related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.

  13. Immunohistochemical Determination of p53 Protein Overexpression for Predicting p53 Gene Mutations in Hepatocellular Carcinoma: A Meta-Analysis

    PubMed Central

    Deng, Miao; Liu, Dechun; Ma, Qingyong; Feng, Xiaoshan

    2016-01-01

    Background Whether increased expression of the tumor suppressor protein p53 indicates a p53 gene mutation in hepatocellular carcinoma (HCC) remains unclear. We conducted a meta-analysis to determine whether p53 protein overexpression detected by immunohistochemistry (IHC) offers a diagnostic prediction for p53 gene mutations in HCC patients. Methods Systematic literature searches were conducted with an end date of December 2015. A meta-analysis was performed to estimate the diagnostic accuracy of IHC-determined p53 protein overexpression in the prediction of p53 gene mutations in HCC. Sensitivity, subgroup, and publication bias analyses were also conducted. Results Thirty-six studies were included in the meta-analysis. The results showed that the overall sensitivity and specificity for IHC-determined p53 overexpression in the diagnostic prediction of p53 mutations in HCC were 0.83 (95% CI: 0.80–0.86) and 0.74 (95% CI: 0.71–0.76), respectively. The summary positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 2.65 (95% CI: 2.21–3.18) and 0.36 (95% CI: 0.26–0.50), respectively. The diagnostic odds ratio (DOR) of IHC-determined p53 overexpression in predicting p53 mutations ranged from 0.56 to 105.00 (pooled, 9.77; 95% CI: 6.35–15.02), with significant heterogeneity between the included studies (I2 = 40.7%, P = 0.0067). Moreover, subgroup and sensitivity analyses did not alter the results of the meta-analysis. However, potential publication bias was present in the current meta-analysis. Conclusion The upregulation of the tumor suppressor protein p53 was indeed linked to p53 gene mutations. IHC determination of p53 overexpression can predict p53 gene mutations in HCC patients. PMID:27428001

  14. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The C-repeat Binding Factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a t...

  15. Overexpression of the AtSHI Gene in Poinsettia, Euphorbia pulcherrima, Results in Compact Plants

    PubMed Central

    Islam, M. Ashraful; Lütken, Henrik; Haugslien, Sissel; Blystad, Dag-Ragnar; Torre, Sissel; Rolcik, Jakub; Rasmussen, Søren K.; Olsen, Jorunn E.; Clarke, Jihong Liu

    2013-01-01

    Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21–52%) and internode lengths (31–49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11–31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced. PMID:23308204

  16. Overexpression of the AtSHI gene in poinsettia, Euphorbia pulcherrima, results in compact plants.

    PubMed

    Islam, M Ashraful; Lütken, Henrik; Haugslien, Sissel; Blystad, Dag-Ragnar; Torre, Sissel; Rolcik, Jakub; Rasmussen, Søren K; Olsen, Jorunn E; Clarke, Jihong Liu

    2013-01-01

    Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21-52%) and internode lengths (31-49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11-31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced. PMID:23308204

  17. Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana

    PubMed Central

    Visscher, Anne M.; Belfield, Eric J.; Vlad, Daniela; Irani, Niloufer; Moore, Ian; Harberd, Nicholas P.

    2015-01-01

    A subset of genes in Arabidopsis thaliana is known to be up-regulated in response to a wide range of different environmental stress factors. However, not all of these genes are characterized as yet with respect to their functions. In this study, we used transgenic knockout, overexpression and reporter gene approaches to try to elucidate the biological roles of five unknown multiple-stress responsive genes in Arabidopsis. The selected genes have the following locus identifiers: At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010. Firstly, T-DNA insertion knockout lines were identified for each locus and screened for altered phenotypes. None of the lines were found to be visually different from wildtype Col-0. Secondly, 35S-driven overexpression lines were generated for each open reading frame. Analysis of these transgenic lines showed altered phenotypes for lines overexpressing the At1g74450 ORF. Plants overexpressing the multiple-stress responsive gene At1g74450 are stunted in height and have reduced male fertility. Alexander staining of anthers from flowers at developmental stage 12–13 showed either an absence or a reduction in viable pollen compared to wildtype Col-0 and At1g74450 knockout lines. Interestingly, the effects of stress on crop productivity are most severe at developmental stages such as male gametophyte development. However, the molecular factors and regulatory networks underlying environmental stress-induced male gametophytic alterations are still largely unknown. Our results indicate that the At1g74450 gene provides a potential link between multiple environmental stresses, plant height and pollen development. In addition, ruthenium red staining analysis showed that At1g74450 may affect the composition of the inner seed coat mucilage layer. Finally, C-terminal GFP fusion proteins for At1g74450 were shown to localise to the cytosol. PMID:26485022

  18. Over-Expression of SlSHN1 Gene Improves Drought Tolerance by Increasing Cuticular Wax Accumulation in Tomato

    PubMed Central

    Al-Abdallat, Ayed M.; Al-Debei, Hmoud S.; Ayad, Jamal Y.; Hasan, Shireen

    2014-01-01

    Increasing cuticular wax accumulation in plants has been associated with improving drought tolerance in plants. In this study, a cDNA clone encoding the SlSHN1 transcription factor, the closest ortholog to WIN/SHN1 gene in Arabidopsis, was isolated from tomato plant. Expression analysis of SlSHN1 indicated that it is induced in response to drought conditions. The over-expression of SlSHN1 in tomato under the control of the constitutive CaMV 35S promoter produced plants that showed mild growth retardation phenotype with shiny and dark green leaves. Scanning electron microscopy showed that the over-expression of SlSHN1 in tomato resulted in higher cuticular wax deposition on leaf epidermial tissue when compared to non-transformed plants. Expression analysis in transgenic lines over-expressing SlSHN1 indicated that several wax-related synthesis genes were induced. Transgenic tomato plants over-expressing SlSHN1 showed higher drought tolerance when compared with wild type plants; this was reflected in delayed wilting of transgenic lines, improved water status and reduced water loss rate when compared with wild type plants. In conclusion, the SlSHN1 gene can modulate wax accumulation and could be utilized to enhance drought tolerance in tomato plant. PMID:25350113

  19. Overexpression of a wheat phospholipase D gene, TaPLDα, enhances tolerance to drought and osmotic stress in Arabidopsis thaliana.

    PubMed

    Wang, Junbin; Ding, Bo; Guo, Yaolin; Li, Ming; Chen, Shuaijun; Huang, Guozhong; Xie, Xiaodong

    2014-07-01

    Phospholipase D (PLD) is crucial for plant responses to stress and signal transduction, however, the regulatory mechanism of PLD in abiotic stress is not completely understood; especially, in crops. In this study, we isolated a gene, TaPLDα, from common wheat (Triticum aestivum L.). Analysis of the amino acid sequence of TaPLDα revealed a highly conserved C2 domain and two characteristic HKD motifs, which is similar to other known PLD family genes. Further characterization revealed that TaPLDα expressed differentially in various organs, such as roots, stems, leaves and spikelets of wheat. After treatment with abscisic acid (ABA), methyl jasmonate, dehydration, polyethylene glycol and NaCl, the expression of TaPLDα was up-regulated in shoots. Subsequently, we generated TaPLDα-overexpressing transgenic Arabidopsis lines under the control of the dexamethasone-inducible 35S promoter. The overexpression of TaPLDα in Arabidopsis resulted in significantly enhanced tolerance to drought, as shown by reduced chlorosis and leaf water loss, higher relative water content and lower relative electrolyte leakage than the wild type. Moreover, the TaPLDα-overexpressing plants exhibited longer roots in response to mannitol treatment. In addition, the seeds of TaPLDα-overexpressing plants showed hypersensitivity to ABA and osmotic stress. Under dehydration, the expression of several stress-related genes, RD29A, RD29B, KIN1 and RAB18, was up-regulated to a higher level in TaPLDα-overexpressing plants than in wild type. Taken together, our results indicated that TaPLDα can enhance tolerance to drought and osmotic stress in Arabidopsis and represents a potential candidate gene to enhance stress tolerance in crops. PMID:24705986

  20. Post-thymic T cell lymphomas frequently overexpress p53 protein but infrequently exhibit p53 gene mutations.

    PubMed Central

    Matsushima, A. Y.; Cesarman, E.; Chadburn, A.; Knowles, D. M.

    1994-01-01

    We recently demonstrated that only one of 36 T-cell neoplasms contained p53 gene mutations. Although p53 gene mutations are known to result in overexpression of the p53 gene product, we also recently discovered that p53 protein overexpression does not correlate with p53 gene mutations, but does correlate with proliferation (r = 0.92), in anaplastic large cell lymphoma. In view of these findings, we investigated 34 non-human T-cell lymphotropic virus type I (HTLV-I) related postthymic T-cell lymphomas immunohistochemically for p53 protein, using monoclonal antibody 1801, and for proliferation, using monoclonal antibody Ki-67, and quantitated the results with the CAS-200 computerized image analysis system. We evaluated the presence of mutations in conserved exons 5 to 9 of the p53 gene using single-strand conformation polymorphism analysis and DNA sequencing. p53 mutations were detected in three of 34 cases, including two that contained deletions. p53 protein overexpression was detected in 17 of 34 cases, including the three mutated cases, with reactivities ranging from 10% to 48%. However, many cases in which a structural alteration could not be detected demonstrated levels of p53 protein expression comparable to those cases that were mutated. Correlation of p53 protein expression and proliferation, as assessed by Ki-67 expression, in this group of lymphomas was poor (r = 0.34). Whether alternative mechanisms of p53 protein inactivation are causing phenotypic overexpression of the p53 protein in these malignant lymphomas is unknown, although preliminary studies do not support a major role for such mechanisms. Therefore, the etiology and the significance of p53 protein overexpression in the cases that lack a demonstrable mutation is unclear. Nevertheless, as in anaplastic large cell lymphoma, overexpression of the p53 gene product is not a reliable predictor of the presence of mutations in conserved portions of the p53 gene in non-HTLV-I associated post-thymic T

  1. Construction of overexpression vectors of Magnaporthe oryzae genes BAS1 and BAS4 fusion to mCherry and screening of overexpression strains.

    PubMed

    Liang, M L; Yan, J L; Yang, Y Q; Liu, L; Li, C Y; Yang, J

    2015-01-01

    The aim of this study was to construct overexpression vectors and selecting strains of the Magnaporthe oryzae effectors BAS1 and BAS4. Primer pairs of BAS1, BAS4, and mCherry were designed based on their known nucleotide sequences. The coding sequences of BAS1 and BAS4 were amplified, and the pXY201 plasmid was selected as a template to amplify the mCherry sequence. Fragments of BAS1 and mCherry, and BAS4 and mCherry were ligated into the pCAMBIA1302 vector. The recombinant pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into E. coli DH5α competent cells. Transformants were screened by PCR, and plasmids from the positive transformants were extracted by enzymatic digestion to obtain pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry. The pCAMBIA-BAS1-mCherry and pCAMBIA-BAS4-mCherry plasmids were transformed into protoplasts of rice blast strains and the transformed strains were screened by PCR using primer pairs against the hygromycin gene. The result showed that the PCR products corresponded with the theoretical sizes. RT-PCR was used to analyze the expression of BAS1 and BAS4 in five transformed strains of BAS1 and BAS4, and the result showed that the higher expression level of the two genes was occurred in five transformant strains comparing to wild-type strain A3467-40 (the strain containing BAS1 and BAS4), but there was no difference among the five overexpression strains. The sporulation and spore germination of transformed strains was higher than in wild type strain, and there was no difference in the germination time. Construction of overexpression vectors and strains of M. oryzae effectors BAS1 and BAS4 provide reference material for other new effectors. PMID:26125917

  2. Enhancing Corynebacterium glutamicum robustness by over-expressing a gene, mshA, for mycothiol glycosyltransferase.

    PubMed

    Liu, Ying-Bao; Chen, Can; Chaudhry, Muhammad Tausif; Si, Mei-Ru; Zhang, Lei; Wang, Yao; Shen, Xi-Hui

    2014-07-01

    Over-expression of the gene, mshA, coding for mycothiol glycosyl transferase improved the robustness of Corynebacterium glutamicum to various stresses. Intracellular mycothiol (MSH) content was increased by 114 % in WT(pXMJ19-mshA) compared to WT(pXMJ19). Survival rates increased by 44, 39, 90, 77, 131, 87, 52, 47, 57, 85 and 33 % as compared to WT(pXMJ19) under stress by H2O2 (40 mM), methylglyoxal (5.8 mM), erythromycin (0.08 mg ml(-1)), streptomycin (0.005 mg ml(-1)), Cd(2+) (0.01 mM), Mn(2+) (2 mM), formic acid (0.05 %), acetic acid (0.15 %), levulinic acid (0.25 %), furfural (7.2 mM), and ethanol (10 % v/v), respectively. Increased MSH content also decreased the concentration of reactive oxygen species in the presence of the above stresses. Our results may open a new avenue for enhancing robustness of industrial bacteria for production of commodity chemicals. PMID:24737070

  3. Overexpression of Meloe Gene in Melanomas Is Controlled Both by Specific Transcription Factors and Hypomethylation

    PubMed Central

    Bobinet, Mathilde; Vignard, Virginie; Florenceau, Laetitia; Lang, Francois

    2013-01-01

    The melanoma antigens MELOE-1 and MELOE-2 are encoded by a messenger, called meloe, overexpressed in melanomas compared with other tumour cell types and healthy tissues. They are both able to elicit melanoma-specific T cell responses in melanoma patients, and MELOE-1-specific CD8 T cells have been involved in melanoma immunosurveillance. With the aim to develop immunotherapies targeting this antigen, we investigated the transcriptional mechanisms leading to the preferential expression of meloe messenger in the melanocytic lineage. We defined the minimal promoter region of meloe gene and identified binding motifs for a set of transcription factors. Using mutagenesis, co-transfection experiments and chromatin immunoprecipitation, we showed that transcription factors involved in meloe promoter activity in melanomas were the melanocytic specific SOX9 and SOX10 proteins together with the activated P-CREB protein. Furthermore, we showed that meloe promoter was hypomethylated in melanomas and melanocytes, and hypermethylated in colon cancer cell lines and mesotheliomas, thus explaining the absence of P-CREB binding in these cell lines. This was a second key to explain the overerexpression of meloe messenger in the melanocytic lineage. To our knowledge, such a dual transcriptional control conferring tissue-specificity has never been described for the expression of tumour antigens. PMID:24086527

  4. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco.

    PubMed

    Zhou, Shiyi; Hu, Wei; Deng, Xiaomin; Ma, Zhanbing; Chen, Lihong; Huang, Chao; Wang, Chen; Wang, Jie; He, Yanzhen; Yang, Guangxiao; He, Guangyuan

    2012-01-01

    Aquaporin (AQP) proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG), abscisic acid (ABA) and H(2)O(2). Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA) and H(2)O(2), and less ion leakage (IL), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities when compared with the wild type (WT) under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants. PMID:23285044

  5. 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.

  6. Gene-dosage dependent overexpression at the 13q amplicon identifies DIS3 as candidate oncogene in colorectal cancer progression.

    PubMed

    de Groen, Florence L M; Krijgsman, Oscar; Tijssen, Marianne; Vriend, Lianne E M; Ylstra, Bauke; Hooijberg, Erik; Meijer, Gerrit A; Steenbergen, Renske D M; Carvalho, Beatriz

    2014-04-01

    Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q-arm of chromosome 13. This aberration occurs in 40%-60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage-dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription-PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q-driven adenoma to carcinoma progression. PMID:24478024

  7. A Genomewide Overexpression Screen Identifies Genes Involved in the Phosphatidylinositol 3-Kinase Pathway in the Human Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Koushik, Amrita B.; Welter, Brenda H.; Rock, Michelle L.

    2014-01-01

    Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. E. histolytica relies on motility, phagocytosis, host cell adhesion, and proteolysis of extracellular matrix for virulence. In eukaryotic cells, these processes are mediated in part by phosphatidylinositol 3-kinase (PI3K) signaling. Thus, PI3K may be critical for virulence. We utilized a functional genomics approach to identify genes whose products may operate in the PI3K pathway in E. histolytica. We treated a population of trophozoites that were overexpressing genes from a cDNA library with a near-lethal dose of the PI3K inhibitor wortmannin. This screen was based on the rationale that survivors would be overexpressing gene products that directly or indirectly function in the PI3K pathway. We sequenced the overexpressed genes in survivors and identified a cDNA encoding a Rap GTPase, a protein previously shown to participate in the PI3K pathway. This supports the validity of our approach. Genes encoding a coactosin-like protein, EhCoactosin, and a serine-rich E. histolytica protein (SREHP) were also identified. Cells overexpressing EhCoactosin or SREHP were also less sensitive to a second PI3K inhibitor, LY294002. This corroborates the link between these proteins and PI3K. Finally, a mutant cell line with an increased level of phosphatidylinositol (3,4,5)-triphosphate, the product of PI3K activity, exhibited increased expression of SREHP and EhCoactosin. This further supports the functional connection between these proteins and PI3K in E. histolytica. To our knowledge, this is the first forward-genetics screen adapted to reveal genes participating in a signal transduction pathway in this pathogen. PMID:24442890

  8. Catalase from the silkworm, Bombyx mori: gene sequence, distribution, and overexpression.

    PubMed

    Yamamoto, Kohji; Banno, Yutaka; Fujii, Hiroshi; Miake, Fumio; Kashige, Nobuhiro; Aso, Yoichi

    2005-04-01

    Living organisms require mechanisms regulating reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion. Catalase is one of the regulatory enzymes and facilitates the degradation of hydrogen peroxide to oxygen and water. Biochemical information on an insect catalase is, however, insufficient. Using mRNA from fat body of the silkworm, Bombyx mori, a cDNA encoding a putative catalase was amplified by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence comprised 507 residues with more than seventy residues forming a scaffold for a heme cofactor conserved. The sequence showed 71% and 66% identities to those of the Drosophila melanogaster and Apis mellifera catalases, respectively; the catalase from B. mori was estimated to be phylogenetically close to that from A. mellifera. The transcripts of the gene and the catalase activity were distributed in diverse tissues of B. mori, suggesting its ubiquitous nature. Using the gene, a recombinant catalase (rCAT) was functionally overexpressed in a soluble form using Escherichia coli, purified to homogeneity, and characterized. The pH-optimum of rCAT was broad around pH 8.0. More than 80% of the original rCAT activity was retained after incubation in the following conditions: at pH 8-11 and 4 degrees C for 24 h; at pH 7 and temperatures below 50 degrees C for 30 min. The Michaelis constant for hydrogen peroxide was evaluated to be 28 mM at pH 6.5 and 30 degrees C. rCAT was suggested to be a member of the typical catalase family. PMID:15763464

  9. LncRNA2Target: a database for differentially expressed genes after lncRNA knockdown or overexpression

    PubMed Central

    Jiang, Qinghua; Wang, Jixuan; Wu, Xiaoliang; Ma, Rui; Zhang, Tianjiao; Jin, Shuilin; Han, Zhijie; Tan, Renjie; Peng, Jiajie; Liu, Guiyou; Li, Yu; Wang, Yadong

    2015-01-01

    Long non-coding RNAs (lncRNAs) have emerged as critical regulators of genes at epigenetic, transcriptional and post-transcriptional levels, yet what genes are regulated by a specific lncRNA remains to be characterized. To assess the effects of the lncRNA on gene expression, an increasing number of researchers profiled the genome-wide or individual gene expression level change after knocking down or overexpressing the lncRNA. Herein, we describe a curated database named LncRNA2Target, which stores lncRNA-to-target genes and is publicly accessible at http://www.lncrna2target.org. A gene was considered as a target of a lncRNA if it is differentially expressed after the lncRNA knockdown or overexpression. LncRNA2Target provides a web interface through which its users can search for the targets of a particular lncRNA or for the lncRNAs that target a particular gene. Both search types are performed either by browsing a provided catalog of lncRNA names or by inserting lncRNA/target gene IDs/names in a search box. PMID:25399422

  10. The overexpression of SIRT1 inhibited osteoarthritic gene expression changes induced by interleukin-1β in human chondrocytes.

    PubMed

    Matsushita, Takehiko; Sasaki, Hiroshi; Takayama, Koji; Ishida, Kazunari; Matsumoto, Tomoyuki; Kubo, Seiji; Matsuzaki, Tokio; Nishida, Kotaro; Kurosaka, Masahiro; Kuroda, Ryosuke

    2013-04-01

    In this study, we examined the effects of overexpression of SIRT1 on IL-1β-induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL-1β. SIRT1 was also inhibited by siRNA under stimulation with IL-1β. Gene expression changes were examined by real-time PCR. The interaction of SIRT1 and p65 (NF-κB) were examined by Western blotting. SIRT1, MMP-13, and ADAMTS-5 expressions in human cartilage were examined by immunohistochemistry. IL-1β stimulation significantly up-regulated MMP-1, 2, 9, and 13 and ADAMTS-5. Overexpression of SIRT1 significantly inhibited the up-regulation of those genes caused by IL-1β while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL-1β-induced acetylation of p65. SIRT1 expression was clearly detected in the non-OA cartilage while MMP-13 and ADAMTS-5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP-13 and ADAMTS-5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL-1β-induced expressions of cartilage-degrading enzymes partially through the modulation of the NF-κB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. PMID:23143889

  11. Enhancement of poly-3-hydroxybutyrate production in Synechocystis sp. PCC 6803 by overexpression of its native biosynthetic genes.

    PubMed

    Khetkorn, Wanthanee; Incharoensakdi, Aran; Lindblad, Peter; Jantaro, Saowarath

    2016-08-01

    Synechocystis sp. PCC 6803 strains overexpressing pha genes were constructed and characterized for poly-3-hydroxybutyrate (PHB) production. These pha overexpressing strains showed slightly reduced growth rates. Under N-deprived condition, the strains overexpressing (OE) phaAB, phaEC and phaABEC showed significantly higher PHB contents than the wild type. The maximum PHB content, a 2.6-fold increase producing 26% PHB (dcw), was observed in OE phaAB cells grown for 9days in N-deprived medium. Under this condition, these OE phaAB cells increased PHB production to 35% PHB (dcw) upon addition of 0.4% (w/v) acetate. Higher PHB granules in OE phaAB cells were clearly visualized by both Nile red staining and TEM imaging. All OE strains under N-deficient condition had increased glgX transcript levels. Overall results demonstrate an enhanced PHB production in Synechocystis cells overexpressing pha genes, particularly phaA and phaB, when grown in N-deprived medium containing 0.4% (w/v) acetate. PMID:27213577

  12. An overexpression screen in Drosophila for genes that restrict growth or cell-cycle progression in the developing eye.

    PubMed Central

    Tseng, Ai-Sun Kelly; Hariharan, Iswar K

    2002-01-01

    We screened for genes that, when overexpressed in the proliferating cells of the eye imaginal disc, result in a reduction in the size of the adult eye. After crossing the collection of 2296 EP lines to the ey-GAL4 driver, we identified 46 lines, corresponding to insertions in 32 different loci, that elicited a small eye phenotype. These lines were classified further by testing for an effect in postmitotic cells using the sev-GAL4 driver, by testing for an effect in the wing using en-GAL4, and by testing for the ability of overexpression of cycE to rescue the small eye phenotype. EP lines identified in the screen encompass known regulators of eye development including hh and dpp, known genes that have not been studied previously with respect to eye development, as well as 19 novel ORFs. Lines with insertions near INCENP, elB, and CG11518 were characterized in more detail with respect to changes in growth, cell-cycle phasing, and doubling times that were elicited by overexpression. RNAi-induced phenotypes were also analyzed in SL2 cells. Thus overexpression screens can be combined with RNAi experiments to identify and characterize new regulators of growth and cell proliferation. PMID:12242236

  13. Overexpression of wheat gene TaMOR improves root system architecture and grain yield in Oryza sativa.

    PubMed

    Li, Bo; Liu, Dan; Li, Qiaoru; Mao, Xinguo; Li, Ang; Wang, Jingyi; Chang, Xiaoping; Jing, Ruilian

    2016-07-01

    Improved root architecture is an effective strategy to increase crop yield. We demonstrate that overexpression of transcription factor gene MORE ROOT (TaMOR) from wheat (Triticum aestivum L.) results in more roots and higher grain yield in rice (Oryza sativa). TaMOR, encoding a plant-specific transcription factor belonging to the ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES (AS2/LOB) protein family, is highly conserved in wheat and its wild relatives. In this study, tissue expression patterns indicated that TaMOR mainly localizes to root initiation sites. The consistent gene expression pattern suggests that TaMOR is involved in root initiation. Exogenous auxin treatment induced TaMOR expression without de novo protein biosynthesis. Both in vivo and in vitro experiments demonstrated that TaMOR interacts with TaMOR-related protein TaMRRP, which contains a four-tandem-pentatricopeptide repeat motif. Overexpression of TaMOR led to more lateral roots in Arabidopsis thaliana, and TaMOR-overexpressing rice plants had more crown roots, a longer main panicle, a higher number of primary branches on the main panicle, a higher grain number per plant, and higher yield per plant than the plants of wild type. In general, TaMOR-D-overexpressing lines had larger root systems in Arabidopsis and rice, and produce a higher grain yield per plant. TaMOR therefore offers an opportunity to improve root architecture and increase yield in crop plants. PMID:27229732

  14. Identification of New SRF Binding Sites in Genes Modulated by SRF Over-Expression in Mouse Hearts

    PubMed Central

    Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Burton, Brian; Huang, Chris; Zhong, Ying; Gu, Xuesong; Fang, Hong; Tong, Weida; Wei, Jeanne Y.

    2011-01-01

    Background: To identify in vivo new cardiac binding sites of serum response factor (SRF) in genes and to study the response of these genes to mild over-expression of SRF, we employed a cardiac-specific, transgenic mouse model, with mild over-expression of SRF (Mild-O SRF Tg). Methodology: Microarray experiments were performed on hearts of Mild-O-SRF Tg at 6 months of age. We identified 207 genes that are important for cardiac function that were differentially expressed in vivo. Among them the promoter region of 192 genes had SRF binding motifs, the classic CArG or CArG-like (CArG-L) elements. Fifty-one of the 56 genes with classic SRF binding sites had not been previously reported. These SRF-modulated genes were grouped into 12 categories based on their function. It was observed that genes associated with cardiac energy metabolism shifted toward that of carbohydrate metabolism and away from that of fatty acid metabolism. The expression of genes that are involved in transcription and ion regulation were decreased, but expression of cytoskeletal genes was significantly increased. Using public databases of mouse models of hemodynamic stress (GEO database), we also found that similar altered expression of the SRF-modulated genes occurred in these hearts with cardiac ischemia or aortic constriction as well. Conclusion and significance: SRF-modulated genes are actively regulated under various physiological and pathological conditions. We have discovered that a large number of cardiac genes have classic SRF binding sites and were significantly modulated in the Mild-O-SRF Tg mouse hearts. Hence, the mild elevation of SRF protein in the heart that is observed during typical adult aging may have a major impact on many SRF-modulated genes, thereby affecting cardiac structure and performance. The results from our study could help to enhance our understanding of SRF regulation of cellular processes in the aged heart. PMID:21792293

  15. Overexpression of SAMDC1 gene in Arabidopsis thaliana increases expression of defense-related genes as well as resistance to Pseudomonas syringae and Hyaloperonospora arabidopsidis

    PubMed Central

    Marco, Francisco; Busó, Enrique; Carrasco, Pedro

    2014-01-01

    It has been previously described that elevation of endogenous spermine levels in Arabidopsis could be achieved by transgenic overexpression of S-Adenosylmethionine decarboxylase (SAMDC) or Spermine synthase (SPMS). In both cases, spermine accumulation had an impact on the plant transcriptome, with up-regulation of a set of genes enriched in functional categories involved in defense-related processes against both biotic and abiotic stresses. In this work, the response of SAMDC1-overexpressing plants against bacterial and oomycete pathogens has been tested. The expression of several pathogen defense-related genes was induced in these plants as well as in wild type plants exposed to an exogenous supply of spermine. SAMDC1-overexpressing plants showed an increased tolerance to infection by Pseudomonas syringae and by Hyaloperonospora arabidopsidis. Both results add more evidence to the hypothesis that spermine plays a key role in plant resistance to biotic stress. PMID:24734036

  16. Overexpression of the CaTIP1-1 Pepper Gene in Tobacco Enhances Resistance to Osmotic Stresses

    PubMed Central

    Yin, Yan-Xu; Wang, Shu-Bin; Xiao, Huai-Juan; Zhang, Huai-Xia; Zhang, Zhen; Jing, Hua; Zhang, Ying-Li; Chen, Ru-Gang; Gong, Zhen-Hui

    2014-01-01

    Both the gene expression and activity of water channel protein can control transmembrane water movement. We have reported the overexpression of CaTIP1-1, which caused a decrease in chilling tolerance in transgenic plants by increasing the size of the stomatal pore. CaTIP1-1 expression was strongly induced by salt and mannitol stresses in pepper (Capsicum annuum). However, its biochemical and physiological functions are still unknown in transgenic tobacco. In this study, transient expression of CaTIP1-1-GFP in tobacco suspension cells revealed that the protein was localized in the tonoplast. CaTIP1-1 overexpressed in radicle exhibited vigorous growth under high salt and mannitol treatments more than wild-type plants. The overexpression of CaTIP1-1 pepper gene in tobacco enhanced the antioxidant enzyme activities and increased transcription levels of reactive oxygen species-related gene expression under osmotic stresses. Moreover, the viability of transgenic tobacco cells was higher than the wild-type after exposure to stress. The pepper plants with silenced CaTIP1-1 in P70 decreased tolerance to salt and osmotic stresses using the detached leaf method. We concluded that the CaTIP1-1 gene plays an important role in response to osmotic stresses in tobacco. PMID:25375192

  17. Over-expression of histone H3K4 demethylase gene JMJ15 enhances salt tolerance in Arabidopsis

    PubMed Central

    Shen, Yuan; Conde e Silva, Natalia; Audonnet, Laure; Servet, Caroline; Wei, Wei; Zhou, Dao-Xiu

    2014-01-01

    Histone H3 lysine 4 trimethylation (H3K4me3) has been shown to be involved in stress-responsive gene expression and gene priming in plants. However, the role of H3K4me3 resetting in the processes is not clear. In this work we studied the expression and function of Arabidopsis H3K4 demethylase gene JMJ15. We show that the expression of JMJ15 was relatively low and was limited to a number of tissues during vegetative growth but was higher in young floral organs. Over-expression of the gene in gain-of-function mutants reduced the plant height with accumulation of lignin in stems, while the loss-of-function mutation did not produce any visible phenotype. The gain-of-function mutants showed enhanced salt tolerance, whereas the loss-of-function mutant was more sensitive to salt compared to the wild type. Transcriptomic analysis revealed that over-expression of JMJ15 down-regulated many genes which are preferentially marked by H3K4me3 and H3K4me2. Many of the down-regulated genes encode transcription regulators involved in stress responses. The data suggest that increased JMJ15 levels may regulate the gene expression program that enhances stress tolerance. PMID:25009544

  18. Induction of pseudohyphal growth by overexpression of PHD1, a Saccharomyces cerevisiae gene related to transcriptional regulators of fungal development.

    PubMed Central

    Gimeno, C J; Fink, G R

    1994-01-01

    When starved for nitrogen, MATa/MAT alpha cells of the budding yeast Saccharomyces cerevisiae undergo a dimorphic transition to pseudohyphal growth. A visual genetic screen, called PHD (pseudohyphal determinant), for S. cerevisiae pseudohyphal growth mutants was developed. The PHD screen was used to identify seven S. cerevisiae genes that when overexpressed in MATa/MAT alpha cells growing on nitrogen starvation medium cause precocious and unusually vigorous pseudohyphal growth. PHD1, a gene whose overexpression induced invasive pseudohyphal growth on a nutritionally rich medium, was characterized. PHD1 maps to chromosome XI and is predicted to encode a 366-amino-acid protein. PHD1 has a SWI4- and MBP1-like DNA binding motif that is 73% identical over 100 amino acids to a region of Aspergillus nidulans StuA. StuA regulates two pseudohyphal growth-like cell divisions during conidiophore morphogenesis. Epitope-tagged PHD1 was localized to the nucleus by indirect immunofluorescence. These facts suggest that PHD1 may function as a transcriptional regulatory protein. Overexpression of PHD1 in wild-type haploid strains does not induce pseudohyphal growth. Interestingly, PHD1 overexpression enhances pseudohyphal growth in a haploid strain that has the diploid polar budding pattern because of a mutation in the BUD4 gene. In addition, wild-type diploid strains lacking PHD1 undergo pseudohyphal growth when starved for nitrogen. The possible functions of PHD1 in pseudohyphal growth and the uses of the PHD screen to identify morphogenetic regulatory genes from heterologous organisms are discussed. Images PMID:8114741

  19. Overexpression of the PSAT1 Gene in Nasopharyngeal Carcinoma Is an Indicator of Poor Prognosis

    PubMed Central

    Liao, Kuang-Ming; Chao, Tung-Bo; Tian, Yu-Feng; Lin, Ching-Yih; Lee, Sung-Wei; Chuang, Hua-Ying; Chan, Ti-Chun; Chen, Tzu-Ju; Hsing, Chung-Hsi; Sheu, Ming-Jen; Li, Chien-Feng

    2016-01-01

    Purpose: Nasopharyngeal carcinoma (NPC) is a common cancer in southern China and Southeast Asia, but risk stratification and treatment outcome in NPC patients remain suboptimal. Our study identified and validated metabolic drivers that are relevant to the pathogenesis of NPC using a published transcriptome. Phosphoserine aminotransferase 1 (PSAT1) is an enzyme that is involved in serine biosynthesis, and its overexpression is associated with colon cancer, non-small cell lung cancer and breast cancer. However, its expression has not been systemically evaluated in patients with NPC. Materials and Methods: We evaluated two public transcriptomes of NPC tissues and benign nasopharyngeal mucosal epithelial tissues that deposited in the NIH Gene Expression Omnibus database under accession number GSE34574 and GSE12452. We also performed immunohistochemical staining and assessment of PSAT1 in a total of 124 NPC patients received radiotherapy and were regularly followed-up until death or loss. The endpoints analyzed were local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS), disease-specific survival (DSS), and overall survival (OS). Results: We retrospectively evaluated 124 patients with NPC and found that high PSAT1 expression was associated with poor prognosis of NPC and indicator of advanced tumor stage. High PSAT1 expression also correlated with an aggressive clinical course, with significantly shorter DSS (HR= 2.856, 95% CI 1.599 to 5.101), DMFS (HR= 3.305, 95% CI 1.720 to 6.347), LRFS (HR= 2.834, 95% CI 1.376 to 5.835), and OS HR= 2.935, 95% CI 1.646-5.234) in multivariate analyses. Conclusions: Our study showed that PSAT1 is a potential prognostic biomarker and higher expression of PSAT1 is associated with a poor prognosis in NPC. PMID:27326252

  20. Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression.

    PubMed

    Li, Dan; Williams, Jon I; Pietras, Richard J

    2002-04-25

    Angiogenesis is important for growth and progression of ovarian cancers. Squalamine is a natural antiangiogenic sterol, and its potential role in treatment of ovarian cancers with or without standard cisplatin chemotherapy was assessed. Since HER-2 gene overexpression is associated with cisplatin resistance in vitro and promotion of tumor angiogenesis in vivo, the response of ovarian cancer cells with or without HER-2 gene overexpression to squalamine and cisplatin was evaluated both in tumor xenograft models and in tissue culture. Ovarian cancer cells with or without HER-2 overexpression were grown as subcutaneous xenografts in nude mice. Animals were treated by intraperitoneal injection with control vehicle, cisplatin, squalamine or cisplatin combined with squalamine. At the end of the experiment, tumors were assessed for tumor growth inhibition and for changes in microvessel density and apoptosis. Additional in vitro studies evaluated effects of squalamine on tumor and endothelial cell growth and on signaling pathways in human endothelial cells. Profound growth inhibition was elicited by squalamine alone and by combined treatment with squalamine and cisplatin for both parental and HER-2-overexpressing ovarian tumor xenografts. Immunohistochemical evaluation of tumors revealed decreased microvessel density and increased apoptosis. Although HER-2-overexpressing tumors had more angiogenic and less apoptotic activity than parental cancers, growth of both tumor types was similarly suppressed by treatment with squalamine combined with cisplatin. In in vitro studies, we found that squalamine does not directly affect proliferation of ovarian cells. However, squalamine significantly blocked VEGF-induced activation of MAP kinase and cell proliferation in human vascular endothelial cells. The results suggest that squalamine is anti-angiogenic for ovarian cancer xenografts and appears to enhance cytotoxic effects of cisplatin chemotherapy independent of HER-2 tumor status

  1. Neuroplasticity and MRI: A perfect match.

    PubMed

    Hamaide, Julie; De Groof, Geert; Van der Linden, Annemie

    2016-05-01

    Numerous studies have illustrated the benefits of physical workout and cognitive exercise on brain function and structure and, more importantly, on decelerating cognitive decline in old age and promoting functional rehabilitation following injury. Despite these behavioral observations, the exact mechanisms underlying these neuroplastic phenomena remain obscure. This gap illustrates the need for carefully designed in-depth studies using valid models and translational tools which allow to uncover the observed events up to the molecular level. We promote the use of in vivo magnetic resonance imaging (MRI) because it is a powerful translational imaging technique able to extract functional, structural, and biochemical information from the entire brain. Advanced processing techniques allow performing voxel-based analyses which are capable of detecting novel loci implicated in specific neuroplastic events beyond traditional regions-of-interest analyses. In addition, its non-invasive character sets it as currently the best global imaging tool for performing dynamic longitudinal studies on the same living subject, allowing thus exploring the effects of experience, training, treatment etc. in parallel to additional measures such as age, cognitive performance scores, hormone levels, and many others. The aim of this review is (i) to introduce how different animal models contributed to extend the knowledge on neuroplasticity in both health and disease, over different life stages and upon various experiences, and (ii) to illustrate how specific MRI techniques can be applied successfully to inform on the fundamental mechanisms underlying experience-dependent or activity-induced neuroplasticity including cognitive processes. PMID:26260430

  2. Improved Acetic Acid Resistance in Saccharomyces cerevisiae by Overexpression of the WHI2 Gene Identified through Inverse Metabolic Engineering.

    PubMed

    Chen, Yingying; Stabryla, Lisa; Wei, Na

    2016-01-01

    Development of acetic acid-resistant Saccharomyces cerevisiae is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge due to limited information on effective genetic perturbation targets for improving acetic acid resistance in the yeast. This study employed a genomic-library-based inverse metabolic engineering approach to successfully identify a novel gene target, WHI2 (encoding a cytoplasmatic globular scaffold protein), which elicited improved acetic acid resistance in S. cerevisiae. Overexpression of WHI2 significantly improved glucose and/or xylose fermentation under acetic acid stress in engineered yeast. The WHI2-overexpressing strain had 5-times-higher specific ethanol productivity than the control in glucose fermentation with acetic acid. Analysis of the expression of WHI2 gene products (including protein and transcript) determined that acetic acid induced endogenous expression of Whi2 in S. cerevisiae. Meanwhile, the whi2Δ mutant strain had substantially higher susceptibility to acetic acid than the wild type, suggesting the important role of Whi2 in the acetic acid response in S. cerevisiae. Additionally, overexpression of WHI2 and of a cognate phosphatase gene, PSR1, had a synergistic effect in improving acetic acid resistance, suggesting that Whi2 might function in combination with Psr1 to elicit the acetic acid resistance mechanism. These results improve our understanding of the yeast response to acetic acid stress and provide a new strategy to breed acetic acid-resistant yeast strains for renewable biofuel production. PMID:26826231

  3. The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis.

    PubMed

    Palmeros-Suárez, Paola A; Massange-Sánchez, Julio A; Martínez-Gallardo, Norma A; Montero-Vargas, Josaphat M; Gómez-Leyva, Juan F; Délano-Frier, John P

    2015-11-01

    Nuclear factor-Y (NF-Y), is a plant heterotrimeric transcription factor constituted by NF-YA, NF-YB and NF-YC subunits. The function of many NF-Y subunits, mostly of the A and B type, has been studied in plants, but knowledge regarding the C subunit remains fragmentary. Here, a water stress-induced NF-YC gene from Amaranthus hypochondriacus (AhNF-YC) was further characterized by its overexpression in transgenic Arabidospis thaliana plants. A role in development was inferred from modified growth rates in root, rosettes and inflorescences recorded in AhNF-YC overexpressing Arabidopsis plants, in addition to a delayed onset of flowering. Also, the overexpression of AhNF-YC caused increased seedling sensitivity to abscisic acid (ABA), and influenced the expression of several genes involved in secondary metabolism, development and ABA-related responses. An altered expression of the latter in water stressed and recovered transgenic plants, together with the observed increase in ABA sensitivity, suggested that their increased water stress resistance was partly ABA-dependent. An untargeted metabolomic analysis also revealed an altered metabolite pattern, both in normal and water stress/recovery conditions. These results suggest that AhNF-YC may play an important regulatory role in both development and stress, and represents a candidate gene for the engineering of abiotic stress resistance in commercial crops. PMID:26475185

  4. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila

    PubMed Central

    Auer, Jasmin S.; Nagel, Anja C.; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-01-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H)MAPK-ko and a phospho-mimetic Su(H)MAPK-ac isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vgBE-lacZ). In general, Su(H)MAPK-ko induced a stronger response than wild-type Su(H), whereas the response to Su(H)MAPK-ac was very weak. Notch target genes cut, wingless and vgBE-lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DERact) or the MAPK (rlSEM) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones. PMID:26702412

  5. Local overexpression of Su(H)-MAPK variants affects Notch target gene expression and adult phenotypes in Drosophila.

    PubMed

    Auer, Jasmin S; Nagel, Anja C; Schulz, Adriana; Wahl, Vanessa; Preiss, Anette

    2015-12-01

    In Drosophila, Notch and EGFR signalling pathways are closely intertwined. Their relationship is mostly antagonistic, and may in part be based on the phosphorylation of the Notch signal transducer Suppressor of Hairless [Su(H)] by MAPK. Su(H) is a transcription factor that together with several cofactors regulates the expression of Notch target genes. Here we address the consequences of a local induction of three Su(H) variants on Notch target gene expression. To this end, wild-type Su(H), a phospho-deficient Su(H) (MAPK-) (ko) and a phospho-mimetic Su(H) (MAPK-ac) isoform were overexpressed in the central domain of the wing anlagen. The expression of the Notch target genes cut, wingless, E(spl)m8-HLH and vestigial, was monitored. For the latter two, reporter genes were used (E(spl)m8-lacZ, vg (BE) -lacZ). In general, Su(H) (MAPK-) (ko) induced a stronger response than wild-type Su(H), whereas the response to Su(H) (MAPK-ac) was very weak. Notch target genes cut, wingless and vg (BE) -lacZ were ectopically activated, whereas E(spl)m8-lacZ was repressed by overexpression of Su(H) proteins. In addition, in epistasis experiments an activated form of the EGF-receptor (DER (act) ) or the MAPK (rl (SEM) ) and individual Su(H) variants were co-overexpressed locally, to compare the resultant phenotypes in adult flies (thorax, wings and eyes) as well as to assay the response of the Notch target gene cut in cell clones. PMID:26702412

  6. A Novel Subset of Human Tumors That Simultaneously Overexpress Multiple E2F-responsive Genes Found in Breast, Ovarian, and Prostate Cancers

    PubMed Central

    Shackney, Stanley E; Chowdhury, Salim Akhter; Schwartz, Russell

    2014-01-01

    Reasoning that overexpression of multiple E2F-responsive genes might be a useful marker for RB1 dysfunction, we compiled a list of E2F-responsive genes from the literature and evaluated their expression in publicly available gene expression microarray data of patients with breast cancer, serous ovarian cancer, and prostate cancer. In breast cancer, a group of tumors was identified, each of which simultaneously overexpressed multiple E2F-responsive genes. Seventy percent of these genes were concerned with cell cycle progression, DNA repair, or mitosis. These E2F-responsive gene overexpressing (ERGO) tumors frequently exhibited additional evidence of Rb/E2F axis dysfunction, were mostly triple negative, and preferentially overexpressed multiple basal cytokeratins, suggesting that they overlapped substantially with the basal-like tumor subset. ERGO tumors were also identified in serous ovarian cancer and prostate cancer. In these cancer types, there was no evidence for a tumor subset comparable to the breast cancer basal-like subset. A core group of about 30 E2F-responsive genes were overexpressed in all three cancer types. Thus, it appears that disorders of the Rb/E2F axis can arise at multiple organ sites and produce tumors that simultaneously overexpress multiple E2F-responsive genes. PMID:25392696

  7. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea

    PubMed Central

    Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

    2015-01-01

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain. PMID:25955649

  8. Gene Overexpression and RNA Silencing Tools for the Genetic Manipulation of the S-(+)-Abscisic Acid Producing Ascomycete Botrytis cinerea.

    PubMed

    Ding, Zhong-Tao; Zhang, Zhi; Luo, Di; Zhou, Jin-Yan; Zhong, Juan; Yang, Jie; Xiao, Liang; Shu, Dan; Tan, Hong

    2015-01-01

    The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain. PMID:25955649

  9. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

    PubMed Central

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

  10. Overexpression of the Trichoderma brevicompactum tri5 Gene: Effect on the Expression of the Trichodermin Biosynthetic Genes and on Tomato Seedlings

    PubMed Central

    Tijerino, Anamariela; Hermosa, Rosa; Cardoza, Rosa E.; Moraga, Javier; Malmierca, Monica G.; Aleu, Josefina; Collado, Isidro G.; Monte, Enrique; Gutierrez, Santiago

    2011-01-01

    Trichoderma brevicompactum IBT 40841 produces trichodermin, a trichothecene-type toxin that shares most of the steps of its biosynthesis with harzianum A, another trichothecene produced by several Trichoderma species. The first specific step in the trichothecene biosynthesis is carried out by a terpene cylcase, trichodiene synthase, that catalyzes the conversion of farnesyl pyrophosphate to trichodiene and that is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin production, but also in an increase in tyrosol and hydroxytyrosol production, two antioxidant compounds that may play a regulatory role in trichothecene biosynthesis, and also in a higher expression of three trichothecene genes, tri4, tri6 and tri10, and of the erg1 gene, which participates in the synthesis of triterpenes. The effect of tri5 overexpression on tomato seedling disease response was also studied. PMID:22069764

  11. Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression.

    PubMed

    Hinds, Terry D; Peck, Bailey; Shek, Evan; Stroup, Steven; Hinson, Jennifer; Arthur, Susan; Marino, Joseph S

    2016-01-01

    Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C12 muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C12 was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids. PMID:26875982

  12. Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

    PubMed Central

    Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

    2014-01-01

    Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars. PMID:25003197

  13. Transgenic tobacco plants overexpressing a grass PpEXP1 gene exhibit enhanced tolerance to heat stress.

    PubMed

    Xu, Qian; Xu, Xiao; Shi, Yang; Xu, Jichen; Huang, Bingru

    2014-01-01

    Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars. PMID:25003197

  14. Over-expression of XIST, the Master Gene for X Chromosome Inactivation, in Females With Major Affective Disorders

    PubMed Central

    Ji, Baohu; Higa, Kerin K.; Kelsoe, John R.; Zhou, Xianjin

    2015-01-01

    Background Psychiatric disorders are common mental disorders without a pathological biomarker. Classic genetic studies found that an extra X chromosome frequently causes psychiatric symptoms in patients with either Klinefelter syndrome (XXY) or Triple X syndrome (XXX). Over-dosage of some X-linked escapee genes was suggested to cause psychiatric disorders. However, relevance of these rare genetic diseases to the pathogenesis of psychiatric disorders in the general population of psychiatric patients is unknown. Methods XIST and several X-linked genes were studied in 36 lymphoblastoid cell lines from healthy females and 60 lymphoblastoid cell lines from female patients with either bipolar disorder or recurrent major depression. XIST and KDM5C expression was also quantified in 48 RNA samples from postmortem human brains of healthy female controls and female psychiatric patients. Findings We found that the XIST gene, a master in control of X chromosome inactivation (XCI), is significantly over-expressed (p = 1 × 10− 7, corrected after multiple comparisons) in the lymphoblastoid cells of female patients with either bipolar disorder or major depression. The X-linked escapee gene KDM5C also displays significant up-regulation (p = 5.3 × 10− 7, corrected after multiple comparisons) in the patients' cells. Expression of XIST and KDM5C is highly correlated (Pearson's coefficient, r = 0.78, p = 1.3 × 10− 13). Studies on human postmortem brains supported over-expression of the XIST gene in female psychiatric patients. Interpretations We propose that over-expression of XIST may cause or result from subtle alteration of XCI, which up-regulates the expression of some X-linked escapee genes including KDM5C. Over-expression of X-linked genes could be a common mechanism for the development of psychiatric disorders between patients with those rare genetic diseases and the general population of female psychiatric patients with XIST over-expression. Our studies

  15. Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene, Leading to Improved Drought Tolerance in Both Arabidopsis and Maize.

    PubMed

    Shi, Jinrui; Habben, Jeffrey E; Archibald, Rayeann L; Drummond, Bruce J; Chamberlin, Mark A; Williams, Robert W; Lafitte, H Renee; Weers, Ben P

    2015-09-01

    Lack of sufficient water is a major limiting factor to crop production worldwide, and the development of drought-tolerant germplasm is needed to improve crop productivity. The phytohormone ethylene modulates plant growth and development as well as plant response to abiotic stress. Recent research has shown that modifying ethylene biosynthesis and signaling can enhance plant drought tolerance. Here, we report novel negative regulators of ethylene signal transduction in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). These regulators are encoded by the ARGOS gene family. In Arabidopsis, overexpression of maize ARGOS1 (ZmARGOS1), ZmARGOS8, Arabidopsis ARGOS homolog ORGAN SIZE RELATED1 (AtOSR1), and AtOSR2 reduced plant sensitivity to ethylene, leading to enhanced drought tolerance. RNA profiling and genetic analysis suggested that the ZmARGOS1 transgene acts between an ethylene receptor and CONSTITUTIVE TRIPLE RESPONSE1 in the ethylene signaling pathway, affecting ethylene perception or the early stages of ethylene signaling. Overexpressed ZmARGOS1 is localized to the endoplasmic reticulum and Golgi membrane, where the ethylene receptors and the ethylene signaling protein ETHYLENE-INSENSITIVE2 and REVERSION-TO-ETHYLENE SENSITIVITY1 reside. In transgenic maize plants, overexpression of ARGOS genes also reduces ethylene sensitivity. Moreover, field testing showed that UBIQUITIN1:ZmARGOS8 maize events had a greater grain yield than nontransgenic controls under both drought stress and well-watered conditions. PMID:26220950

  16. The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene

    PubMed Central

    2012-01-01

    Background Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance. Methods The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated. Results The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated

  17. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    PubMed

    Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas. PMID:27411057

  18. Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs

    PubMed Central

    Svoboda, Laurie K; Harris, Ashley; Bailey, Natashay J; Schwentner, Raphaela; Tomazou, Eleni; von Levetzow, Cornelia; Magnuson, Brian; Ljungman, Mats; Kovar, Heinrich; Lawlor, Elizabeth R

    2014-01-01

    The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1. PMID:25625846

  19. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance

    PubMed Central

    Pandey, Sonika; Patel, Manish Kumar; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas. PMID:27411057

  20. Overexpression of the Circadian Clock Gene Rev-erbα Affects Murine Bone Mesenchymal Stem Cell Proliferation and Osteogenesis

    PubMed Central

    He, Yao; Lin, Fuwei; Chen, Yaqun; Tan, Zhen; Bai, Ding

    2015-01-01

    Bone mesenchymal stem cell (BMSC) age-related changes include decreased osteogenesis and increased adipogenesis. Rev-erbα and the Wnt/β-catenin signaling pathway were known to play important roles in BMSC aging. In this study, we have aimed to elucidate whether Rev-erbα and Wnt/β-catenin signaling interact during BMSC proliferation and osteogenesis. Our results showed that Rev-erbα expression gradually dropped during BMSC osteogenesis, and overexpression of Rev-erbα in BMSCs inhibited cell proliferation and osteogenesis. The inhibition of cell proliferation induced by Rev-erbα overexpression was partially reversed when Wnt/β-catenin signaling was activated. These results suggested that Rev-erbα could promote BMSC aging and may be the negative regulator during the late stage of osteogenesis. The clock gene Rev-erbα and Wnt/β-catenin signaling interact in the regulation of cell proliferation. PMID:25539035

  1. Overexpression of a CYP94 family gene CYP94C2b increases internode length and plant height in rice

    PubMed Central

    Kurotani, Ken-Ich; Hattori, Tsukaho; Takeda, Shin

    2015-01-01

    Plant growth is controlled by intrinsic developmental programmes and environmental cues. Jasmonate (JA) has important roles in both processes, by regulating cell division and differentiation, as well as in defense responses and senescence. We report an increase in rice plant height caused by overexpression of a gene encoding a cytochrome P450 enzyme, CYP94C2b, which promoted deactivation of JA-Ile. The height increase occurred through enhanced elongation of internodes in the absence of concomitant cell elongation, unlike previous findings with coi1 knock-down plants. Thus, modulating JA metabolism can increase the number of elongated cells in an internode. Based on these and previous findings, we discuss the difference in the effects of CYP94C2b overexpression vs. coi1 knock-down. PMID:26251886

  2. Transcriptional regulation of receptor-like protein genes by environmental stresses and hormones and their overexpression activities in Arabidopsis thaliana.

    PubMed

    Wu, Jinbin; Liu, Zhijun; Zhang, Zhao; Lv, Yanting; Yang, Nan; Zhang, Guohua; Wu, Menyao; Lv, Shuo; Pan, Lixia; Joosten, Matthieu H A J; Wang, Guodong

    2016-05-01

    Receptor-like proteins (RLPs) have been implicated in multiple biological processes, including plant development and immunity to microbial infection. Fifty-seven AtRLP genes have been identified in Arabidopsis, whereas only a few have been functionally characterized. This is due to the lack of suitable physiological screening conditions and the high degree of functional redundancy among AtRLP genes. To overcome the functional redundancy and further understand the role of AtRLP genes, we studied the evolution of AtRLP genes and compiled a comprehensive profile of the transcriptional regulation of AtRLP genes upon exposure to a range of environmental stresses and different hormones. These results indicate that the majority of AtRLP genes are differentially expressed under various conditions that were tested, an observation that will help to select certain AtRLP genes involved in a specific biological process for further experimental studies to eventually dissect their function. A large number of AtRLP genes were found to respond to more than one treatment, suggesting that one single AtRLP gene may be involved in multiple physiological processes. In addition, we performed a genome-wide cloning of the AtRLP genes, and generated and characterized transgenic Arabidopsis plants overexpressing the individual AtRLP genes, presenting new insight into the roles of AtRLP genes, as exemplified by AtRLP3, AtRLP11 and AtRLP28 Our study provides an overview of biological processes in which AtRLP genes may be involved, and presents valuable resources for future investigations into the function of these genes. PMID:27099374

  3. Transcriptional regulation of receptor-like protein genes by environmental stresses and hormones and their overexpression activities in Arabidopsis thaliana

    PubMed Central

    Wu, Jinbin; Liu, Zhijun; Zhang, Zhao; Lv, Yanting; Yang, Nan; Zhang, Guohua; Wu, Menyao; Lv, Shuo; Pan, Lixia; Joosten, Matthieu H. A. J.; Wang, Guodong

    2016-01-01

    Receptor-like proteins (RLPs) have been implicated in multiple biological processes, including plant development and immunity to microbial infection. Fifty-seven AtRLP genes have been identified in Arabidopsis, whereas only a few have been functionally characterized. This is due to the lack of suitable physiological screening conditions and the high degree of functional redundancy among AtRLP genes. To overcome the functional redundancy and further understand the role of AtRLP genes, we studied the evolution of AtRLP genes and compiled a comprehensive profile of the transcriptional regulation of AtRLP genes upon exposure to a range of environmental stresses and different hormones. These results indicate that the majority of AtRLP genes are differentially expressed under various conditions that were tested, an observation that will help to select certain AtRLP genes involved in a specific biological process for further experimental studies to eventually dissect their function. A large number of AtRLP genes were found to respond to more than one treatment, suggesting that one single AtRLP gene may be involved in multiple physiological processes. In addition, we performed a genome-wide cloning of the AtRLP genes, and generated and characterized transgenic Arabidopsis plants overexpressing the individual AtRLP genes, presenting new insight into the roles of AtRLP genes, as exemplified by AtRLP3, AtRLP11 and AtRLP28. Our study provides an overview of biological processes in which AtRLP genes may be involved, and presents valuable resources for future investigations into the function of these genes. PMID:27099374

  4. Overexpression of Wnt-1 in thyrocytes enhances cellular growth but suppresses transcription of the thyroperoxidase gene via different signaling mechanisms.

    PubMed

    Kim, Won Bae; Lewis, Christopher J; McCall, Kelly D; Malgor, Ramiro; Kohn, Aimee D; Moon, Randall T; Kohn, Leonard D

    2007-04-01

    Wnt binding to cell surface receptors can activate a 'canonical' pathway that increases cellular beta-catenin or a 'noncanonical' Ca(++) pathway which can increase protein kinase C (PKC) activity. Although components of both Wnt/beta-catenin-signaling pathways exist in thyrocytes, their biological role is largely unknown. In evaluating the biological role of Wnt signaling in differentiated FRTL-5 thyroid cells, we showed that TSH increased canonical Wnt-1 but, surprisingly, decreased the active form of beta-catenin. Transient overexpression of Wnt-1 or beta-catenin in FRTL-5 cells increased active beta-catenin (ABC), decreased thyroperoxidase (TPO) mRNA, and suppressed TPO-promoter activity. The target of beta-catenin suppressive action was a consensus T cell factor/lymphoid enhancing factor (TCF/LEF)-binding site 5'-A/T A/T CAAAG-3', -137 to -129 bp on the rat TPO promoter. beta-Catenin overexpression significantly increased complex formation between beta-catenin/TCF-1 and an oligonucleotide containing the TCF/LEF sequence, suggesting that the beta-catenin/TCF-1 complex acts as a transcriptional repressor of the TPO gene. Stable over-expression of Wnt-1 in FRTL-5 cells significantly increased the growth rate without increasing beta-catenin levels. Increased growth was blunted by a PKC inhibitor, staurosporin. Wnt-1 overexpression increased serine phosphorylation, without affecting tyrosine phosphorylation, of signal transducers and activators of transcription 3 (STAT3) protein. In addition, these final results suggest that TSH-induced increase in Wnt-1 levels in thyrocytes contributes to enhanced cellular growth via a PKC pathway that increases STAT3 serine phosphorylation and activation, whereas TSH-induced decrease in activation of beta-catenin simultaneously relieves transcriptional suppression of TPO. We hypothesize that Wnt signaling contributes to the ability of TSH to simultaneously increase cell growth and functional, thyroid-specific, gene expression

  5. Overexpression of a Homeobox Gene, LeT6, Reveals Indeterminate Features in the Tomato Compound Leaf1

    PubMed Central

    Janssen, Bart-Jan; Lund, Lance; Sinha, Neelima

    1998-01-01

    The cultivated tomato (Lycopersicon esculentum) has a unipinnate compound leaf. In the developing leaf primordium, major leaflet initiation is basipetal, and lobe formation and early vascular differentiation are acropetal. We show that engineered alterations in the expression of a tomato homeobox gene, LeT6, can cause dramatic changes in leaf morphology. The morphological states are variable and unstable and the phenotypes produced indicate that the tomato leaf has an inherent level of indeterminacy. This is manifested by the production of multiple orders of compounding in the leaf, by numerous shoot, inflorescence, and floral meristems on leaves, and by the conversion of rachis-petiolule junctions into “axillary” positions where floral buds can arise. Overexpression of a heterologous homeobox transgene, kn1, does not produce such phenotypic variability. This indicates that LeT6 may differ from the heterologous kn1 gene in the effects manifested on overexpression, and that 35S-LeT6 plants may be subject to alterations in expression of both the introduced and endogenous LeT6 genes. The expression patterns of LeT6 argue in favor of a fundamental role for LeT6 in morphogenesis of leaves in tomato and also suggest that variability in homeobox gene expression may account for some of the diversity in leaf form seen in nature. PMID:9662520

  6. Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

    PubMed Central

    Choi, Min Ji; Song, Kang-Moon; Park, Jin-Mi; Kwon, Mi-Hye; Kwon, Ki-Dong; Park, Soo-Hwan; Ryu, Dong-Soo; Ryu, Ji-Kan; Suh, Jun-Kyu

    2015-01-01

    Transforming growth factor-β1 (TGF-β1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD. PMID:25532569

  7. Overexpression of two chrysanthemum DgDREB1 group genes causing delayed flowering or dwarfism in Arabidopsis.

    PubMed

    Tong, Zheng; Hong, Bo; Yang, Yingjie; Li, Qiuhua; Ma, Nan; Ma, Chao; Gao, Junping

    2009-09-01

    We isolated 13 DREB1 (dehydration responsive element binding factor 1) genes from chrysanthemum and further divided them into three groups, DgDREB1A, DgDREB1B and DgDREB1C, based on the phylogenetic analysis. Each group showed their unique expression patterns under cold, dehydration and salt stress conditions. Arabidopsis plants overexpressing DgDREB1A (1A plants) exhibited significantly stronger tolerance to freezing and drought than those overexpressing DgDREB1B (1B plants) and the control plants. In addition, 1A plants showed delayed flowering, but not dwarfism; while 1B plants showed dwarfism, but not delayed flowering. In 1A plants, the expression of three stress-related DREB1-downstream genes, COR47, COR15A, and RD29A, was strongly induced while the expression of CO and FT, two photoperiod responsive flowering-time genes, was inhibited. In 1B plants, the expression of GA2ox7, a GA-deactivation enzyme gene, was dramatically enhanced. The results above strongly suggest that members from different DgDREB1 groups may have distinct effects on plant development: DgDREB1A may be involved in photoperiod-related flowering-time determination and DgDREB1B in GA-mediated plant development. PMID:19544047

  8. Overexpression of lycopene ε-cyclase gene from lycium chinense confers tolerance to chilling stress in Arabidopsis thaliana.

    PubMed

    Song, Xinyu; Diao, Jinjin; Ji, Jing; Wang, Gang; Li, Zhaodi; Wu, Jiang; Josine, Tchouopou Lontchi; Wang, Yurong

    2016-01-15

    Lutein plays an important role in protecting the photosynthetic apparatus from photodamage and eliminating ROS to render normal physiological function of cells. As a rate-limiting step for lutein synthesis in plants, lycopene ε-cyclase catalyzes lycopene to δ-carotene. We cloned a lycopene ε-cyclase gene (Lcε-LYC) from Lycium chinense (L. chinense), a deciduous woody perennial halophyte growing in various environmental conditions. The Lcε-LYC gene has an ORF of 1569bp encoding a protein of 522 aa. The deduced amino acid sequence of Lcε-LYC gene has higher homology with LycEs in other plants, such as Nicotiana tabacum and Solanum tuberosum. When L. chinense was exposed to chilling stress, relative expression of Lcε-LYC increased. To study the protective role of Lcε-LYC against chilling stress, we overexpressed the Lcε-LYC gene in Arabidopsis thaliana. Lcε-LYC overexpression led to an increase of lutein accumulation in transgenic A. thaliana, and the content of lutein decreased when transgenics were under cold conditions. In addition, the transgenic plants under chilling stress displayed higher activities of superoxide dismutase (SOD) and peroxidase (POD) and less H2O2 and malondialdehyde (MDA) than the control. Moreover, the photosynthesis rate, photosystem II activity (Fv/fm), and Non-photochemical quenching (NPQ) also increased in the transgenetic plants. On the whole, overexpression of Lcε-LYC ameliorates photoinhibition and photooxidation, and decreases the sensitivity of photosynthesis to chilling stress in transgenic plants. PMID:26526130

  9. Transcriptional Profiling of Canker-Resistant Transgenic Sweet Orange (Citrus sinensis Osbeck) Constitutively Overexpressing a Spermidine Synthase Gene

    PubMed Central

    Fu, Xing-Zheng; Liu, Ji-Hong

    2013-01-01

    Citrus canker disease caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases affecting the citrus industry worldwide. In our previous study, the canker-resistant transgenic sweet orange (Citrus sinensis Osbeck) plants were produced via constitutively overexpressing a spermidine synthase. To unravel the molecular mechanisms underlying Xcc resistance of the transgenic plants, in the present study global transcriptional profiling was compared between untransformed line (WT) and the transgenic line (TG9) by hybridizing with Affymetrix Citrus GeneChip. In total, 666 differentially expressed genes (DEGs) were identified, 448 upregulated, and 218 downregulated. The DEGs were classified into 33 categories after Gene ontology (GO) annotation, in which 68 genes are in response to stimulus and involved in immune system process, 12 genes are related to cell wall, and 13 genes belong to transcription factors. These genes and those related to starch and sucrose metabolism, glutathione metabolism, biosynthesis of phenylpropanoids, and plant hormones were hypothesized to play major roles in the canker resistance of TG9. Semiquantitative RT-PCR analysis showed that the transcript levels of several candidate genes in TG9 were significantly higher than in WT both before and after Xcc inoculation, indicating their potential association with canker disease. PMID:23509803

  10. Physical exercise, neuroplasticity, spatial learning and memory.

    PubMed

    Cassilhas, Ricardo C; Tufik, Sergio; de Mello, Marco Túlio

    2016-03-01

    There has long been discussion regarding the positive effects of physical exercise on brain activity. However, physical exercise has only recently begun to receive the attention of the scientific community, with major interest in its effects on the cognitive functions, spatial learning and memory, as a non-drug method of maintaining brain health and treating neurodegenerative and/or psychiatric conditions. In humans, several studies have shown the beneficial effects of aerobic and resistance exercises in adult and geriatric populations. More recently, studies employing animal models have attempted to elucidate the mechanisms underlying neuroplasticity related to physical exercise-induced spatial learning and memory improvement, even under neurodegenerative conditions. In an attempt to clarify these issues, the present review aims to discuss the role of physical exercise in the improvement of spatial learning and memory and the cellular and molecular mechanisms involved in neuroplasticity. PMID:26646070

  11. Neuroplasticity and Predictors of Alcohol Recovery

    PubMed Central

    Seo, Dongju; Sinha, Rajita

    2015-01-01

    Chronic alcohol-related neuroadaptations in key neural circuits of emotional and cognitive control play a critical role in the development of, and recovery from, alcoholism. Converging evidence in the neurobiological literature indicates that neuroplastic changes in the prefrontal–striatal–limbic circuit, which governs emotion regulation and decisionmaking and controls physiological responses in the autonomic nervous system and hypothalamic–pituitary–adrenal axis system, contribute to chronic alcoholism and also are significant predictors of relapse and recovery. This paper reviews recent evidence on the neuroplasticity associated with alcoholism in humans, including acute and chronic effects, and how these neurobiological adaptations contribute to alcohol recovery, along with the discussion of relevant clinical implications and future research directions. PMID:26259094

  12. Abiotic and biotic stress tolerance in Arabidopsis overexpressing the multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene.

    PubMed

    Kim, Min-Jung; Lim, Gah-Hyun; Kim, Eun-Seon; Ko, Chang-Beom; Yang, Kwang-Yeol; Jeong, Jin-An; Lee, Myung-Chul; Kim, Cheol Soo

    2007-03-01

    We conducted a genetic yeast screen to identify salt tolerance (SAT) genes in a maize kernel cDNA library. During the screening, we identified a maize clone (SAT41) that seemed to confer elevated salt tolerance in comparison to control cells. SAT41 cDNA encodes a 16-kDa protein which is 82.4% identical to the Arabidopsis Multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene. To further examine salinity tolerance in Arabidopsis, we functionally characterized the MBF1a gene and found that dehydration as well as heightened glucose (Glc) induced MBF1a expression. Constitutive expression of MBF1a in Arabidopsis led to elevated salt tolerance in transgenic lines. Interestingly, plants overexpressing MBF1a exhibited insensitivity to Glc and resistance to fungal disease. Our results suggest that MBF1a is involved in stress tolerance as well as in ethylene and Glc signaling in Arabidopsis. PMID:17234157

  13. Neuroplasticity: Insights from Patients Harboring Gliomas

    PubMed Central

    2016-01-01

    Neuroplasticity is the ability of the brain to reorganize itself during normal development and in response to illness. Recent advances in neuroimaging and direct cortical stimulation in human subjects have given neuroscientists a window into the timing and functional anatomy of brain networks underlying this dynamic process. This review will discuss the current knowledge about the mechanisms underlying neuroplasticity, with a particular emphasis on reorganization following CNS pathology. First, traditional mechanisms of neuroplasticity, most relevant to learning and memory, will be addressed, followed by a review of adaptive mechanisms in response to pathology, particularly the recruitment of perilesional cortical regions and unmasking of latent connections. Next, we discuss the utility and limitations of various investigative techniques, such as direct electrocortical stimulation (DES), functional magnetic resonance imaging (fMRI), corticocortical evoked potential (CCEP), and diffusion tensor imaging (DTI). Finally, the clinical utility of these results will be highlighted as well as possible future studies aimed at better understanding of the plastic potential of the brain with the ultimate goal of improving quality of life for patients with neurologic injury. PMID:27478645

  14. Neuroplasticity: Insights from Patients Harboring Gliomas.

    PubMed

    Kong, Nathan W; Gibb, William R; Tate, Matthew C

    2016-01-01

    Neuroplasticity is the ability of the brain to reorganize itself during normal development and in response to illness. Recent advances in neuroimaging and direct cortical stimulation in human subjects have given neuroscientists a window into the timing and functional anatomy of brain networks underlying this dynamic process. This review will discuss the current knowledge about the mechanisms underlying neuroplasticity, with a particular emphasis on reorganization following CNS pathology. First, traditional mechanisms of neuroplasticity, most relevant to learning and memory, will be addressed, followed by a review of adaptive mechanisms in response to pathology, particularly the recruitment of perilesional cortical regions and unmasking of latent connections. Next, we discuss the utility and limitations of various investigative techniques, such as direct electrocortical stimulation (DES), functional magnetic resonance imaging (fMRI), corticocortical evoked potential (CCEP), and diffusion tensor imaging (DTI). Finally, the clinical utility of these results will be highlighted as well as possible future studies aimed at better understanding of the plastic potential of the brain with the ultimate goal of improving quality of life for patients with neurologic injury. PMID:27478645

  15. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster

    PubMed Central

    Bai, Chang; Connolly, Brett; Metzker, Michael L.; Hilliard, Catherine A.; Liu, Xiaomei; Sandig, Volker; Soderman, Avery; Galloway, Sheila M.; Liu, Qingyun; Austin, Christopher P.; Caskey, C. Thomas

    2000-01-01

    Fas-mediated apoptosis is an important regulator of cell survival, and abnormalities in this system have been shown to result in a number of human pathological conditions. A secreted member of the tumor necrosis factor receptor superfamily, DcR3, was recently reported to be amplified in human lung and colon cancers as a negative regulator of Fas-mediated apoptosis. We identified this gene, which we call M68. M68 genomic DNA, mRNA, and protein levels were examined in a series of human gastrointestinal tract tumors. Using M68 immunohistochemistry and a scoring system similar to that used for HER-2/neu, we found that M68 protein was overexpressed in 30 of 68 (44%) human adenocarcinomas of the esophagus, stomach, colon, and rectum. Tumors examined by Northern blot revealed M68 mRNA highly elevated in a similar fraction of primary tumors from the same gastrointestinal tract regions, as well as in the colon adenocarcinoma cell lines SW480 and SW1116. Further, we found M68 protein to be overexpressed in a substantial number of tumors in which gene amplification could not be detected by fluorescence in situ hybridization or quantitative genomic PCR, suggesting that overexpression of M68 may precede amplification in tumors. Finally, we find that M68 lies within a four-gene cluster that includes a novel helicase-like gene (NHL) related to RAD3/ERCC2, a plasma membrane Ras-related GTPase and a member of the stathmin family, amplification or overexpression of which may also contribute to cell growth and tumor progression. PMID:10655513

  16. Identification of four novel human genes amplified and overexpressed in breast carcinoma and localized to the q11-q21.3 region of chromosome 17

    SciTech Connect

    Tomasetto, C.; Regnier, C.; Basset, P.

    1995-08-10

    We have performed differential screening of a human metastatic lymph node cDNA library to identify genes possibly involved during breast cancer progression. We have identified four novel genes overexpressed in malignant tissues. They were all located between q11 and q21.3, a region known to contain the c-erbB-2 oncogene and the BRCA1 breast carcinomas, and overexpression of three of them was dependent on gene amplification in breast cancer cell lines. These findings further support the concept that human chromosome 17 specifically carries genes possibly involved in breast cancer progression. 61 refs., 3 figs., 4 tabs.

  17. CTNNB1 mutations and overexpression of Wnt/beta-catenin target genes in WT1-mutant Wilms' tumors.

    PubMed

    Li, Chi-Ming; Kim, Connie E; Margolin, Adam A; Guo, Meirong; Zhu, Jimmy; Mason, Jacqueline M; Hensle, Terrence W; Murty, Vundavalli V V S; Grundy, Paul E; Fearon, Eric R; D'Agati, Vivette; Licht, Jonathan D; Tycko, Benjamin

    2004-12-01

    Gain-of-function mutations in exon 3 of beta-catenin (CTNNB1) are specific for Wilms' tumors that have lost WT1, but 50% of WT1-mutant cases lack such "hot spot" mutations. To ask whether stabilization of beta-catenin might be essential after WT1 loss, and to identify downstream target genes, we compared expression profiles in WT1-mutant versus WT1 wild-type Wilms' tumors. Supervised and nonsupervised hierarchical clustering of the expression data separated these two classes of Wilms' tumor. The WT1-mutant tumors overexpressed genes encoding myogenic and other transcription factors (MOX2, LBX1, SIM2), signaling molecules (TGFB2, FST, BMP2A), extracellular Wnt inhibitors (WIF1, SFRP4), and known beta-catenin/TCF targets (FST, CSPG2, CMYC). Beta-Catenin/TCF target genes were overexpressed in the WT1-mutant tumors even in the absence of CTNNB1 exon 3 mutations, and complete sequencing revealed gain-of-function mutations elsewhere in the CTNNB1 gene in some of these tumors, increasing the overall mutation frequency to 75%. Lastly, we identified and validated a novel direct beta-catenin target gene, GAD1, among the WT1-mutant signature genes. These data highlight two molecular classes of Wilms' tumor, and indicate strong selection for stabilization of beta-catenin in the WT1-mutant class. Beta-Catenin stabilization can initiate tumorigenesis in other systems, and this mechanism is likely critical in tumor formation after loss of WT1. PMID:15579438

  18. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    PubMed Central

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-01-01

    AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

  19. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

    PubMed Central

    Shi, Yanmei; Guo, Jinggong; Zhang, Wei; Jin, Lifeng; Liu, Pingping; Chen, Xia; Li, Feng; Wei, Pan; Li, Zefeng; Li, Wenzheng; Wei, Chunyang; Zheng, Qingxia; Chen, Qiansi; Zhang, Jianfeng; Lin, Fucheng; Qu, Lingbo; Snyder, John Hugh; Wang, Ran

    2015-01-01

    Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum. PMID:26703579

  20. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

    PubMed

    Shi, Yanmei; Guo, Jinggong; Zhang, Wei; Jin, Lifeng; Liu, Pingping; Chen, Xia; Li, Feng; Wei, Pan; Li, Zefeng; Li, Wenzheng; Wei, Chunyang; Zheng, Qingxia; Chen, Qiansi; Zhang, Jianfeng; Lin, Fucheng; Qu, Lingbo; Snyder, John Hugh; Wang, Ran

    2015-01-01

    Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum. PMID:26703579

  1. Overexpression of MuHSP70 gene from Macrotyloma uniflorum confers multiple abiotic stress tolerance in transgenic Arabidopsis thaliana.

    PubMed

    Masand, Shikha; Yadav, Sudesh Kumar

    2016-02-01

    A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H2O2, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program. PMID:26694324

  2. A screening method to identify genes commonly overexpressed in carcinomas and the identification of a novel complementary DNA sequence.

    PubMed

    Byrne, J A; Tomasetto, C; Garnier, J M; Rouyer, N; Mattei, M G; Bellocq, J P; Rio, M C; Basset, P

    1995-07-01

    We describe a differential screening method for cDNA libraries which used a combination of subtracted and PCR-amplified cDNA probes, and which can be applied to the selection of genes expressed in multiple tissues. This technique was used to identify genes commonly overexpressed in breast and basal cell carcinomas. These represent stromally dependent, invasive tumors with and without metastatic capacity. Thus, this screening sought to identify genes involved in the early stages of tumor progression. We identified a total of 16 genes, including c-erbB-2 and tissue inhibitor of metalloproteinases 3 whose products have been implicated in tumorigenesis or invasion. We also identified a novel sequence (D52) showing little homology with others described in any species, which maps to the human chromosomal band 8q21. In situ RNA hybridizations of breast carcinoma sections indicated that the D52 gene was expressed in cancer cells, whereas other genes identified in the differential screening were expressed in fibroblastic or inflammatory cells within the tumor stroma. Thus, the procedure developed in this study selected genes expressed in a diversity of cell types, indicating its potential usefulness in other systems. PMID:7796418

  3. Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.

    PubMed Central

    Brocca, S.; Schmidt-Dannert, C.; Lotti, M.; Alberghina, L.; Schmid, R. D.

    1998-01-01

    The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms. PMID:9655346

  4. Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

    PubMed Central

    Yang, Qingyong; Cheng, Yan; Ma, Ming; Kliebenstein, Daniel J.; Zhou, Yongming

    2015-01-01

    Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies. PMID:26465156

  5. Genetic variants in the HER2 gene: Influence on HER2 overexpression and loss of heterozygosity in breast cancer.

    PubMed

    Cresti, Nicola; Lee, Joanne; Rourke, Emma; Televantou, Despina; Jamieson, David; Verrill, Mark; Boddy, Alan V

    2016-03-01

    Human epidermal growth factor receptor 2 (HER2) overexpression in breast cancer is an indicator of poor prognosis and is the pre-requisite for treatment with the agents targeting this member of the epidermal growth factor receptor family. In order to determine the influence of these common single-nucleotide polymorphisms (SNPs) in the HER2 gene, genomic DNA was obtained from 361 patients with breast cancer, aged between 29 and 82 years. Samples of tumour tissue were obtained from 241 (66%) patients and material for extraction of DNA is isolated from surrounding normal tissue by laser capture microdissection. Genotyping was performed using the Taqman fluorogenic 5' nuclease assay. Of the 360 patients with definitive determination of HER2 status, 49% were positive. The Ile655Val SNP had no influence on the frequency of HER2 expression. However, the proline allele of the Ala1170Pro SNP was associated with a higher frequency of HER2 overexpression (56% versus 43%, p = 0.015). Where the germline genotype was homozygous, the tumour genotype was identical in every case and for both SNPs. In HER2-positive tumours, heterozygosity was maintained in only 15% and 18% of the Ile655Val and Ala1170Pro SNPs, respectively. This was lower than in the HER2-negative tumours (46% and 43%, respectively). Normal breast tissue (n = 23) retained the germline genotype in all but one case. The underlying link between the Ala1170Pro SNP and HER2 positivity is not known, nor is the significance of HER2 overexpression and loss of heterozygosity in breast cancer. However, these results illustrate the complexity of HER2 genotype and overexpression in this disease. PMID:26773371

  6. Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

    PubMed

    Zhang, Yuanyuan; Huai, Dongxin; Yang, Qingyong; Cheng, Yan; Ma, Ming; Kliebenstein, Daniel J; Zhou, Yongming

    2015-01-01

    Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies. PMID:26465156

  7. Overexpression of miR-506 suppresses proliferation and promotes apoptosis of osteosarcoma cells by targeting astrocyte elevated gene-1

    PubMed Central

    Yao, Jie; Qin, Li; Miao, Sen; Wang, Xiangshan; Wu, Xuejian

    2016-01-01

    There is increasing evidence that microRNAs (miRs) are implicated in tumor development and progression; however, their specific roles in osteosarcoma are not well understood. The aim of the present study was to investigate the role of miR-506 in the pathogenesis of osteosarcoma. The expression levels of miR-506 and astrocyte elevated gene-1 (AEG-1) mRNA were detected using quantitative polymerase chain reaction, and the protein levels of AEG-1, β-catenin, c-myc and cyclin D1 were determined using western blot analysis. The effects of miR-506 and AEG-1 on cell viability, colony forming ability and apoptosis were assessed using MTT assay, colony formation assay, and flow cytometry, respectively. Lucifer reporter assays were used to demonstrate whether AEG-1 is a direct target of miR-506. The present study identified that miR-506 was downregulated in osteosarcoma tissues and cells. Overexpression of miR-506 suppressed the proliferation and induced apoptosis in osteosarcoma cells in vitro and inhibited tumor formation in vivo. Overexpression of miR-506 significantly inhibited the luciferase activity of AEG-1 with a wild-type 3′-untranslated region, providing clear evidence that AEG-1 was a direct and functional downstream target of miR-506. Similar to the overexpression of miR-506, downregulation of AEG-1 lead to an inhibitory effect on osteosarcoma in vitro. Furthermore, overexpression of miR-506 or downregulation of AEG-1 inhibited the Wnt/β-catenin signaling pathway, and inhibition of this pathway by β-catenin small interfering RNA or CGP049090, a small molecule inhibitor, suppressed cell proliferation and induced apoptosis in vitro. Overall, the present data indicated that miR-506 functions as a tumor suppressor by targeting AEG-1 in osteosarcoma via the regulation of the Wnt/β-catenin signaling pathway. PMID:27602115

  8. Overexpression of Pleomorphic Adenoma Gene-Like 2 Is a Novel Poor Prognostic Marker of Prostate Cancer

    PubMed Central

    Guo, Jia; Wang, Min; Wang, Zhishun; Liu, Xiuheng

    2016-01-01

    Pleomorphic adenoma gene like-2 (PLAGL2) is a member of the PLAG gene family. Previous studies have revealed that overexpression of PLAGL2 is associated with many human cancers. However, it has been reported that PLAGL2 also plays as a tumor suppressor. The precise role of PLAGL2 in prostate cancer (PCa) is still unknown. The aim of this study was to investigate the expression and prognostic value of PLAGL2 in PCa. Data from microarray datasets demonstrated that the DNA copy number and mRNA level of PLAGL2 were significantly increased in PCa compared with normal prostate. qRT-PCR and western blot analysis from paired PCa samples and prostate cell lines confirmed upregulated mRNA and protein expression levels in PCa. Immunohistochemistry analysis showed that staining of PLAGL2 in PCa tissues was significantly higher than that in benign prostatic hyperplasia (BPH) tissues. In addition, the high expression of PLAGL2 was only involved in preoperative PSA, but was not related to age, Gleason score, seminal vesicle invasion, surgical margin status, clinical stage and positive lymph node metastasis. Moreover, our results showed that PLAGL2 was an independent prognostic factor for biochemical recurrence (BCR)-free survival and overall survival (OS) of PCa patients, and overexpressed PLAGL2 was related to early development of BCR and poor OS. In conclusion, our findings suggest that PLAGL2 is overexpressed in PCa. The increased expression of PLAGL2 correlates to PCa progression following radical prostatectomy and may serve as a novel poor prognostic marker for PCa. PMID:27537362

  9. Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1)

    PubMed Central

    Sancisi, Valentina; Germinario, Elena; Esposito, Alessandra; Morini, Elisabetta; Peron, Samantha; Moggio, Maurizio; Tomelleri, Giuliano; Danieli-Betto, Daniela

    2013-01-01

    Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca2+. We demonstrate that the decrease of Ca2+ sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression. PMID:24305066

  10. Gene overexpression as a tool for identifying new trans-acting factors involved in translation termination in Saccharomyces cerevisiae.

    PubMed Central

    Namy, Olivier; Hatin, Isabelle; Stahl, Guillaume; Liu, Hongmei; Barnay, Stephanie; Bidou, Laure; Rousset, Jean-Pierre

    2002-01-01

    In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process. PMID:12072456

  11. A Histologically Distinctive Interstitial Pneumonia Induced by Overexpression of the Interleukin 6, Transforming Growth Factor β1, or Platelet-Derived Growth Factor B Gene

    NASA Astrophysics Data System (ADS)

    Yoshida, Mitsuhiro; Sakuma, Junko; Hayashi, Seiji; Abe, Kin'ya; Saito, Izumu; Harada, Shizuko; Sakatani, Mitsunoir; Yamamoto, Satoru; Matsumoto, Norinao; Kaneda, Yasufumi; Kishmoto, Tadamitsu

    1995-10-01

    Interstitial pneumonia is characterized by alveolitis with resulting fibrosis of the interstitium. To determine the relevance of humoral factors in the pathogenesis of interstitial pneumonia, we introduced expression vectors into Wistar rats via the trachea to locally overexpress humoral factors in the lungs. Human interleukin (IL) 6 and IL-6 receptor genes induced lymphocytic alveolitis without marked fibroblast proliferation. In contrast, overexpression of human transforming growth factor β1 or human platelet-derived growth factor B gene induced only mild or apparent cellular infiltration in the alveoli, respectively. However, both factors induced significant proliferation of fibroblasts and deposition of collagen fibrils. These histopathologic changes induced by the transforming growth factor β1 and platelet-derived growth factor B gene are partly akin to those changes seen in lung tissues from patients with pulmonary fibrosis and markedly contrast with the changes induced by overexpression of the IL-6 and IL-6 receptor genes that mimics lymphocytic interstitial pneumonia.

  12. Process and genes for expression and overexpression of active [FeFe] hydrogenases

    DOEpatents

    Seibert, Michael; King, Paul W; Ghirardi, Maria Lucia; Posewitz, Matthew C; Smolinski, Sharon L

    2014-09-16

    A process for expression of active [FeFe]-hydrogenase in a host organism that does not contain either the structural gene(s) for [FeFe]-hydrogenases and/or homologues for the maturation genes HydE, HydF and HyG, comprising: cloning the structural hydrogenase gene(s) and/or the maturation genes HydE, HydF and HydG from an organisms that contains these genes into expression plasmids; transferring the plasmids into an organism that lacks a native [FeFe]-hydrogenase or that has a disrupted [FeFe]-hydrogenase and culturing it aerobically; and inducing anaerobiosis to provide [FeFe] hydrogenase biosynthesis and H?2#191 production.

  13. Overexpression of MdbHLH104 gene enhances the tolerance to iron deficiency in apple.

    PubMed

    Zhao, Qiang; Ren, Yi-Ran; Wang, Qing-Jie; Yao, Yu-Xin; You, Chun-Xiang; Hao, Yu-Jin

    2016-07-01

    Fe deficiency is a widespread nutritional disorder in plants. The basic helix-loop-helix (bHLH) transcription factors (TFs), especially Ib subgroup bHLH TFs which are involved in iron uptake, have been identified. In this study, an IVc subgroup bHLH TF MdbHLH104 was identified and characterized as a key component in the response to Fe deficiency in apple. The overexpression of the MdbHLH104 gene noticeably increased the H(+) -ATPase activity under iron limitation conditions and the tolerance to Fe deficiency in transgenic apple plants and calli. Further investigation showed that MdbHLH104 proteins bonded directly to the promoter of the MdAHA8 gene, thereby positively regulating its expression, the plasma membrane (PM) H(+) -ATPase activity and Fe uptake. Similarly, MdbHLH104 directly modulated the expression of three Fe-responsive bHLH genes, MdbHLH38, MdbHLH39 and MdPYE. In addition, MdbHLH104 interacted with 5 other IVc subgroup bHLH proteins to coregulate the expression of the MdAHA8 gene, the activity of PM H(+) -ATPase and the content of Fe in apple calli. Therefore, MdbHLH104 acts together with other apple bHLH TFs to regulate Fe uptake by modulating the expression of the MdAHA8 gene and the activity of PM H(+) -ATPase in apple. PMID:26801352

  14. Genomic loss of EZH2 leads to epigenetic modifications and overexpression of the HOX gene clusters in myelodysplastic syndrome

    PubMed Central

    Chang, Chun-Kang; He, Qi; Wu, Ling-Yun; Zhang, Zheng; Shi, Wen-Hui; Guo, Juan; Zhu, Yang; Zhao, You-Shan; Gu, Shu-Cheng; Fei, Cheng-Ming; Li, Xiao

    2016-01-01

    The role of EZH2 in cancer is complex and may vary depending on cancer type or stage. We examined the effect of altered EZH2 levels on H3K27 methylation, HOX gene expression, and malignant phenotype in myelodysplastic syndrome (MDS) cell lines and an in vivo xenograft model. We also studied links between EZH2 expression and prognosis in MDS patients. Patients with high-grade MDS exhibited lower levels of EZH2 expression than those with low-grade MDS. Low EZH2 expression was associated with high percentages of blasts, shorter survival, and increased transformation of MDS into acute myeloid leukemia (AML). MDS patients frequently had reductions in EZH2 copy number. EZH2 knockdown increased tumor growth capacity and reduced H3K27me3 levels in both MDS-derived leukemia cells and in a xenograft model. H3K27me3 levels were reduced and HOX gene cluster expression was increased in MDS patients. EZH2 knockdown also increased HOX gene cluster expression by reducing H3K27me3, and H3K27 demethylating agents increased HOX gene cluster expression in MDS-derived cell lines. These findings suggest genomic loss of EZH2 contributes to overexpression of the HOX gene clusters in MDS through epigenetic modifications. PMID:26812882

  15. Neuroplasticity Signaling Pathways Linked to the Pathophysiology of Schizophrenia

    PubMed Central

    Balu, Darrick T.; Coyle, Joseph T.

    2010-01-01

    Schizophrenia is a severe mental illness that afflicts nearly 1% of the world's population. One of the cardinal pathological features of schizophrenia is perturbation in synaptic connectivity. Although the etiology of schizophrenia is unknown, it appears to be a developmental disorder involving the interaction of a potentially large number of risk genes, with no one gene producing a strong effect except rare, highly penetrant copy number variants. The purpose of this review is to detail how putative schizophrenia risk genes (DISC-1, neuregulin/ErbB4, dysbindin, Akt1, BDNF, and NMDA receptor) are involved in regulating neuroplasticity and how alterations in their expression may contribute to the disconnectivity observed in schizophrenia. Moreover, this review highlights how many of these risk genes converge to regulate common neurotransmitter systems and signaling pathways. Future studies aimed at elucidating the functions of these risk genes will provide new insights into the pathophysiology of schizophrenia and will likely lead to the nomination of novel therapeutic targets for restoring proper synaptic connectivity in the brain in schizophrenia and related disorders. PMID:20951727

  16. RNA Interference Improves Myopathic Phenotypes in Mice Over-expressing FSHD Region Gene 1 (FRG1)

    PubMed Central

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

    2011-01-01

    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. PMID:21730972

  17. 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. PMID:21730972

  18. Harnessing the power of neuroplasticity for intervention

    PubMed Central

    Kolb, Bryan; Muhammad, Arif

    2014-01-01

    A fundamental property of the brain is its capacity to change with a wide variety of experiences, including injury. Although there are spontaneous reparative changes following injury, these changes are rarely sufficient to support significant functional recovery. Research on the basic principles of brain plasticity is leading to new approaches to treating the injured brain. We review factors that affect synaptic organization in the normal brain, evidence of spontaneous neuroplasticity after injury, and the evidence that factors including postinjury experience, pharmacotherapy, and cell-based therapies, can form the basis of rehabilitation strategies after brain injuries early in life and in adulthood. PMID:25018713

  19. Dental Occlusal Changes Induce Motor Cortex Neuroplasticity.

    PubMed

    Avivi-Arber, L; Lee, J-C; Sessle, B J

    2015-12-01

    Modification to the dental occlusion may alter oral sensorimotor functions. Restorative treatments aim to restore sensorimotor functions; however, it is unclear why some patients fail to adapt to the restoration and remain with sensorimotor complaints. The face primary motor cortex (face-M1) is involved in the generation and control of orofacial movements. Altered sensory inputs or motor function can induce face-M1 neuroplasticity. We took advantage of the continuous eruption of the incisors in Sprague-Dawley rats and used intracortical microstimulation (ICMS) to map the jaw and tongue motor representations in face-M1. Specifically, we tested the hypothesis that multiple trimming of the right mandibular incisor, to keep it out of occlusal contacts for 7 d, and subsequent incisor eruption and restoration of occlusal contacts, can alter the ICMS-defined features of jaw and tongue motor representations (i.e., neuroplasticity). On days 1, 3, 5, and 7, the trim and trim-recovered groups had 1 to 2 mm of incisal trimming of the incisor; a sham trim group had buccal surface trimming with no occlusal changes; and a naive group had no treatment. Systematic mapping was performed on day 8 in the naive, trim, and sham trim groups and on day 14 in the trim-recovered group. In the trim group, the tongue onset latency was shorter in the left face-M1 than in the right face-M1 (P < .001). In the trim-recovered group, the number of tongue sites and jaw/tongue overlapping sites was greater in the left face-M1 than in the right face-M1 (P = 0.0032, 0.0016, respectively), and the center of gravity was deeper in the left than in the right face-M1 (P = 0.026). Therefore, incisor trimming and subsequent restoration of occlusal contacts induced face-M1 neuroplasticity, reflected in significant disparities between the left and right face-M1 in some ICMS-defined features of the tongue motor representations. Such neuroplasticity may reflect or contribute to subjects' ability to adapt their

  20. Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus

    PubMed Central

    Flaherty, J. E.; Payne, G. A.

    1997-01-01

    The aflatoxin biosynthetic pathway regulatory gene, aflR, encodes a putative 47-kDa protein containing a zinc cluster DNA binding motif. It is required for the transcription of all of the characterized aflatoxin pathway genes in both Aspergillus flavus and Aspergillus parasiticus. The objective of this study was to examine the effects of aflR overexpression on temporal gene expression, aflatoxin production, and nitrate inhibition of aflatoxin biosynthesis in A. flavus. An inducible expression construct was made by fusing the coding region of aflR to the promoter region of the A. flavus adh1 gene. This construct was transformed into A. flavus 656-2 (FGSC A1010), a strain mutated at the aflR locus. Strain 656-2 containing the adh1(p)::aflR construct had induced transcription of two early aflatoxin pathway genes, nor-1 and pksA, and produced wild-type concentrations of aflatoxin in a temporal pattern similar to that of wild-type strains of A. flavus. Strains 656-2 and 86-10 (FGSC A1009) an aflatoxigenic strain, were transformed with a construct containing the constitutive promoter gpdA driving aflR. Transformants of these strains constitutively expressed aflR, fas-1A, pksA, nor-1, and omtA but did not constitutively produce aflatoxin. Strain 86-10 containing the gpdA(p)::aflR construct produced 50 times more aflatoxin than 86-10, but the temporal pattern of aflatoxin production was the same as for 86-10, and aflatoxin production was also induced by sucrose. The addition of 10 g of nitrate per liter to sucrose low salts medium inhibited aflatoxin production by both strain 86-10 and a transformant of 86-10 containing the gpdA(p)::aflR construct, indicating that nitrate inhibition of aflatoxin biosynthesis does not occur solely at the level of aflR transcription. These studies show that constitutive overexpression of the pathway transcriptional regulatory gene aflR leads to higher transcript accumulation of pathway genes and increased aflatoxin production but that the

  1. Liposome-mediated in vivo E1A gene transfer suppressed dissemination of ovarian cancer cells that overexpress HER-2/neu.

    PubMed

    Yu, D; Matin, A; Xia, W; Sorgi, F; Huang, L; Hung, M C

    1995-10-01

    The HER-2/neu proto-oncogene is frequently amplified or overexpressed in many different types of human cancers, a phenomenon that has been shown to correlate with shorter survival time and lower survival rate in ovarian cancer patients. We previously reported that increased HER-2/neu expression led to more severe malignancy and increased metastatic potential in animal models and that the adenovirus 5 E1A gene repressed HER-2/neu gene expression at transcriptional level and was able to suppress tumor growth when stably transfected into human ovarian cancer SKOV-3 cells which overexpress HER-2/neu. To investigate whether the E1A gene may be used as a therapeutic agent for HER-2/neu-overexpressing human cancers in living hosts, we first developed tumor-bearing mice by injecting SKOV-3 cells that overexpress HER-2/neu intraperitonealy into female nu/nu mice. Five days later, we used cationic liposomes to directly deliver the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice that received the SKOV-3 cell injection. We found that liposome-mediated E1A gene transfer significantly inhibited growth and dissemination of ovarian cancer cells that overexpress HER-2/neu in the treated mice; about 70% of these mice survived at least 365 days, whereas all the control mice that did not receive the gene therapy developed severe tumor symptoms and died within 160 days. The results suggest that liposome-mediated E1A gene transfer may serve as an effective therapy for human ovarian cancers that overexpress HER-2/neu by directly targeting the HER-2/neu oncogene. PMID:7478560

  2. A screen of apoptosis and senescence regulatory genes for life span effects when over-expressed in Drosophila

    PubMed Central

    Shen, Jie; Curtis, Christina; Tavaré, Simon; Tower, John

    2009-01-01

    Conditional expression of transgenes in Drosophila was produced using the Geneswitch system, wherein feeding the drug RU486/Mifepristone activates the artificial transcription factor Geneswitch. Geneswitch was expressed using the Actin5C promoter and this was found to yield conditional, tissue-general expression of a target transgene (UAS-GFP) in both larvae and adult flies. Nervous system-specific (Elav-GS) and fat body-specific Geneswitch drivers were also characterized using UAS-GFP. Fourteen genes implicated in growth, apoptosis and senescence regulatory pathways were over-expressed in adult flies or during larval development, and assayed for effects on adult fly life span. Over-expression of a dominant p53 allele (p53-259H) in adult flies using the ubiquitous driver produced increased life span in females but not males, consistent with previous studies. Both wingless and Ras activated form transgenes were lethal when expressed in larvae, and reduced life span when expressed in adults, consistent with results from other model systems indicating that the wingless and Ras pathways can promote senescence. Over-expression of the caspase inhibitor baculovirus p35 during larval development reduced the mean life span of male and female adults, and also produced a subset of females with increased life span. These experiments suggest that baculovirus p35 and the wingless and Ras pathways can have sex-specific and developmental stage-specific effects on adult Drosophila life span, and these reagents should be useful for the further analysis of the role of these conserved pathways in aging. PMID:20157509

  3. Overexpression of GbRLK, a putative receptor-like kinase gene, improved cotton tolerance to Verticillium wilt

    PubMed Central

    Jun, Zhao; Zhang, Zhiyuan; Gao, Yulong; Zhou, Lei; Fang, Lei; Chen, Xiangdong; Ning, Zhiyuan; Chen, Tianzi; Guo, Wangzhen; Zhang, Tianzhen

    2015-01-01

    Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression. PMID:26446555

  4. 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+). PMID:24594398

  5. Overexpression of folate biosynthesis genes in rice (Oryza sativa L.) and evaluation of their impact on seed folate content.

    PubMed

    Dong, Wei; Cheng, Zhi-jun; Lei, Cai-lin; Wang, Xiao-le; Wang, Jiu-lin; Wang, Jie; Wu, Fu-qing; Zhang, Xin; Guo, Xiu-ping; Zhai, Hu-qu; Wan, Jian-min

    2014-12-01

    Folate (vitamin B9) deficiency is a global health problem especially in developing countries where the major staple foods such as rice contain extremely low folates. Biofortification of rice could be an alternative complement way to fight folate deficiency. In this study, we evaluated the availability of the genes in each step of folate biosynthesis pathway for rice folate enhancement in the japonica variety kitaake genetic background. The first enzymes GTP cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase (ADCS) in the pterin and para-aminobenzoate branches resulted in significant increase in seed folate content, respectively (P < 0.01). Overexpression of two closely related enzymes dihydrofolate synthase (DHFS) and folypolyglutamate synthase (FPGS), which perform the first and further additions of glutamates, produced slightly increase in seed folate content separately. The GTPCHI transgene was combined with each of the other transgenes except ADCS to investigate the effects of gene stacking on seed folate accumulation. Seed folate contents in the gene-stacked plants were higher than the individual low-folate transgenic parents, but lower than the high-folate GTPCHI transgenic lines, pointing to an inadequate supply of para-aminobenzoic acid (PABA) precursor initiated by ADCS in constraining folate overproduction in gene-stacked plants. PMID:25432789

  6. Field performance of transgenic 'M.26' apples overexpressing a peach CBF gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability to cold acclimate and undergo a period of dormancy is essential to temperate woody plants in order to survive freezing winter temperatures. CBF genes have been shown to regulate a large number of cold-regulated (COR) genes whose products are thought to contribute to freezing tolerance. ...

  7. 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. PMID:26712321

  8. Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1

    PubMed Central

    Brunetti, Patrizia; Zanella, Letizia; Proia, Alessandra; De Paolis, Angelo; Falasca, Giuseppina; Altamura, Maria Maddalena; Sanità di Toppi, Luigi; Costantino, Paolo; Cardarelli, Maura

    2011-01-01

    Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length. PMID:21841172

  9. Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1.

    PubMed

    Brunetti, Patrizia; Zanella, Letizia; Proia, Alessandra; De Paolis, Angelo; Falasca, Giuseppina; Altamura, Maria Maddalena; Sanità di Toppi, Luigi; Costantino, Paolo; Cardarelli, Maura

    2011-11-01

    Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length. PMID:21841172

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

    PubMed Central

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

    2012-01-01

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

  11. Cloning a glutathione peroxidase gene from Nelumbo nucifera and enhanced salt tolerance by overexpressing in rice.

    PubMed

    Diao, Ying; Xu, Huaxue; Li, Guolin; Yu, Aiqing; Yu, Xia; Hu, Wanling; Zheng, Xingfei; Li, Shaoqing; Wang, Youwei; Hu, Zhongli

    2014-08-01

    A full-length cDNA clone encoding an 866 bp-length glutathione peroxidase protein (NnGPX) was isolated from lotus (Nelumbo nucifera L.). The deduced amino acid sequence of the NnGPX gene had significant homology with ATGPX6. A 3D structural model of the NnGPX was constructed by homology modeling. The cloned NnGPX gene was expressed in Escherichia coli, and a fusion protein of about 40 kDa was detected after isopropyl thiogalactoside induction. Under different concentrations of Na2SeO3 treatments, NnGPX was found to be an enzyme that does not contain selenium. Real-time PCR analysis showed that the NnGPX gene was expressed in all organs of lotus, and its high expression mainly occurred in organs with active metabolisms. NnGPX transcript increased remarkably in response to cold, heat, mechanical damage, and salt treatment. Subsequently, the NnGPX gene was introduced in Oryza sativa cv. Yuetai B. PCR results verified the integration of this gene into the genome of rice and reverse transcription-PCR verified that this gene had been expressed in transgenic rice. The transgenic plants were significantly more tolerant to salt stress compared with the wild-type. PMID:24715609

  12. Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

    PubMed

    Nagy, Zsolt; Acs, Bence; Butz, Henriett; Feldman, Karolina; Marta, Alexa; Szabo, Peter M; Baghy, Kornelia; Pazmany, Tamas; Racz, Karoly; Liko, Istvan; Patocs, Attila

    2016-01-01

    The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies. PMID:26480216

  13. Frequent methylation of the KLOTHO gene and overexpression of the FGFR4 receptor in invasive ductal carcinoma of the breast.

    PubMed

    Dallol, Ashraf; Buhmeida, Abdelbaset; Merdad, Adnan; Al-Maghrabi, Jaudah; Gari, Mamdooh A; Abu-Elmagd, Muhammad M; Elaimi, Aisha; Assidi, Mourad; Chaudhary, Adeel G; Abuzenadah, Adel M; Nedjadi, Taoufik; Ermiah, Eramah; Alkhayyat, Shadi S; Al-Qahtani, Mohammed H

    2015-12-01

    Invasive ductal carcinoma of the breast is the most common cancer affecting women worldwide. The marked heterogeneity of breast cancer is matched only with the heterogeneity in its associated or causative factors. Breast cancer in Saudi Arabia is apparently an early onset with many of the affected females diagnosed before they reach the age of 50 years. One possible rationale underlying this observation is that consanguinity, which is widely spread in the Saudi community, is causing the accumulation of yet undetermined cancer susceptibility mutations. Another factor could be the accumulation of epigenetic aberrations caused by the shift toward a Western-like lifestyle in the past two decades. In order to shed some light into the molecular mechanisms underlying breast cancer in the Saudi community, we identified KLOTHO (KL) as a tumor-specific methylated gene using genome-wide methylation analysis of primary breast tumors utilizing the MBD-seq approach. KL methylation was frequent as it was detected in 55.3 % of breast cancer cases from Saudi Arabia (n = 179) using MethyLight assay. Furthermore, KL is downregulated in breast tumors with its expression induced following treatment with 5-azacytidine. The involvement of KL in breast cancer led us to investigate its relationship in the context of breast cancer, with one of the protagonists of its function, fibroblast growth factor receptor 4 (FGFR4). Overexpression of FGFR4 in breast cancer is frequent in our cohort and this overexpression is associated with poor overall survival. Interestingly, FGFR4 expression is higher in the absence of KL methylation and lower when KL is methylated and presumably silenced, which is suggestive of an intricate relationship between the two factors. In conclusion, our findings further implicate "metabolic" genes or pathways in breast cancer that are disrupted by epigenetic mechanisms and could provide new avenues for understanding this disease in a new context. PMID:26152288

  14. Enhanced accumulation of carotenoids in sweetpotato plants overexpressing IbOr-Ins gene in purple-fleshed sweetpotato cultivar.

    PubMed

    Park, Sung-Chul; Kim, Sun Ha; Park, Seyeon; Lee, Hyeong-Un; Lee, Joon Seol; Park, Woo Sung; Ahn, Mi-Jeong; Kim, Yun-Hee; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

    2015-01-01

    Sweetpotato [Ipomoea batatas (L.) Lam] is an important root crop that produces low molecular weight antioxidants such as carotenoids and anthocyanin. The sweetpotato orange (IbOr) protein is involved in the accumulation of carotenoids. To increase the levels of carotenoids in the storage roots of sweetpotato, we generated transgenic sweetpotato plants overexpressing IbOr-Ins under the control of the cauliflower mosaic virus (CaMV) 35S promoter in an anthocyanin-rich purple-fleshed cultivar (referred to as IbOr plants). IbOr plants exhibited increased carotenoid levels (up to 7-fold) in their storage roots compared to wild type (WT) plants, as revealed by HPLC analysis. The carotenoid contents of IbOr plants were positively correlated with IbOr transcript levels. The levels of zeaxanthin were ∼ 12 times elevated in IbOr plants, whereas β-carotene increased ∼ 1.75 times higher than those of WT. Quantitative RT-PCR analysis revealed that most carotenoid biosynthetic pathway genes were up-regulated in the IbOr plants, including PDS, ZDS, LCY-β, CHY-β, ZEP and Pftf, whereas LCY-ɛ was down-regulated. Interestingly, CCD1, CCD4 and NCED, which are related to the degradation of carotenoids, were also up-regulated in the IbOr plants. Anthocyanin contents and transcription levels of associated biosynthetic genes seemed to be altered in the IbOr plants. The yields of storage roots and aerial parts of IbOr plants and WT plants were not significantly different under field cultivation. Taken together, these results indicate that overexpression of IbOr-Ins can increase the carotenoid contents of sweetpotato storage roots. PMID:25438140

  15. Overexpression of ShDHN, a dehydrin gene from Solanum habrochaites enhances tolerance to multiple abiotic stresses in tomato.

    PubMed

    Liu, Hui; Yu, Chuying; Li, Hanxia; Ouyang, Bo; Wang, Taotao; Zhang, Junhong; Wang, Xin; Ye, Zhibiao

    2015-02-01

    Dehydrins (DHNs) play important roles in plant adaptation to abiotic stress. In this study, a cold-induced SK3-type DHN gene (ShDHN) isolated from wild tomato species Solanum habrochaites was characterized for its function in abiotic stress tolerance. ShDHN was constitutively expressed in root, leaf, stem, flower and fruit. ShDHN was continuously up-regulated during cold stress and showed higher expression level in the cold-tolerant S. habrochaites than in the susceptible S. lycopersicum. Moreover, ShDHN expression was also regulated by drought, salt, osmotic stress, and exogenous signaling molecules. Overexpression of ShDHN in cultivated tomato increased tolerance to cold and drought stresses and improved seedling growth under salt and osmotic stresses. Compared with the wild-type, the transgenic plants accumulated more proline, maintained higher enzymatic activities of superoxide dismutase and catalase, and suffered less membrane damage under cold and drought stresses. Moreover, the transgenic plants accumulated lower levels of H2O2 and O2(-) under cold stress, and had higher relative water contents and lower water loss rates under dehydration conditions. Furthermore, overexpression of ShDHN in tomato led to the up- or down-regulated expression of several genes involved in ROS scavenging and JA signaling pathway, including SOD1, GST, POD, LOX, PR1 and PR2. Taken together, these results indicate that ShDHN has pleiotropic effects on improving plant adaptation to abiotic stresses and that it possesses potential usefulness in genetic improvement of stress tolerance in tomato. PMID:25576005

  16. MicroRNA Gene Expression Signature Driven by miR-9 Overexpression in Ovarian Clear Cell Carcinoma.

    PubMed

    Yanaihara, Nozomu; Noguchi, Yukiko; Saito, Misato; Takenaka, Masataka; Takakura, Satoshi; Yamada, Kyosuke; Okamoto, Aikou

    2016-01-01

    Previous studies have identified microRNA (miRNA) involvement in human cancers. This study aimed to elucidate potential clinical and biological associations of ovarian cancer-related miRNA gene expression profiles in high-grade serous carcinoma (HGSC) and ovarian clear cell carcinoma (OCCC). Accordingly, we investigated 27 patients with ovarian cancer (12 HGSC and 15 OCCC cases) using quantitative real-time reverse transcription polymerase chain reaction to determine the cancer-related miRNA expressions. Gene Cluster 3.0 was used for hierarchical clustering analysis, and differentially expressed miRNAs between HGSC and OCCC were identified by the class comparison analysis using BRB-ArrayTools. An unsupervised hierarchical clustering analysis identified two distinct miRNA expression clusters, with histological subtype-related significant differences in the associations between clusters and clinicopathological features. A comparison of miRNA expression in HGSCs and OCCCs identified five miRNAs (miR-132, miR-9, miR-126, miR-34a, and miR-21), with OCCCs demonstrating a statistically higher expression. Further investigation of the biological significance of miR-9 overexpression in OCCC revealed that miR-9 inhibition reduced the cell invasion ability and upregulated E-cadherin expression. Using a luciferase reporter assay, we further demonstrated the direct binding of miR-9 to E-cadherin. Global cancer-related miRNA expression analysis identified statistically unique profiles that could discriminate ovarian cancer histotypes. In OCCC, miR-9 overexpression may affect pathogenesis by targeting E-cadherin, thereby inducing an epithelial-mesenchymal transition. Therefore, miR-9 may be a promising therapeutic target strategy for OCCC. PMID:27612152

  17. Neuroplasticity, Psychosocial Genomics, and the Biopsychosocial Paradigm in the 21st Century

    PubMed Central

    Garland, Eric; Howard, Matthew Owen

    2010-01-01

    The biopsychosocial perspective is a foundation of social work theory and practice. Recent research on neuroplasticity and psychosocial genomics lends compelling support to this perspective by elucidating mechanisms through which psychosocial forces shape neurobiology. Investigations of neuroplasticity demonstrate that the adult brain can continue to form novel neural connections and grow new neurons in response to learning or training even into old age. These findings are complemented by the contributions of psychosocial genomics, a field of scientific inquiry that explores the modulating effects of experience on gene expression. Findings from these new sciences provide external validation for the biopsychosocial perspective and offer important insights into the manifold means by which socioenvironmental experiences influence neurobiological structure and function across the life course. PMID:19728478

  18. PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions.

    PubMed

    Corbeil-Girard, Louis-Philippe; Klein, Arnaud F; Sasseville, A Marie-Josée; Lavoie, Hugo; Dicaire, Marie-Josée; Saint-Denis, Anik; Pagé, Martin; Duranceau, André; Codère, François; Bouchard, Jean-Pierre; Karpati, George; Rouleau, Guy A; Massie, Bernard; Langelier, Yves; Brais, Bernard

    2005-04-01

    Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function. PMID:15755682

  19. Overexpression of a novel MADS-box gene SlFYFL delays senescence, fruit ripening and abscission in tomato

    NASA Astrophysics Data System (ADS)

    Xie, Qiaoli; Hu, Zongli; Zhu, Zhiguo; Dong, Tingting; Zhao, Zhiping; Cui, Baolu; Chen, Guoping

    2014-03-01

    MADS-domain proteins are important transcription factors involved in many biological processes of plants. In our study, a tomato MADS-box gene, SlFYFL, was isolated. SlFYFL is expressed in all tissues of tomato and significantly higher in mature leave, fruit of different stages, AZ (abscission zone) and sepal. Delayed leaf senescence and fruit ripening, increased storability and longer sepals were observed in 35S:FYFL tomato. The accumulation of carotenoid was reduced, and ethylene content, ethylene biosynthetic and responsive genes were down-regulated in 35S:FYFL fruits. Abscission zone (AZ) did not form normally and abscission zone development related genes were declined in AZs of 35S:FYFL plants. Yeast two-hybrid assay revealed that SlFYFL protein could interact with SlMADS-RIN, SlMADS1 and SlJOINTLESS, respectively. These results suggest that overexpression of SlFYFL regulate fruit ripening and development of AZ via interactions with the ripening and abscission zone-related MADS box proteins.

  20. Human coronary artery perivascular adipocytes overexpress genes responsible for regulating vascular morphology, inflammation, and hemostasis

    PubMed Central

    Aronow, Bruce J.; Tong, Wilson S.; Manka, David; Tang, Yaoliang; Bogdanov, Vladimir Y.; Unruh, Dusten; Blomkalns, Andra L.; Piegore, Mark G.; Weintraub, Daniel S.; Rudich, Steven M.; Kuhel, David G.; Hui, David Y.; Weintraub, Neal L.

    2013-01-01

    Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells. PMID:23737535

  1. Effect of overexpression of HOX genes on its invasive tendency in cerebral glioma

    PubMed Central

    GUO, YUN-BAO; SHAO, YI-MENG; CHEN, JING; XU, SONG-BAI; ZHANG, XING-DONG; WANG, MAO-REN; LIU, HAI-YAN

    2016-01-01

    Transcription factors encoded by HOX genes are vital in the determination of cell fate and identity during embryonic development. In certain malignancies, HOX genes also behave as oncogenes. The present study demonstrated suppression of the invasive tendency of glioblastoma multiforme U-118 and U-138 cells by the introduction of the antisense fragments of HOXA6 and B13 genes using electroporation. The invasion index indicated 79 and 72% reductions in the invasive ability of antisense HOXA6 and B13, respectively. No significant differences in the invasive index of the parental and mock cells of each HOX gene were observed (invasive index, 0.75–0.91; P=0.05). A reduction in invasion tendency was also observed following betulinic acid (BA) treatment: The results from the matrigel assay analysis clearly demonstrated a significant inhibition in the invasive behaviour of U-118 and U-138 cell lines from day 15 following BA treatment, with a maximum effect on day 30. The invasion index demonstrated 62 and 65% reductions in invasion ability in the U-118 and U-138 cell lines, respectively. The suppression of HOXC6 and B13 expression by the introduction of the corresponding antisense fragments in addition to BA reduced invasion tendency in U-118 and U-138 cell lines. The mechanism underlying the association between the HOX gene and invasive behavior in glioma cells is yet to be understood. However, the anti-invasive behavior of BA may aid understanding of the mechanism in future studies. PMID:26870170

  2. Overexpression of a Stress-Responsive NAC Transcription Factor Gene ONAC022 Improves Drought and Salt Tolerance in Rice.

    PubMed

    Hong, Yongbo; Zhang, Huijuan; Huang, Lei; Li, Dayong; Song, Fengming

    2016-01-01

    The NAC transcription factors play critical roles in regulating stress responses in plants. However, the functions for many of the NAC family members in rice are yet to be identified. In the present study, a novel stress-responsive rice NAC gene, ONAC022, was identified. Expression of ONAC022 was induced by drought, high salinity, and abscisic acid (ABA). The ONAC022 protein was found to bind specifically to a canonical NAC recognition cis-element sequence and showed transactivation activity at its C-terminus in yeast. The ONAC022 protein was localized to nucleus when transiently expressed in Nicotiana benthamiana. Three independent transgenic rice lines with overexpression of ONAC022 were generated and used to explore the function of ONAC022 in drought and salt stress tolerance. Under drought stress condition in greenhouse, soil-grown ONAC022-overexpressing (N22oe) transgenic rice plants showed an increased drought tolerance, leading to higher survival ratios and better growth than wild-type (WT) plants. When grown hydroponically in Hogland solution supplemented with 150 mM NaCl, the N22oe plants displayed an enhanced salt tolerance and accumulated less Na(+) in roots and shoots as compared to WT plants. Under drought stress condition, the N22oe plants exhibited decreased rates of water loss and transpiration, reduced percentage of open stomata and increased contents of proline and soluble sugars. However, the N22oe lines showed increased sensitivity to exogenous ABA at seed germination and seedling growth stages but contained higher level of endogenous ABA. Expression of some ABA biosynthetic genes (OsNCEDs and OsPSY), signaling and regulatory genes (OsPP2C02, OsPP2C49, OsPP2C68, OsbZIP23, OsAP37, OsDREB2a, and OsMYB2), and late stress-responsive genes (OsRAB21, OsLEA3, and OsP5CS1) was upregulated in N22oe plants. Our data demonstrate that ONAC022 functions as a stress-responsive NAC with transcriptional activator activity and plays a positive role in drought

  3. Creating Drought- and Salt-Tolerant Crops by Overexpressing a Vacuolar Pyrophosphatase Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proto...

  4. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in s...

  5. Mutant and overexpression analysis of a C2H2 single zinc finger gene of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The goal of this work was to characterize an Arabidopsis C2H2 single zinc finger gene, named AtZFP11, that is similar to SUPERMAN (SUP) and RABBIT EARS (RBE). No altered phenotype was observed in mutants analyzed that were derived through TILLING, nor a T-DNA insertion into the exon of AtZFP11. Pl...

  6. Phenotypic and molecular consequences of overexpression of metal-homeostasis genes

    PubMed Central

    Antosiewicz, Danuta M.; Barabasz, Anna; Siemianowski, Oskar

    2014-01-01

    Metal hyperaccumulating plants are able to store very large amounts of metals in their shoots. There are a number of reasons why it is important to be able to introduce metal hyperaccumulation traits into non-accumulating species (e.g., phytoremediation or biofortification in minerals) and to engineer a desired level of accumulation and distribution of metals. Metal homeostasis genes have therefore been used for these purposes. Engineered accumulation levels, however, have often been far from expected, and transgenic plants frequently display phenotypic features not related to the physiological function of the introduced gene. In this review, we focus on an aspect often neglected in research on plants expressing metal homeostasis genes: the specific regulation of endogenous metal homeostasis genes of the host plant in response to the transgene-induced imbalance of the metal status. These modifications constitute one of the major mechanisms involved in the generation of the plant's phenotype, including unexpected characteristics. Interestingly, activation of so-called “metal cross-homeostasis” has emerged as a factor of primary importance. PMID:24639682

  7. Overcoming of multidrug resistance by introducing the apoptosis gene, bcl-Xs, into MRP-overexpressing drug resistant cells.

    PubMed

    Ohi, Y; Kim, R; Toge, T

    2000-05-01

    Multidrug resistance associated protein (MRP) is one of drug transport membranes that confer multidrug resistance in cancer cells. Multidrug resistance has been known to be associated with resistance to apoptosis. In this study, using MRP overexpressing multidrug resistant nasopharyngeal cancer cells, we examined the expression of apoptosis related genes including p53, p21WAF1, bax and bcl-Xs between drug sensitive KB and its resistant KB/7D cells. We also examined whether the introduction of apoptosis related gene could increase the sensitivity to anticancer drugs in association with apoptotic cell death. The relative resistances to anticancer drugs in KB/7D cells evaluated by IC50 values were 3.6, 61.3, 10.4 and 10.5 to adriamycin (ADM), etoposide (VP-16), vincristine (VCR) and vindesine (VDS), respectively. The resistance to anticancer drugs in KB/7D cells was associated with the attenuation of internucleosomal DNA ladder formation in apoptosis. Of important, the mRNA expression of bcl-Xs gene in KB/7D cells was decreased in one-fourth as compared to that of KB cells among the apoptosis genes. The mRNA expression of bcl-Xs gene in a bcl-Xs transfected clone (KB/7Dbcl-Xs) was increased about 2-fold compared to that of KB/7Dneo cells, while the mRNA expression of MRP gene was not significantly different in KB/7bcl-Xs and KB/7Dneo cells. The sensitivities to anticancer drugs including ADM, VCR and VDS except VP-16 were increased in KB/7Dbcl-Xs cells, in turn, the relative resistance in KB/7Dbcl-Xs cells was decreased to 1.4, 4.0, and 3.0 in ADM, VCR and VDS, respectively, as compared to those of KB/7Dneo cells. Of interest, the studies on the accumulation of [3H]VCR showed that the decrease of [3H]VCR accumulation in KB/7Dbcl-Xs was not significantly different from that of KB/7Dneo cells. Collectively, these results indicated that the mechanism(s) of drug resistance in KB/7D cells could be explained at least by two factors: a) reduced drug accumulation mediated by

  8. Neuroplasticity and Successful Cognitive Aging: A Brief Overview for Nursing

    PubMed Central

    Vance, David E.; Kaur, Jaspreet; Fazeli, Pariya L.; Talley, Michele H.; Yuen, Hon K.; Kitchin, Beth; Lin, Feng

    2013-01-01

    The brain remains dynamic even in older age and can benefit from mental exercise. Thus, it is important to understand the concepts of positive neuroplasticity and negative neuroplasticity and how these mechanisms either support or detract from cognitive reserve. This article provides a brief review of these key concepts using four exemplary studies that clearly demonstrate the effects these neurological mechanisms exert on cognitive reserve and cognitive functioning. From this review, a working knowledge of how neuroplasticity and cognitive reserve are expressed in patients will be provided along with how this information can be incorporated into nursing practice and research. PMID:22743813

  9. Diffuse low-grade gliomas and neuroplasticity.

    PubMed

    Duffau, H

    2014-10-01

    The traditional approach in neuro-oncology is to study the tumor in great detail and ultimately give little consideration to the brain itself. Choosing the best treatment strategy for each patient with a diffuse low-grade glioma, in other words optimizing the oncologic and functional balance, implies not only a full knowledge of the natural history of this chronic disease, but also an understanding of the adaptation of the brain in response to growth and spread of the glioma. The aim of this review is to examine the mechanisms underlying this neuroplasticity, allowing functional compensation when the tumor progresses, and opening the way to new treatments with the principle of shifting towards "functional personalized neuro-oncology", improving both median survival and quality of life. PMID:25218490

  10. A neuroplastic deafferentation hypothesis for bipolar disorder

    PubMed Central

    Rogers, Jonathan; Mirams, Jamie; Patel, Rashmi

    2015-01-01

    Bipolar disorder, characterised by extreme cyclical variations in mood between depression and mania, is a common, debilitating and sometimes fatal psychiatric condition with an unclear aetiology. In this paper we propose a hypothesis for the development of bipolar disorder through which neuroplastic changes in response to an index depressive episode leads to the amplification of subthreshold pleasurable stimuli that then drive conversion into a manic state. This ‘pleasure deafferentation hypothesis’ is reached through a discussion of the neuroscientific basis of deafferentation at the level of the neuron and its role in the development of various neurological and psychiatric phenomena before a case for deafferentation as applied to bipolar disorder is justified and its implications discussed. PMID:26459976

  11. Overexpression of HOXB7 homeobox gene in oral cancer induces cellular proliferation and is associated with poor prognosis.

    PubMed

    De Souza Setubal Destro, Maria Fernanda; Bitu, Carolina Cavalcanti; Zecchin, Karina G; Graner, Edgard; Lopes, Marcio A; Kowalski, Luis Paulo; Coletta, Ricardo D

    2010-01-01

    A growing body of evidence has confirmed the involvement of dysregulated expression of HOX genes in cancer. HOX genes are a family of 39 transcription factors, divided in 4 clusters (HOXA to HOXD), that during normal development regulate cell proliferation and specific cell fate. In the present study it was investigated whether genes of the HOXB cluster play a role in oral cancer. We showed that most of the genes in the HOXB network are inactive in oral tissues, with exception of HOXB2, HOXB7 and HOXB13. Expression of HOXB7 was significantly higher in oral squamous cell carcinomas (OSCC) compared to normal oral mucosas. We further demonstrated that HOXB7 overexpression in HaCAT human epithelial cell line promoted proliferation, whereas downregulation of HOXB7 endogenous levels in human oral carcinoma cells (SCC9 cells) decreased proliferation. In OSCCs, expression of HOXB7 and Ki67, a marker of proliferation, correlate strongly with each other (rs=0.79, p<0.006). High immunohistochemical expression of HOXB7 was correlated with T stage (p=0.06), N stage (p=0.07), disease stage (p=0.09) and Ki67 expression (p=0.01), and patients with tumors showing high number of HOXB7-positive cells had shorter overall survival (p=0.08) and shorter disease-free survival after treatment (p=0.10) compared with patients with tumors exhibiting low amount of HOXB7-positive cells. Our data suggest that HOXB7 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and imply that HOXB7 may be an important determinant of OSCC patient prognosis. PMID:19956843

  12. HES6 gene is selectively overexpressed in glioma and represents an important transcriptional regulator of glioma proliferation.

    PubMed

    Haapa-Paananen, S; Kiviluoto, S; Waltari, M; Puputti, M; Mpindi, J P; Kohonen, P; Tynninen, O; Haapasalo, H; Joensuu, H; Perälä, M; Kallioniemi, O

    2012-03-01

    Malignant glioma is the most common brain tumor with 16,000 new cases diagnosed annually in the United States. We performed a systematic large-scale transcriptomics data mining study of 9783 tissue samples from the GeneSapiens database to systematically identify genes that are most glioma-specific. We searched for genes that were highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared with 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (hairy and enhancer of split 6) emerged as the most glioma-specific gene. Immunostaining of a tissue microarray showed HES6 expression in 335 (98.8%) out of the 339 glioma samples. HES6 was expressed in endothelial cells of the normal brain and glioma tissue. Recurrent grade 2 astrocytomas and grade 2 or 3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. High HES6 mRNA expression correlated with the proneural subtype that generally has a favorable outcome but is prone to recur. Functional studies suggested an important role for HES6 in supporting survival of glioma cells, as evidenced by reduction of cancer cell proliferation and migration after HES6 silencing. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which implicated a role for HES6 in p53, c-myc and nuclear factor-κB transcriptional networks. We conclude that HES6 is important for glioma cell proliferation and migration, and may have a role in angiogenesis. PMID:21785461

  13. C-kit overexpression correlates with KIT gene copy numbers increases in phyllodes tumors of the breast.

    PubMed

    Liu, Junjun; Liu, Xiaozhen; Feng, Xiaolong; Liu, Jian; Lv, Shuhua; Zhang, Wei; Niu, Yun

    2015-01-01

    We determined c-kit expression in the stroma and epithelia of benign, borderline, and malignant phyllodes tumors (PTs), respectively, as well as the relationship between c-kit expression in stromal elements and KIT gene copy number variations (CNVs). To assess c-kit expression and KIT CNVs, 348 PT cases were studied: 120 (34.4 %) benign cases, 115 (33.1 %) borderline cases, and 113 (32.5 %) malignant cases. All of these cases were evaluated for c-kit (CD117) expression using immunohistochemistry. Forty-two cases (29 c-kit-positive in the stromal cells cases and 13 negative cases) were investigated for KIT gene CNVs via genomic polymerase chain reaction (PCR). The overall rate of c-kit positivity in the stroma was 46.8 %, as well as 24.2, 53.1, and 64.6 %, respectively, in PTs of three different grades. However, in the majority of cases, the epithelia were c-kit positive (98.2 %), and the positivity was 100, 99.1, and 95 % in PTs of three different grades, respectively. There was a significant change in the expression of c-kit in the stroma and epithelia according to grade (P < 0.001, P = 0.014). From the genomic PCR results, we can confirm that c-kit positivity in the stroma is directly correlated with KIT gene copy numbers increases (P = 0.003, P = 0.041). We demonstrated that c-kit expression in the stroma of PTs is positively associated with malignancy. c-Kit epithelial positivity was inversely correlated with PTs malignancy. c-Kit overexpression in the stroma was related to KIT gene copy numbers increases. PMID:25534827

  14. Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene.

    PubMed

    Wang, Yong; Boghigian, Brett A; Pfeifer, Blaine A

    2007-11-01

    An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l(-1) OD(600)(-1). In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds. PMID:17876579

  15. Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco.

    PubMed

    Meng, Chen; Zhang, Song; Deng, Yong-Sheng; Wang, Guo-Dong; Kong, Fan-Ying

    2015-11-01

    Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3-hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was up-regulated under chilling (4 °C), heat (42 °C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O2(·-) levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress. PMID:26372946

  16. Overexpression of piRNA Pathway Genes in Epithelial Ovarian Cancer

    PubMed Central

    Lim, Shu Ly; Ricciardelli, Carmela; Oehler, Martin K.; De Arao Tan, Izza M. D.; Russell, Darryl; Grützner, Frank

    2014-01-01

    The importance of the Piwi-interacting RNA (piRNA) pathway for germ cell maintenance, genome integrity, DNA methylation and retrotransposon control raises possible roles of this pathway in cancer. Indeed aberrant expression of human PIWI orthologs and Maelstrom has been observed in various cancers. In this study we explored the expression and function of piRNA pathway genes in human ovarian cancer, based on our recent work, which showed widespread expression of piRNA pathway genes in the mammalian. Our work shows that PIWIL1 and MAEL expression is significantly increased in malignant EOC (n = 25) compared to benign tumor tissues (n = 19) and normal ovarian tissue (n = 8). The expression of PIWIL3 is lower in malignant and benign tissues when compared to normal ovary. Sequencing of PIWIL1 transcript revealed that in many tumors deletion of exon 17 leads to the introduction of a premature stop codon in the PIWI domain, likely due to a splicing error. In situ hybridization on tumor sections revealed that L1, PIWIL1, 2 and MAEL are specifically expressed in epithelial cells (cancerous cells) of EOC. Furthermore, PIWIL2 and MAEL are co-expressed in the stromal cells adjacent to tumor cells. Since PIWIL1 and MAEL are up regulated in malignant EOC and expressed in the epithelial cells, we investigated if these two genes affect invasiveness of ovarian cancer cell lines that do not normally express these genes. PIWIL1 and MAEL were transiently over expressed in the ovarian cancer cell line SKOV3, followed by real-time measurements of cell invasiveness. Surprisingly both PIWIL1 and MAEL over expression decreased the invasiveness of SKOV3 cells. Our findings support a growing body of evidence that shows that genes in this pathway are upregulated in cancer. In ovarian cancer we show for the first time that Piwil1 transcript may often be abnormal result in non functional product. In contrast to what has been observed in other cell types, we found that PIWIL1 and

  17. Overexpression of a tomato carotenoid ε-hydroxylase gene alleviates sensitivity to chilling stress in transgenic tobacco.

    PubMed

    Zhou, Bin; Deng, Yong-Sheng; Kong, Fan-Ying; Li, Bin; Meng, Qing-Wei

    2013-09-01

    Chilling is one of the most serious environmental stresses that disrupt the metabolic balance of cells and enhance the production of reactive oxygen species (ROS). Lutein plays important roles in dissipating excess excitation energy and eliminating ROS to maintain the normal physiological function of cells. A tomato carotenoid epsilon-ring hydroxylase gene (LeLUT1) was isolated, and the LeLUT1-GFP fusion protein was localized in the chloroplast of Arabidopsis mesophyll protoplast. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the expression of LeLUT1 was the highest in the leaves and was down-regulated by various abiotic stresses in tomato. The transgenic tobacco plants overexpressing LeLUT1 had higher lutein content, which was decreased in cold condition. Under chilling stress, the non-photochemical quenching (NPQ) values were higher in the transgenic plants than in the wild type (WT) plants. Compared with the WT plants, the transgenic plants showed lower levels of hydrogen peroxide (H2O2), superoxide radical (O2(·-)), relative electrical conductivity, and malondialdehyde content (MDA), and relatively higher values of maximal photochemical efficiency of photosystem II (Fv/Fm), oxidizable P700 of PSI, and net photosynthetic rate (Pn). Therefore, the transgenic seedlings were less suppressed in growth and lost less cotyledon chlorophyll than the WT seedlings. These results suggested that the overexpression of LeLUT1 had a key function in alleviating photoinhibition and photooxidation, and decreased the sensitivity of photosynthesis to chilling stress. PMID:23796723

  18. Analysis of lung tumor initiation and progression in transgenic mice for Cre-inducible overexpression of Cul4A gene

    DOE PAGESBeta

    Wang, Yang; Xu, Zhidong; Mao, Jian -Hua; Hung, Ming -Szu; Hsieh, David; Au, Alfred; Jablons, David M.; You, Liang

    2015-06-08

    Background: Lung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Methods:more » Viral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. Results: Here we show that the use of a recombinant adenovirus expressing Cre-recombinase (“AdenoCre”) to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well. Conclusion: Our findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.« less

  19. Analysis of lung tumor initiation and progression in transgenic mice for Cre-inducible overexpression of Cul4A gene

    SciTech Connect

    Wang, Yang; Xu, Zhidong; Mao, Jian -Hua; Hung, Ming -Szu; Hsieh, David; Au, Alfred; Jablons, David M.; You, Liang

    2015-06-08

    Background: Lung cancer is the leading cause of morbidity and death worldwide. Although the available lung cancer animal models have been informative and further propel our understanding of human lung cancer, they still do not fully recapitulate the complexities of human lung cancer. The pathogenesis of lung cancer remains highly elusive because of its aggressive biologic nature and considerable heterogeneity, compared to other cancers. The association of Cul4A amplification with aggressive tumor growth and poor prognosis has been suggested. Our previous study suggested that Cul4A is oncogenic in vitro, but its oncogenic role in vivo has not been studied. Methods: Viral delivery approaches have been used extensively to model cancer in mouse models. In our experiments, we used Cre-recombinase induced overexpression of the Cul4A gene in transgenic mice to study the role of Cul4A on lung tumor initiation and progression and have developed a new model of lung tumor development in mice harboring a conditionally expressed allele of Cul4A. Results: Here we show that the use of a recombinant adenovirus expressing Cre-recombinase (“AdenoCre”) to induce Cul4A overexpression in the lungs of mice allows controls of the timing and multiplicity of tumor initiation. Following our mouse models, we are able to study the potential role of Cul4A in the development and progression in pulmonary adenocarcinoma as well. Conclusion: Our findings indicate that Cul4A is oncogenic in vivo, and this mouse model is a tool in understanding the mechanisms of Cul4A in human cancers and for testing experimental therapies targeting Cul4A.

  20. MAL Overexpression Leads to Disturbed Expression of Genes That Influence Cytoskeletal Organization and Differentiation of Schwann Cells

    PubMed Central

    Schmid, Daniela; Zeis, Thomas; Sobrio, Monia

    2014-01-01

    In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0) and the low-affinity neurotrophin receptor p75NTR. This study shows that MAL overexpression leads to a significant reduction of Mpz and p75NTR expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB) and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination. PMID:25290060

  1. Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcus thermophilus and Their Effects on Metabolite Formation.

    PubMed

    Akyol, Ismail; Ozcelik, Fatma Gul; Karakas-Sen, Asuman; Ozkose, Emin; Gezginc, Yekta; Ekinci, M Sait

    2015-10-01

    Streptococcus thermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp. bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level. PMID:26280324

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Further increased production of free fatty acids by overexpressing a predicted transketolase gene of the pentose phosphate pathway in Aspergillus oryzae faaA disruptant.

    PubMed

    Tamano, Koichi; Miura, Ai

    2016-09-01

    Free fatty acids are useful as source materials for the production of biodiesel fuel and various chemicals such as pharmaceuticals and dietary supplements. Previously, we attained a 9.2-fold increase in free fatty acid productivity by disrupting a predicted acyl-CoA synthetase gene (faaA, AO090011000642) in Aspergillus oryzae. In this study, we achieved further increase in the productivity by overexpressing a predicted transketolase gene of the pentose phosphate pathway in the faaA disruptant. The A. oryzae genome is predicted to have three transketolase genes and overexpression of AO090023000345, one of the three genes, resulted in phenotypic change and further increase (corresponding to an increased production of 0.38 mmol/g dry cell weight) in free fatty acids at 1.4-fold compared to the faaA disruptant. Additionally, the biomass of hyphae increased at 1.2-fold by the overexpression. As a result, free fatty acid production yield per liter of liquid culture increased at 1.7-fold by the overexpression. PMID:26998626

  4. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines.

    PubMed

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K

    2013-01-30

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. PMID:23262422

  5. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines

    PubMed Central

    Wang, Zhiying; Pal, Dhananjay; Patel, Ashaben; Kwatra, Deep; Mitra, Ashim K.

    2013-01-01

    The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [3H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [3H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes. PMID:23262422

  6. Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein.

    PubMed

    Matsumura, P; Rydel, J J; Linzmeier, R; Vacante, D

    1984-10-01

    We overexpressed the CheY protein by fusing the cheY gene to the tryptophan promoter from Serratia marcescens. Expression of the trp promoter-cheY fusion and subsequent purification of the protein resulted in the isolation of up to 20 mg of homogeneously pure CheY protein from 100 mg of the cytoplasmic supernatant fraction. Purification of the CheY protein was accomplished by exploiting the affinity of CheY protein to cibacron blue dye and molecular sieve chromatography. Preliminary biochemical characterization of the pure CheY protein revealed specific interactions with S-adenosylmethionine and cibacron blue dye. Additional kinetic analysis showed that CheY protein inhibits EcoRI methyltransferase. The amino acid composition of the CheY protein predicted by the DNA sequence of the cheY gene and the amino acid analysis of the CheY protein were in agreement, confirming the authenticity of the purified CheY protein. PMID:6090423

  7. Overexpression and sequence of the Escherichia coli cheY gene and biochemical activities of the CheY protein.

    PubMed Central

    Matsumura, P; Rydel, J J; Linzmeier, R; Vacante, D

    1984-01-01

    We overexpressed the CheY protein by fusing the cheY gene to the tryptophan promoter from Serratia marcescens. Expression of the trp promoter-cheY fusion and subsequent purification of the protein resulted in the isolation of up to 20 mg of homogeneously pure CheY protein from 100 mg of the cytoplasmic supernatant fraction. Purification of the CheY protein was accomplished by exploiting the affinity of CheY protein to cibacron blue dye and molecular sieve chromatography. Preliminary biochemical characterization of the pure CheY protein revealed specific interactions with S-adenosylmethionine and cibacron blue dye. Additional kinetic analysis showed that CheY protein inhibits EcoRI methyltransferase. The amino acid composition of the CheY protein predicted by the DNA sequence of the cheY gene and the amino acid analysis of the CheY protein were in agreement, confirming the authenticity of the purified CheY protein. Images PMID:6090423

  8. Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

    PubMed Central

    Zhang, Xiaolian; Wang, Ning; Chen, Pei; Gao, Mengmeng; Liu, Juge; Wang, Yufeng; Zhao, Tuanjie; Li, Yan; Gai, Junyi

    2014-01-01

    Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress. PMID:25364908

  9. Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance

    PubMed Central

    Sahni, Sangita; Prasad, Bishun D.; Liu, Qing; Grbic, Vojislava; Sharpe, Andrew; Singh, Surinder P.; Krishna, Priti

    2016-01-01

    As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR–related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions. PMID:27324083

  10. Overexpression of the brassinosteroid biosynthetic gene DWF4 in Brassica napus simultaneously increases seed yield and stress tolerance.

    PubMed

    Sahni, Sangita; Prasad, Bishun D; Liu, Qing; Grbic, Vojislava; Sharpe, Andrew; Singh, Surinder P; Krishna, Priti

    2016-01-01

    As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the Arabidopsis BR biosynthetic gene AtDWF4 in the oilseed plant Brassica napus and scored growth and stress response phenotypes. The transgenic B. napus plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens Leptosphaeria maculans and Sclerotinia sclerotiorum. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR-related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions. PMID:27324083

  11. Overexpression of HMGA2-LPP fusion transcripts promotes expression of the {alpha} 2 type XI collagen gene

    SciTech Connect

    Kubo, Takahiro; Matsui, Yoshito . E-mail: ymatsui@sb4.so-net.ne.jp; Goto, Tomohiro; Yukata, Kiminori; Yasui, Natsuo

    2006-02-10

    In a subset of human lipomas, a specific t (3; 12) chromosome translocation gives rise to HMGA2-LPP fusion protein, containing the amino (N)-terminal DNA binding domains of HMGA2 fused to the carboxyl (C)-terminal LIM domains of LPP. In addition to its role in adipogenesis, several observations suggest that HMGA2-LPP is linked to chondrogenesis. Here, we analyzed whether HMGA2-LPP promotes chondrogenic differentiation, a marker of which is transactivation of the {alpha} 2 type XI collagen gene (Col11a2). Real-time PCR analysis showed that HMGA2-LPP and COL11A2 were co-expressed. Luciferase assay demonstrated that either of HMGA2-LPP, wild-type HMGA2 or the N-terminal HMGA2 transactivated the Col11a2 promoter in HeLa cells, while the C-terminal LPP did not. RT-PCR analysis revealed that HMGA2-LPP transcripts in lipomas with the fusion were 591-fold of full-length HMGA2 transcripts in lipomas without the fusion. These results indicate that in vivo overexpression of HMGA2-LPP promotes chondrogenesis by upregulating cartilage-specific collagen gene expression through the N-terminal DNA binding domains.

  12. Age and gene overexpression interact to abolish nesting behavior in Tg2576 amyloid precursor protein (APP) mice.

    PubMed

    Wesson, Daniel W; Wilson, Donald A

    2011-01-01

    Elucidating the modulators of social behavioral is important in understanding the neural basis of behavior and in developing methods to enhance behavior in cases of disorder. The work here stems from the observation that the Alzheimer's disease mouse model Tg2576, overexpressing human mutations of the amyloid-β precursor protein (APP), fails to construct nests when supplied paper towels in their home cages. Experiments using commercially available cotton nesting material found similar results. Additional experiments revealed that the genotype effect is progressively modulated by age in APP mice but not their WT counterparts. There was no effect of sex on nesting behavior in any group. Finally, this effect was independent of ambient temperature - even when subjected to a cold environment, APP mice fail to build nests whereas WT mice do. These results suggest that the APP gene plays a role in affiliative behaviors and are discussed in relation to disorders characteristic of mutations in the APP gene and in affective dysfunction, including Alzheimer's disease. PMID:20804789

  13. Nicotianamine synthase overexpression positively modulates iron homeostasis-related genes in high iron rice

    PubMed Central

    Wang, Meng; Gruissem, Wilhelm; Bhullar, Navreet K.

    2013-01-01

    Nearly one-third of the world population, mostly women and children, suffer from iron malnutrition and its consequences, such as anemia or impaired mental development. Biofortification of rice, which is a staple crop for nearly half of the world's population, can significantly contribute in alleviating iron deficiency. NFP rice (transgenic rice expressing nicotianamine synthase, ferritin and phytase genes) has a more than six-fold increase in iron content in polished rice grains, resulting from the synergistic action of nicotianamine synthase (NAS) and ferritin transgenes. We investigated iron homeostasis in NFP plants by analyzing the expression of 28 endogenous rice genes known to be involved in the homeostasis of iron and other metals, in iron-deficient and iron-sufficient conditions. RNA was collected from different tissues (roots, flag leaves, grains) and at three developmental stages during grain filling. NFP plants showed increased sensitivity to iron-deficiency conditions and changes in the expression of endogenous genes involved in nicotianamine (NA) metabolism, in comparison to their non-transgenic siblings (NTS). Elevated transcript levels were detected in NFP plants for several iron transporters. In contrast, expression of OsYSL2, which encodes a member of yellow stripe like protein family, and a transporter of the NA-Fe(II) complex was reduced in NFP plants under low iron conditions, indicating that expression of OsYSL2 is regulated by the endogenous iron status. Expression of the transgenes did not significantly affect overall iron homeostasis in NFP plants, which establishes the engineered push-pull mechanism as a suitable strategy to increase rice endosperm iron content. PMID:23755054

  14. Characterization of a lily anther-specific gene encoding cytoskeleton-binding glycoproteins and overexpression of the gene causes severe inhibition of pollen tube growth.

    PubMed

    Wang, Bing-Jyun; Hsu, Yi-Feng; Chen, Yun-Chu; Wang, Co-Shine

    2014-09-01

    This work characterizes an anther/pollen-specific gene that encodes potential intermediate filament (IF)-binding glycoproteins in lily (Lilium longiflorum Thunb. cv. Snow Queen) anthers during the development and pollen germination. LLP13 is a single gene that encodes a polypeptide of 807 amino acids, and a calculated molecular mass of 91 kDa. The protein contains a predicted transmembrane domain at the N-terminus and a conserved domain of unknown function (DUF)593 at the C-terminal half of the polypeptide. Sequence analysis revealed that LLP13 shares significant identity (37-41 %) with two intermediate filament antigen-binding proteins, representing a unique subgroup of DUF593 domain proteins from known rice and Arabidopsis species. The expression of LLP13 gene is anther-specific, and the transcript accumulates only at the stage of pollen maturation. Both premature drying and abscisic acid (ABA) treatment of developing pollen indicated that LLP13 was not induced by desiccation and ABA, but by other developmental cues. Antiserum was raised against the overexpressed LLP13C fragment of the protein in Escherichia coli and affinity-purified antibodies were prepared. Immunoblot analyses revealed that the LLP13 protein was a heterogeneous, anther-specific glycoprotein that accumulated only at the stage of pollen maturation. The protein is not heat-soluble. The level of LLP13 protein remained for 24 h during germination in vitro. Overexpression of LLP13-GFP or GFP-LLP13 in lily pollen tubes caused severe inhibition of tube elongation. The LLP13 protein codistributed with mTalin in growing tubes, suggesting that it apparently decorates actin cytoskeleton and is likely a cytoskeleton-binding protein that binds with IFs that potentially exist in pollen tubes. PMID:24944111

  15. Probing the Role of HDACs and Mechanisms of Chromatin-Mediated Neuroplasticity

    PubMed Central

    Haggarty, Stephen J.; Tsai, Li-Huei

    2011-01-01

    Advancing our understanding of neuroplasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of a myriad of nervous system disorders. Epigenetic mechanisms of neuroplasticity involve the post-translational modification of chromatin and the recruitment or loss of macromolecular complexes that control neuronal activity-dependent gene expression. While over a century after Ramón y Cajal first described nuclear subcompartments and foci that we now know correspond to sites of active transcription with acetylated histones that are under epigenetic control, the rate and extent to which epigenetic processes act in a dynamic and combinatorial fashion to shape experience-dependent phenotypic and behavioral plasticity in response to various types of neuronal stimuli over a range of time scales is only now coming into focus. With growing recognition that a subset of human diseases involving cognitive dysfunction can be classified as ‘chromatinopathies’, in which aberrant chromatin-mediated neuroplasticity plays a causal role in the underlying disease pathophysiology, understanding the molecular nature of epigenetic mechanisms in the nervous system may provide important new avenues for the development of novel therapeutics. In this review, we discuss the chemistry and neurobiology of the histone deacetylase (HDAC) family of chromatin-modifying enzymes, outline the role of HDACs in the epigenetic control of neuronal function, and discuss the potential relevance of these epigenetic mechanisms to the development of therapeutics aiming to enhance memory and neuroplasticity. Finally, open questions, challenges, and critical needs for the field of ‘neuroepigenetics’ in the years to come will be summarized. PMID:21545841

  16. Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

    PubMed Central

    Lawrence, M S; Ho, D Y; Dash, R; Sapolsky, R M

    1995-01-01

    We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypoglycemia. Microinfusion of GT vectors into the rat hippocampus also reduces kainic acid-induced seizure damage in the CA3 cell field. Furthermore, delivery of the vector even after onset of the seizure is protective, suggesting that HSV-mediated gene transfer for neuroprotection need not be carried out in anticipation of neurologic crises. Using the bicistronic vector v alpha 22 beta gal alpha 4GT, which coexpresses both GT and the Escherichia coli lacZ marker gene, we further demonstrate an inverse correlation between the extent of vector expression in the dentate and the amount of CA3 damage resulting from the simultaneous delivery of kainic acid. Images Fig. 2 Fig. 5 PMID:7638175

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

    PubMed

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

    2016-10-01

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

  18. Cyclic hair-loss and regrowth in transgenic mice overexpressing an intermediate filament gene.

    PubMed Central

    Powell, B C; Rogers, G E

    1990-01-01

    We have produced transgenic mice containing up to 250 copies of a sheep wool intermediate filament keratin gene to study the effect of its expression on hair structure and development. Several transgenic lines expressed the gene and in the one containing 250 transgenes, a pattern of hair-loss and regrowth was stably established. Successive waves of hair growth follow periods of denuding like the natural progression of hairs in the mouse hair cycle. By in situ hybridization we have shown that the sheep transgenes are expressed at the correct stage in mouse hair development and at a high level. The transgenic hairs contain not only an elevated level of intermediate filament keratin protein but also a decreased level of the filament-associated proteins. This imbalance disrupts the normal ordered array of these proteins in the cells of the hair cortex and leads to weakened fibres which are prematurely lost. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1691707

  19. Multiplex gene expression analysis for high-throughput drug discovery: screening and analysis of compounds affecting genes overexpressed in cancer cells.

    PubMed

    Johnson, Paul H; Walker, Roger P; Jones, Steven W; Stephens, Kathy; Meurer, Janet; Zajchowski, Deborah A; Luke, May M; Eeckman, Frank; Tan, Yuping; Wong, Linda; Parry, Gordon; Morgan, Thomas K; McCarrick, Meg A; Monforte, Joseph

    2002-12-01

    Drug discovery strategies are needed that can rapidly exploit multiple therapeutic targets associated with the complex gene expression changes that characterize a polygenic disease such as cancer. We report a new cell-based high-throughput technology for screening chemical libraries against several potential cancer target genes in parallel. Multiplex gene expression (MGE) analysis provides direct and quantitative measurement of multiple endogenous mRNAs using a multiplexed detection system coupled to reverse transcription-PCR. A multiplex assay for six genes overexpressed in cancer cells was used to screen 9000 chemicals and known drugs in the human prostate cancer cell line PC-3. Active compounds that modulated gene expression levels were identified, and IC50 values were determined for compounds that bind DNA, cell surface receptors, and components of intracellular signaling pathways. A class of steroids related to the cardiac glycosides was identified that potently inhibited the plasma membrane Na(+)K(+)-ATPase resulting in the inhibition of four of the prostate target genes including transcription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclear factor-3alpha, and the inhibitor of apoptosis, survivin. Representative compounds selectively induced apoptosis in PC-3 cells compared with the nonmetastatic cell line BPH-1. The multiplex assay distinguished potencies among structural variants, enabling structure-activity analysis suitable for chemical optimization studies. A second multiplex assay for five toxicological markers, Hsp70, Gadd153, Gadd45, O6-methylguanine-DNA methyltransferase, and cyclophilin, detected compounds that caused DNA damage and cellular stress and was a more sensitive and specific indicator of potential toxicity than measurement of cell viability. MGE analysis facilitates rapid drug screening and compound optimization, the simultaneous measurement of toxicological end points, and gene function analysis. PMID:12516962

  20. New Deferoxamine Glycoconjugates Produced upon Overexpression of Pathway-Specific Regulatory Gene in the Marine Sponge-Derived Streptomyces albus PVA94-07.

    PubMed

    Sekurova, Olga N; Pérez-Victoria, Ignacio; Martín, Jesús; Degnes, Kristin F; Sletta, Håvard; Reyes, Fernando; Zotchev, Sergey B

    2016-01-01

    Activation of silent biosynthetic gene clusters in Streptomyces bacteria via overexpression of cluster-specific regulatory genes is a promising strategy for the discovery of novel bioactive secondary metabolites. This approach was used in an attempt to activate a cryptic gene cluster in a marine sponge-derived Streptomyces albus PVA94-07 presumably governing the biosynthesis of peptide-based secondary metabolites. While no new peptide-based metabolites were detected in the recombinant strain, it was shown to produce at least four new analogues of deferoxamine with additional acyl and sugar moieties, for which chemical structures were fully elucidated. Biological activity tests of two of the new deferoxamine analogues revealed weak activity against Escherichia coli. The gene knockout experiment in the gene cluster targeted for activation, as well as overexpression of certain genes from this cluster did not have an effect on the production of these compounds by the strain overexpressing the regulator. It seems plausible that the production of such compounds is a response to stress imposed by the production of an as-yet unidentified metabolite specified by the cryptic cluster. PMID:27618884

  1. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

    PubMed

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-06-30

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  2. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis

    PubMed Central

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-01-01

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  3. In vivo evidence for neuroplasticity in older adults

    PubMed Central

    de Gobbi Porto, Fábio Henrique; Fox, Anne Murphy; Tusch, Erich Stephen; Sorond, Farzaneh; Mohammed, Abdul H; Daffner, Kirk R

    2015-01-01

    Neuroplasticity can be conceptualized as an intrinsic property of the brain that enables modification of function and structure in response to environmental demands. Neuroplastic strengthening of synapses is believed to serve as a critical mechanism underlying learning, memory, and other cognitive functions. Ex vivo work investigating neuroplasticity has been done on hippocampal slices using high frequency stimulation. However, in vivo neuroplasticity in humans has been difficult to demonstrate. Recently, a long-term potentiation-like phenomenon, a form of neuroplastic change, was identified in young adults by differences in visual evoked potentials (VEPs) that were measured before and after tetanic visual stimulation (TVS). The current study investigated whether neuroplastic changes in the visual pathway can persist in older adults. Seventeen healthy subjects, 65 years and older, were recruited from the community. Subjects had a mean age of 77.4 years, mean education of 17 years, mean MMSE of 29.1, and demonstrated normal performance on neuropsychological tests. 1Hz checkerboard stimulation, presented randomly to the right or left visual hemi-field, was followed by two minutes of 9Hz stimulation (TVS) to one hemi-field. After two minutes of rest, 1Hz stimulation was repeated. Temporospatial principal component analysis was used to identify the N1b component of the VEPs, at lateral occipital locations, in response to 1Hz stimulation pre- and post-TVS. Results showed that the amplitude of factors representing the early and late N1b component was substantially larger after tetanic stimulation. These findings indicate that high frequency visual stimulation can enhance the N1b in cognitively high functioning old adults, suggesting that neuroplastic changes in visual pathways can continue into late life. Future studies are needed to determine the extent to which this marker of neuroplasticity is sustained over a longer period of time, and is influenced by age, cognitive

  4. Developmental neuroplasticity and the origin of neurodegenerative diseases.

    PubMed

    Schaefers, Andrea T U; Teuchert-Noodt, Gertraud

    2013-05-24

    Objectives. Neurodegenerative diseases like Alzheimer's and Parkinson's Disease, marked by characteristic protein aggregations, are more and more accepted to be synaptic disorders and to arise from a combination of genetic and environmental factors. In this review we propose our concept that neuroplasticity might constitute a link between early life challenges and neurodegeneration. Methods. After introducing the general principles of neuroplasticity, we show how adverse environmental stimuli during development impact adult neuroplasticity and might lead to neurodegenerative processes. Results. There are significant overlaps between neurodevelopmental and neurodegenerative processes. Proteins that represent hallmarks of neurodegeneration are involved in plastic processes under physiological conditions. Brain regions - particularly the hippocampus - that retain life-long plastic capacities are the key targets of neurodegeneration. Neuroplasticity is highest in young age making the brain more susceptible to external influences than later in life. Impacts during critical periods have life-long consequences on neuroplasticity and structural self-organization and are known to be common risk factors for neurodegenerative diseases. Conclusions. Several lines of evidence support a link between developmental neuroplasticity and neurodegenerative processes later in life. A deeper insight into these processes is necessary to design strategies to mitigate or even prevent neurodegenerative pathologies. PMID:23705632

  5. Effects of chronic growth hormone overexpression on appetite-regulating brain gene expression in coho salmon.

    PubMed

    Kim, Jin-Hyoung; Leggatt, Rosalind A; Chan, Michelle; Volkoff, Hélène; Devlin, Robert H

    2015-09-15

    Organisms must carefully regulate energy intake and expenditure to balance growth and trade-offs with other physiological processes. This regulation is influenced by key pathways controlling appetite, feeding behaviour and energy homeostasis. Growth hormone (GH) transgenesis provides a model where food intake can be elevated, and is associated with dramatic modifications of growth, metabolism, and feeding behaviour, particularly in fish. RNA-Seq and qPCR analyses were used to compare the expression of multiple genes important in appetite regulation within brain regions and the pituitary gland (PIT) of GH transgenic (fed fully to satiation or restricted to a wild-type ration throughout their lifetime) and wild-type coho salmon (Oncorhynchus kisutch). RNA-Seq results showed that differences in both genotype and ration levels resulted in differentially expressed genes associated with appetite regulation in transgenic fish, including elevated Agrp1 in hypothalamus (HYP) and reduced Mch in PIT. Altered mRNA levels for Agrp1, Npy, Gh, Ghr, Igf1, Mch and Pomc were also assessed using qPCR analysis. Levels of mRNA for Agrp1, Gh, and Ghr were higher in transgenic than wild-type fish in HYP and in the preoptic area (POA), with Agrp1 more than 7-fold higher in POA and 12-fold higher in HYP of transgenic salmon compared to wild-type fish. These data are consistent with the known roles of orexigenic factors on foraging behaviour acting via GH and through MC4R receptor-mediated signalling. Igf1 mRNA was elevated in fully-fed transgenic fish in HYP and POA, but not in ration-restricted fish, yet both of these types of transgenic animals have very pronounced feeding behaviour relative to wild-type fish, suggesting IGF1 is not playing a direct role in appetite stimulation acting via paracrine or autocrine mechanisms. The present findings provide new insights on mechanisms ruling altered appetite regulation in response to chronically elevated GH, and on potential pathways by which

  6. Allelic mutations in noncoding genomic sequences construct novel transcription factor binding sites that promote gene overexpression.

    PubMed

    Tian, Erming; Børset, Magne; Sawyer, Jeffrey R; Brede, Gaute; Våtsveen, Thea K; Hov, Håkon; Waage, Anders; Barlogie, Bart; Shaughnessy, John D; Epstein, Joshua; Sundan, Anders

    2015-11-01

    The growth and survival factor hepatocyte growth factor (HGF) is expressed at high levels in multiple myeloma (MM) cells. We report here that elevated HGF transcription in MM was traced to DNA mutations in the promoter alleles of HGF. Sequence analysis revealed a previously undiscovered single-nucleotide polymorphism (SNP) and crucial single-nucleotide variants (SNVs) in the promoters of myeloma cells that produce large amounts of HGF. The allele-specific mutations functionally reassembled wild-type sequences into the motifs that affiliate with endogenous transcription factors NFKB (nuclear factor kappa-B), MZF1 (myeloid zinc finger 1), and NRF-2 (nuclear factor erythroid 2-related factor 2). In vitro, a mutant allele that gained novel NFKB-binding sites directly responded to transcriptional signaling induced by tumor necrosis factor alpha (TNFα) to promote high levels of luciferase reporter. Given the recent discovery by genome-wide sequencing (GWS) of numerous non-coding mutations in myeloma genomes, our data provide evidence that heterogeneous SNVs in the gene regulatory regions may frequently transform wild-type alleles into novel transcription factor binding properties to aberrantly interact with dysregulated transcriptional signals in MM and other cancer cells. PMID:26220195

  7. Transgenic barley overexpressing a cytokinin dehydrogenase gene shows greater tolerance to drought stress.

    PubMed

    Pospíšilová, Hana; Jiskrová, Eva; Vojta, Petr; Mrízová, Katarína; Kokáš, Filip; Čudejková, Mária Majeská; Bergougnoux, Veronique; Plíhal, Ondřej; Klimešová, Jana; Novák, Ondřej; Dzurová, Lenka; Frébort, Ivo; Galuszka, Petr

    2016-09-25

    Together with auxins, cytokinins are the main plant hormones involved in many different physiological processes. Given this knowledge, cytokinin levels can be manipulated by genetic modification in order to improve agronomic parameters of cereals in relation to, for example, morphology, yield, and tolerance to various stresses. The barley (Hordeum vulgare) cultivar Golden Promise was transformed using the cytokinin dehydrogenase 1 gene from Arabidopsis thaliana (AtCKX1) under the control of mild root-specific β-glucosidase promoter from maize. Increased cytokinin degradation activity was observed positively to affect the number and length of lateral roots. The impact on morphology depended upon the recombinant protein's subcellular compartmentation. While assumed cytosolic and vacuolar targeting of AtCKX1 had negligible effect on shoot growth, secretion of AtCKX1 protein to the apoplast had a negative effect on development of the aerial part and yield. Upon the application of severe drought stress, all transgenic genotypes maintained higher water content and showed better growth and yield parameters during revitalization. Higher tolerance to drought stress was most caused by altered root morphology resulting in better dehydration avoidance. PMID:26773738

  8. Cytotoxicity associated with artemis overexpression after lentiviral vector-mediated gene transfer.

    PubMed

    Multhaup, Megan; Karlen, Andrea D; Swanson, Debra L; Wilber, Andrew; Somia, Nikunj V; Cowan, Morton J; McIvor, R Scott

    2010-07-01

    Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1alpha promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T(-)B(-) phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  9. Cytotoxicity Associated with Artemis Overexpression After Lentiviral Vector-Mediated Gene Transfer

    PubMed Central

    Multhaup, Megan; Karlen, Andrea D.; Swanson, Debra L.; Wilber, Andrew; Somia, Nikunj V.; Cowan, Morton J.

    2010-01-01

    Abstract Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1α promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T−B− phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A. PMID:20163250

  10. Over-expression of VvWRKY1 in grapevines induces expression of jasmonic acid pathway-related genes and confers higher tolerance to the downy mildew.

    PubMed

    Marchive, Chloé; Léon, Céline; Kappel, Christian; Coutos-Thévenot, Pierre; Corio-Costet, Marie-France; Delrot, Serge; Lauvergeat, Virginie

    2013-01-01

    Most WRKY transcription factors activate expression of defence genes in a salicylic acid- and/or jasmonic acid-dependent signalling pathway. We previously identified a WRKY gene, VvWRKY1, which is able to enhance tolerance to fungal pathogens when it is overexpressed in tobacco. The present work analyzes the effects of VvWRKY1 overexpression in grapevine. Microarray analysis showed that genes encoding defence-related proteins were up-regulated in the leaves of transgenic 35S::VvWRKY1 grapevines. Quantitative RT-PCR analysis confirmed that three genes putatively involved in jasmonic acid signalling pathway were overexpressed in the transgenic grapes. The ability of VvWRKY1 to trans-activate the promoters of these genes was demonstrated by transient expression in grape protoplasts. The resistance to the causal agent of downy mildew, Plasmopara viticola, was enhanced in the transgenic plants. These results show that VvWRKY1 can increase resistance of grapevine against the downy mildew through transcriptional reprogramming leading to activation of the jasmonic acid signalling pathway. PMID:23342101