Science.gov

Sample records for agrobacterium-mediated transient expression

  1. Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves.

    PubMed

    Wu, Su-Li; Yang, Xiao-Bing; Liu, Li-Qun; Jiang, Tao; Wu, Hai; Su, Chao; Qian, Yong-Hua; Jiao, Feng

    2015-01-01

    To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD600 of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry. PMID:26024368

  2. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression.

    PubMed

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area-time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  3. Environment Control to Improve Recombinant Protein Yields in Plants Based on Agrobacterium-Mediated Transient Gene Expression

    PubMed Central

    Fujiuchi, Naomichi; Matoba, Nobuyuki; Matsuda, Ryo

    2016-01-01

    Agrobacterium-mediated transient expression systems enable plants to produce a wide range of recombinant proteins on a rapid timescale. To achieve economically feasible upstream production and downstream processing, two yield parameters should be considered: (1) recombinant protein content per unit biomass and (2) recombinant protein productivity per unit area–time at the end of the upstream production. Because environmental factors in the upstream production have impacts on these parameters, environment control is important to maximize the recombinant protein yield. In this review, we summarize the effects of pre- and postinoculation environmental factors in the upstream production on the yield parameters and discuss the basic concept of environment control for plant-based transient expression systems. Preinoculation environmental factors associated with planting density, light quality, and nutrient supply affect plant characteristics, such as biomass and morphology, which in turn affect recombinant protein content and productivity. Accordingly, environment control for such plant characteristics has significant implications to achieve a high yield. On the other hand, postinoculation environmental factors, such as temperature, light intensity, and humidity, have been shown to affect recombinant protein content. Considering that recombinant protein production in Agrobacterium-mediated transient expression systems is a result of a series of complex biological events starting from T-DNA transfer from Agrobacterium tumefaciens to protein biosynthesis and accumulation in leaf tissue, we propose that dynamic environment control during the postinoculation process, i.e., changing environmental conditions at an appropriate timing for each event, may be a promising approach to obtain a high yield. Detailed descriptions of plant growth conditions and careful examination of environmental effects will significantly contribute to our knowledge to stably obtain high recombinant

  4. A Novel Phenolic Compound, Chloroxynil, Improves Agrobacterium-Mediated Transient Transformation in Lotus japonicus

    PubMed Central

    Kimura, Mitsuhiro; Cutler, Sean; Isobe, Sachiko

    2015-01-01

    Agrobacterium-mediated transformation is a commonly used method for plant genetic engineering. However, the limitations of Agrobacterium host-plant interactions and the complexity of plant tissue culture often make the production of transgenic plants difficult. Transformation efficiency in many legume species, including soybean and the common bean, has been reported to be quite low. To improve the transformation procedure in legumes, we screened for chemicals that increase the transformation efficiency of Lotus japonicus, a model legume species. A Chemical library was screened and chemicals that increase in transient transformation efficiency of L. japonicus accession, Miyakojima MG-20 were identified. The transient transformation efficiency was quantified by reporter activity in which an intron-containing reporter gene produces the GUS protein only when the T-DNA is expressed in the plant nuclei. We identified a phenolic compound, chloroxynil, which increased the genetic transformation of L. japonicus by Agrobacterium tumefaciens strain EHA105. Characterization of the mode of chloroxynil action indicated that it enhanced Agrobacterium-mediated transformation through the activation of the Agrobacterium vir gene expression, similar to acetosyringone, a phenolic compound known to improve Agrobacterium-mediated transformation efficiency. Transient transformation efficiency of L. japonicus with 5 μM chloroxynil was 60- and 6- fold higher than that of the control and acetosyringone treatment, respectively. In addition, transgenic L. japonicus lines were successfully generated by 5 μM chloroxynil treatment.Furthermore, we show that chloroxynil improves L. japonicus transformation by Agrobacterium strain GV3101 and rice transformation. Our results demonstrate that chloroxynil significantly improves Agrobacterium tumefaciens-mediated transformation efficiency of various agriculturally important crops. PMID:26176780

  5. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

    PubMed

    Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

    2014-01-01

    Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

  6. Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation.

    PubMed

    Shcherbak, N; Kishchenko, O; Sakhno, L; Komarnytsky, I; Kuchuk, M

    2013-01-01

    Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. PMID:23821951

  7. Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay

    PubMed Central

    Srivastava, Vineet Kumar; Raikwar, Shailendra; Tuteja, Narendra

    2014-01-01

    PsSEOF1, a SEO (sieve element occlusion) gene family protein (forisome) is calcium powered motor protein and is located close to plasma membrane of sieve element. In sieve element (SE) it senses the calcium ion levels and undergoes ATP-independent conformational shifts. Forisome, meaning gate-bodies (Latin foris: wing of a gate; Greek soma: body). Recent reports show that SEO gene family protein can prevent the loss of nutrient rich photoassimilate upon wound injury. The regulation of SEO protein forisome under abiotic/ biotic stress is still unknown. The analysis of cis-regulatory element present in the upstream region is not well understood. Tissue specific promoters guarantee correct expression when it perceives particular stimuli. Here we report isolation of tissue specific promoter of PsSEOF1 was isolated by gene walking PCR from P. sativum (pea) genomic DNA library constructed by BD genome walker kit. In silico analysis revealed several putative cis element within this promoter sequence like wound response, cold, dehydration. Putative elements which might be required for its vascular tissue specificity has also been identified. The GUS activities of PsSEOF1 promoter-GUS chimeric construct in the agroinfiltrated leaves under different environmental stress abiotic and biotic like wound, cold, salt and phytohormones has shown high level of GUS activity. To identify the activity of PsSEOF1 promoter under different stress condition an Agrobacterium-mediated transient expression of tobacco plants were subjected to histochemical GUS staining. Stress-inducible nature of PsSEOF1 promoter opens possibility for the study of the PsSEOF1 gene regulation under stress condition. The isolated promoter sequence could serve as an important candidate for tissue specific promoter in genetic engineering of plant under stress conditions. PMID:25763698

  8. Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos.

    PubMed

    Uçarlı, C; Tufan, F; Gürel, F

    2015-01-01

    Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study. PMID:25730049

  9. Cadophora finlandia and Phialocephala fortinii: Agrobacterium-mediated transformation and functional GFP expression.

    PubMed

    Gorfer, Markus; Klaubauf, Sylvia; Bandian, Dragana; Strauss, Joseph

    2007-07-01

    Hygromycin B resistance was transferred to the sterile mycelia of Cadophora finlandia and Phialocephala fortinii by co-cultivation with Agrobacterium tumefaciens. Constitutively expressed green fluorescent protein (GFP) was also introduced using the same vector. Confocal laser scanning microscopy (CLSM) revealed strong fluorescence of transformants. Both traits were mitotically stable during one year of subculturing on non-selective growth medium. Southern blot analysis showed that the majority of the transformants contained single-copy integrations at random sites in the genome. PMID:17662587

  10. Functional Characterization of a Bidirectional Plant Promoter from Cotton Leaf Curl Burewala Virus Using an Agrobacterium-Mediated Transient Assay

    PubMed Central

    Ashraf, Muhammad Aleem; Shahid, Ahmad Ali; Rao, Abdul Qayyum; Bajwa, Kamran Shehzad; Husnain, Tayyab

    2014-01-01

    The C1 promoter expressing the AC1 gene, and V1 promoter expressing the AV1 gene are located in opposite orientations in the large intergenic region of the Cotton leaf curl Burewala virus (CLCuBuV) genome. Agro-infiltration was used to transiently express putative promoter constructs in Nicotiana tabacum and Gossypium hirsutum leaves, which was monitored by a GUS reporter gene, and revealed that the bidirectional promoter of CLCuBuV transcriptionally regulates both the AC1 and AV1 genes. The CLCuBuV C1 gene promoter showed a strong, consistent transient expression of the reporter gene (GUS) in N. tabacum and G. hirsutum leaves and exhibited GUS activity two- to three-fold higher than the CaMV 35S promoter. The CLCuBuV bidirectional genepromoter is a nearly constitutive promoter that contains basic conserved elements. Many cis-regulatory elements (CREs) were also analyzed within the bidirectional plant promoters of CLCuBuV and closely related geminiviruses, which may be helpful in understanding the transcriptional regulation of both the virus and host plant. PMID:24424501

  11. Transient down-regulation of the RNA silencing machinery increases efficiency of Agrobacterium-mediated transformation of Arabidopsis.

    PubMed

    Bilichak, Andriy; Yao, Youli; Kovalchuk, Igor

    2014-06-01

    Agrobacterium tumefaciens is a plant pathogen that is widely used in plant transformation. As the process of transgenesis includes the delivery of single-stranded T-DNA molecule, we hypothesized that transformation rate may negatively correlate with the efficiency of the RNA-silencing machinery. Using mutants compromised in either the transcriptional or post-transcriptional gene-silencing pathways, two inhibitors of stable transformation were revealed-AGO2 and NRPD1a. Furthermore, an immunoprecipitation experiment has shown that NRPD1, a subunit of Pol IV, directly interacts with Agrobacterium T-DNA in planta. Using the Tobacco rattle virus (TRV)--based virus-induced gene silencing (VIGS) technique, we demonstrated that the transient down-regulation of the expression of either AGO2 or NRPD1a genes in reproductive organs of Arabidopsis, leads to an increase in transformation rate. We observed a 6.0- and 3.5-fold increase in transformation rate upon transient downregulation of either AGO2 or NRPD1a genes, respectively. This is the first report demonstrating the increase in the plant transformation rate via VIGS-mediated transient down-regulation of the components of epigenetic machinery in reproductive tissue. PMID:24472037

  12. Agrobacterium-mediated transformation of three freshwater microalgal strains.

    PubMed

    Sanitha, Mary; Radha, Sudhakar; Fatima, Anwar Aliya; Devi, Selvaraju Gayathri; Ramya, Mohandass

    2014-01-01

    Microalgal transformation has gained interest in recent years. Agrobacterium-mediated transformation remains as the most efficient method for the development of transgenic plants and microalgae due to its wide host range, inexpensive procedure and transfer of large segments of DNA. In the present study, three different microalgal species were isolated from freshwater environment and identified based on the morphological characteristics and ITS-2 region amplification. Agrobacterium-mediated transformation was successful for the isolates Chlorella sp., Ankistrodesmus sp and Scenedesmus bajacalifornicus. Gene integration and expression was confirmed by PCR amplification of hptII and GUS histochemical assay. A. tumifaciens contamination was checked by amplification of npt II gene (kanamycin resistant) which lies outside the T-border. Based on GUS assay, transformation efficiencies were found to be 12.25% for Chlorella sp. 2.96% for Scenedesmus bajacalifornicus and 3.5% for Ankistrodesmus sp. PMID:25804057

  13. Agrobacterium-mediated transformation of Fusarium proliferatum.

    PubMed

    Bernardi-Wenzel, J; Quecine, M C; Azevedo, J L; Pamphile, J A

    2016-01-01

    Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process. PMID:27323127

  14. High-efficiency Agrobacterium-mediated transformation of Norway spruce (Picea abies) and loblolly pine (Pinus taeda)

    NASA Technical Reports Server (NTRS)

    Wenck, A. R.; Quinn, M.; Whetten, R. W.; Pullman, G.; Sederoff, R.; Brown, C. S. (Principal Investigator)

    1999-01-01

    Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology laboratories; however, large-scale use of this organism in conifer transformation has been limited by difficult propagation of explant material, selection efficiencies and low transformation frequency. We have analyzed co-cultivation conditions and different disarmed strains of Agrobacterium to improve transformation. Additional copies of virulence genes were added to three common disarmed strains. These extra virulence genes included either a constitutively active virG or extra copies of virG and virB, both from pTiBo542. In experiments with Norway spruce, we increased transformation efficiencies 1000-fold from initial experiments where little or no transient expression was detected. Over 100 transformed lines expressing the marker gene beta-glucuronidase (GUS) were generated from rapidly dividing embryogenic suspension-cultured cells co-cultivated with Agrobacterium. GUS activity was used to monitor transient expression and to further test lines selected on kanamycin-containing medium. In loblolly pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Agrobacterium-mediated gene transfer is a useful technique for large-scale generation of transgenic Norway spruce and may prove useful for other conifer species.

  15. Transient expression systems for plant-derived biopharmaceuticals.

    PubMed

    Komarova, Tatiana V; Baschieri, Selene; Donini, Marcello; Marusic, Carla; Benvenuto, Eugenio; Dorokhov, Yuri L

    2010-08-01

    In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems. PMID:20673010

  16. Is VIP1 important for Agrobacterium-mediated transformation?

    PubMed

    Shi, Yong; Lee, Lan-Ying; Gelvin, Stanton B

    2014-09-01

    Agrobacterium genetically transforms plants by transferring and integrating T-(transferred) DNA into the host genome. This process requires both Agrobacterium and host proteins. VirE2 interacting protein 1 (VIP1), an Arabidopsis bZIP protein, has been suggested to mediate transformation through interaction with and targeting of VirE2 to nuclei. We examined the susceptibility of Arabidopsis vip1 mutant and VIP1 overexpressing plants to transformation by numerous Agrobacterium strains. In no instance could we detect altered transformation susceptibility. We also used confocal microscopy to examine the subcellular localization of Venus-tagged VirE2 or Venus-tagged VIP1, in the presence or absence of the other untagged protein, in different plant cell systems. We found that VIP1-Venus localized in both the cytoplasm and the nucleus of Arabidopsis roots, agroinfiltrated Nicotiana benthamiana leaves, Arabidopsis mesophyll protoplasts and tobacco BY-2 protoplasts, regardless of whether VirE2 was co-expressed. VirE2 localized exclusively to the cytoplasm of tobacco and Arabidopsis protoplasts, whether in the absence or presence of VIP1 overexpression. In transgenic Arabidopsis plants and agroinfiltrated N. benthamina leaves we could occasionally detect small aggregates of the Venus signal in nuclei, but these were likely to be imagining artifacts. The vast majority of VirE2 remained in the cytoplasm. We conclude that VIP1 is not important for Agrobacterium-mediated transformation or VirE2 subcellular localization. PMID:24953893

  17. Agrobacterium-mediated transformation of tomato with the ICE1 transcription factor gene.

    PubMed

    Juan, J X; Yu, X H; Jiang, X M; Gao, Z; Zhang, Y; Li, W; Duan, Y D; Yang, G

    2015-01-01

    ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower. PMID:25729995

  18. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun.

    PubMed

    Gao, Caixia; Nielsen, Klaus K

    2013-01-01

    Agrobacterium-mediated transformation and direct gene transfer using the gene gun (microparticle -bombardment) are the two most widely used methods for plant genetic modification. The Agrobacterium method has been successfully practiced in dicots for many years, but only recently have efficient protocols been developed for grasses. Microparticle bombardment has evolved as a method delivering exogenous nucleic acids into plant genome and is a commonly employed technique in plant science. Here these two systems are compared for transformation efficiency, transgene integration, and transgene expression when used to transform tall fescue (Festuca arundinacea Schreb.). The tall fescue transformation protocols lead to the production of large numbers of fertile, independent transgenic lines. PMID:23104329

  19. Agrobacterium-Mediated Transformation of the Recalcitrant Vanda Kasem's Delight Orchid with Higher Efficiency

    PubMed Central

    Gnasekaran, Pavallekoodi; James Antony, Jessica Jeyanthi; Uddain, Jasim; Subramaniam, Sreeramanan

    2014-01-01

    The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes. PMID:24977213

  20. A simple agroinfiltration method for transient gene expression in plant leaf discs.

    PubMed

    Matsuo, Kouki; Fukuzawa, Noriho; Matsumura, Takeshi

    2016-09-01

    In the present study, we developed a simple transient gene expression system based on Agrobacterium-mediated transformation. Vacuum infiltration was applied to leaf discs from Nicotiana benthamiana plants with Agrobacterium suspension solution under conventional vacuum conditions in a needleless plastic syringe. Model proteins, green fluorescent protein, β-glucuronidase, mouse granulocyte-macrophage colony-stimulating factor, and human fibroblast growth factor 1 were successfully expressed in leaf discs within 4 days after infiltration. In addition, the functional evaluation of viral RNA silencing suppressors, Artichoke mottled crinkle virus p19 protein, was also performed. Using this method, the contamination and diffusion of genetically modified bacterium to the environment and important transgenic plants were prevented. This method can be conducted without specialized apparatuses or large amounts of Agrobacterium suspension solutions; thus, the simultaneous evaluation of multiple vectors will be easily possible. PMID:26995064

  1. Agrobacterium-mediated transformation of maize (Zea mays) immature embryos.

    PubMed

    Lee, Hyeyoung; Zhang, Zhanyuan J

    2014-01-01

    Agrobacterium tumefaciens-mediated transformation is one of the most efficient and simple gene delivery systems for genetic improvement and biology studies in maize. This system has become more widely used by both public and private laboratories. However, transformation efficiencies vary greatly from laboratory to laboratory for the same genotype. Here, we illustrate our advanced Agrobacterium-mediated transformation method in Hi-II maize using simple binary vectors. The protocol utilizes immature embryos as starting explants and the bar gene as a selectable marker coupled with bialaphos as a selective agent. The protocol offers efficient transformation results with high reproducibility, provided that some experimental conditions are well controlled. This transformation method, with minor modifications, can be also employed to transform certain maize inbreds. PMID:24243211

  2. Agrobacterium-mediated transformation of durum wheat (Triticum turgidum L. var. durum cv Stewart) with improved efficiency

    PubMed Central

    He, Y.; Jones, H. D.; Chen, S.; Chen, X. M.; Wang, D. W.; Li, K. X.; Wang, D. S.; Xia, L. Q.

    2010-01-01

    An efficient Agrobacterium-mediated durum wheat transformation system has been developed for the production of 121 independent transgenic lines. This improved system used Agrobacterium strain AGL1 containing the superbinary pGreen/pSoup vector system and durum wheat cv Stewart as the recipient plant. Acetosyringone at 400 μM was added to both the inoculation and cultivation medium, and picloram at 10 mg l−1 and 2 mg l−1 was used in the cultivation and induction medium, respectively. Compared with 200 μM in the inoculation and cultivation media, the increased acetosyringone concentration led to significantly higher GUS (β-glucuronidase) transient expression and T-DNA delivery efficiency. However, no evident effects of acetosyringone concentration on regeneration frequency were observed. The higher acetosyringone concentration led to an improvement in average final transformation efficiency from 4.7% to 6.3%. Furthermore, the concentration of picloram in the co-cultivation medium had significant effects on callus induction and regeneration. Compared with 2 mg l−1 picloram in the co-cultivation medium, increasing the concentration to 10 mg l−1 picloram resulted in improved final transformation frequency from 2.8% to 6.3%, with the highest frequency of 12.3% reached in one particular experiment, although statistical analysis showed that this difference in final transformation efficiency had a low level of significance. Stable integration of foreign genes, their expression, and inheritance were confirmed by Southern blot analyses, GUS assay, and genetic analysis. Analysis of T1 progeny showed that, of the 31 transgenic lines randomly selected, nearly one-third had a segregation ratio of 3:1, while the remainder had ratios typical of two or three independently segregating loci. PMID:20202997

  3. Development of an Efficient Agrobacterium-Mediated Transformation System and Production of Herbicide-Resistant Transgenic Plants in Garlic (Allium sativum L.)

    PubMed Central

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-01-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst non-transgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

  4. Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

    PubMed

    Ahn, Yul-Kyun; Yoon, Moo-Kyoung; Jeon, Jong-Seong

    2013-08-01

    The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits. PMID:23832764

  5. Aboveground insect infestation attenuates belowground Agrobacterium-mediated genetic transformation.

    PubMed

    Song, Geun Cheol; Lee, Soohyun; Hong, Jaehwa; Choi, Hye Kyung; Hong, Gun Hyong; Bae, Dong-Won; Mysore, Kirankumar S; Park, Yong-Soon; Ryu, Choong-Min

    2015-07-01

    Agrobacterium tumefaciens causes crown gall disease. Although Agrobacterium can be popularly used for genetic engineering, the influence of aboveground insect infestation on Agrobacterium induced gall formation has not been investigated. Nicotiana benthamiana leaves were exposed to a sucking insect (whitefly) infestation and benzothiadiazole (BTH) for 7 d, and these exposed plants were inoculated with a tumorigenic Agrobacterium strain. We evaluated, both in planta and in vitro, how whitefly infestation affects crown gall disease. Whitefly-infested plants exhibited at least a two-fold reduction in gall formation on both stem and crown root. Silencing of isochorismate synthase 1 (ICS1), required for salicylic acid (SA) synthesis, compromised gall formation indicating an involvement of SA in whitefly-derived plant defence against Agrobacterium. Endogenous SA content was augmented in whitefly-infested plants upon Agrobacterium inoculation. In addition, SA concentration was three times higher in root exudates from whitefly-infested plants. As a consequence, Agrobacterium-mediated transformation of roots of whitefly-infested plants was clearly inhibited when compared to control plants. These results suggest that aboveground whitefly infestation elicits systemic defence responses throughout the plant. Our findings provide new insights into insect-mediated leaf-root intra-communication and a framework to understand interactions between three organisms: whitefly, N. benthamiana and Agrobacterium. PMID:25676198

  6. AGROBACTERIUM-MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

    PubMed

    Kathiresan, S; Chandrashekar, A; Ravishankar, G A; Sarada, R

    2009-06-01

    The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (β-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed β-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries. PMID:27034041

  7. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    PubMed

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016. PMID:26871543

  8. Agrobacterium-mediated transformation for the investigation of somatic recombination in the fungal pathogen Armillaria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The honey fungus Armillaria mellea is a destructive soil-borne pathogen that affects over 300 plant species, and is of increasing interest due to its ability to decompose lignin. Here we report the transformation of this fungus. A range of techniques was evaluated, and Agrobacterium-mediated trans...

  9. A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus

    PubMed Central

    Chen, Xi; Stone, Michelle; Schlagnhaufer, Carl; Romaine, C. Peter

    2000-01-01

    We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species. PMID:11010906

  10. Agrobacterium-mediated genetic transformation of Pogostemon cablin (Blanco) Benth. Using leaf explants: bactericidal effect of leaf extracts and counteracting strategies.

    PubMed

    Paul, Anamika; Bakshi, Souvika; Sahoo, Debee Prasad; Kalita, Mohan Chandra; Sahoo, Lingaraj

    2012-04-01

    An optimized protocol for Agrobacterium tumefaciens-mediated transformation of patchouli using leaf disk explants is reported. In vitro antibacterial activity of leaf extracts of the plants revealed Agrobacterium sensitivity to the extracts. Fluorometric assay of bacterial cell viability indicated dose-dependent cytotoxic activity of callus extract against Agrobacterium cells. Addition of 0.1% Tween 20 and 2 g/l L-glutamine to Agrobacterium infection medium counteracted the bactericidal effect and significantly increased the T-DNA delivery to explants. A short preculture of explants for 2 days followed by infection with Agrobacterium in medium containing 150 μM of acetosyringone were found essential for efficient T-DNA delivery. Cocultivation for 3 days at 22 °C in conjunction with other optimized factors resulted in maximum T-DNA delivery. The Agrobacterium-mediated transformation of leaf disk explants were found significantly related to physiological age of the explants, age and origin of the of the donor plant. Leaf explants from second node of the 3-month-old in vivo plants showed highest transformation efficiency (94.3%) revealed by transient GUS expression assay. Plants selected on medium containing 20 mg/l kanamycin showed stable GUS expression in leaves and stem. The elongated shoots readily developed roots on kanamycin-free rooting medium and on transfer to soil, plants were successfully established. Polymerase chain reaction (PCR) and reverse-transcriptase PCR analysis in putative plants confirmed their transgenic nature. The established transformation method should provide new opportunities for the genetic improvement of patchouli for desirable trait. PMID:22434351

  11. Multiple host-cell recombination pathways act in Agrobacterium-mediated transformation of plant cells.

    PubMed

    Mestiri, Imen; Norre, Frédéric; Gallego, Maria E; White, Charles I

    2014-02-01

    Using floral-dip, tumorigenesis and root callus transformation assays of both germline and somatic cells, we present here results implicating the four major non-homologous and homologous recombination pathways in Agrobacterium-mediated transformation of Arabidopsis thaliana. All four single mutant lines showed similar mild reductions in transformability, but knocking out three of four pathways severely compromised Agrobacterium-mediated transformation. Although integration of T-DNA into the plant genome is severely compromised in the absence of known DNA double-strand break repair pathways, it does still occur, suggesting the existence of other pathways involved in T-DNA integration. Our results highlight the functional redundancy of the four major plant recombination pathways in transformation, and provide an explanation for the lack of strong effects observed in previous studies on the roles of plant recombination functions in transformation. PMID:24299074

  12. Strategies to improve low copy transgenic events in Agrobacterium-mediated transformation of maize.

    PubMed

    Sivamani, Elumalai; Li, Xianggan; Nalapalli, Samson; Barron, Yoshimi; Prairie, Anna; Bradley, David; Doyle, Michele; Que, Qiudeng

    2015-12-01

    Transgenic plants containing low copy transgene insertion free of vector backbone are highly desired for many biotechnological applications. We have investigated two different strategies for increasing the percentage of low copy events in Agrobacterium-mediated transformation experiments in maize. One of the strategies is to use a binary vector with two separate T-DNAs, one T-DNA containing an intact E.coli manA gene encoding phosphomannose isomerase (PMI) as selectable marker gene cassette and another T-DNA containing an RNAi cassette of PMI sequences. By using this strategy, low copy transgenic events containing the transgenes were increased from 43 to 60 % in maize. An alternate strategy is using selectable marker gene cassettes containing regulatory or coding sequences derived from essential plant genes such as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or MADS box transcription factor. In this paper we demonstrate that higher percentage of low copy transgenic events can be obtained in Agrobacterium-mediated maize transformation experiments using both strategies. We propose that the above two strategies can be used independently or in combination to increase transgenic events that contain low copy transgene insertion in Agrobacterium-mediated transformation experiments. PMID:26338266

  13. Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation.

    PubMed

    Renovell, Agueda; Gago, Selma; Ruiz-Ruiz, Susana; Velázquez, Karelia; Navarro, Luis; Moreno, Pedro; Vives, Mari Carmen; Guerri, José

    2010-10-25

    Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides -67 and +50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the +1 guanylate and the +2 adenylate are important for CP-sgRNA synthesis. PMID:20708769

  14. Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics.

    PubMed

    Grimaldi, Benedetto; de Raaf, Michiel A; Filetici, Patrizia; Ottonello, Simone; Ballario, Paola

    2005-07-01

    Mycorrhizal ascomycetes are ecologically and commercially important fungi that have proved impervious to genetic transformation so far. We report here on the successful transient transformation of Tuber borchii, an ectomycorrhizal ascomycete that colonizes a variety of trees and produces highly prized hypogeous fruitbodies known as "truffles". A hypervirulent Agrobacterium tumefaciens strain bearing the binary plasmid pBGgHg was used for transformation. The genes for hygromycin resistance and the enhanced green fluorescent protein (EGFP), both under the control of vector-borne promoters, were employed as selection markers. Patches of dark and fluorescent hyphae were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The presence of EGFP was confirmed by both confocal microscopy and PCR analysis. The lack in the transformed mycelia of the DNA coding for kanamicin resistance (a trait encoded by a vector-borne gene located outside of the T-DNA region) indicates that Agrobacterium-mediated gene transfer correctly occurred in T. borchii. PMID:15868150

  15. Improved Agrobacterium-mediated transformation and high efficiency of root formation from hypocotyl meristem of spring Brassica napus 'Precocity' cultivar.

    PubMed

    Liu, X X; Lang, S R; Su, L Q; Liu, X; Wang, X F

    2015-01-01

    Rape seed (Brassica napus L.) is one of the most important oil seed crops in the world. Genetic manipulation of rapeseed requires a suitable tissue culture system and an efficient method for plant regeneration, as well as an efficient transformation procedure. However, development of transgenic B. napus has been problematic, and current studies are limited to cultivated varieties. In this study, we report a protocol for regeneration of transgenic rape after Agrobacterium-mediated transformation of hypocotyls from the spring B. napus 'Precocity' cultivar. We analyzed the effects of plant growth regulators in the medium on regeneration. Additionally, factors affecting the transformation efficiency, including seedling age, Agrobacterium concentration, infection time, and co-cultivation time, were assessed by monitoring GUS expression. Results from these experiments revealed that transformation was optimized when the meristematic parts of the hypocotyls were taken from 8 day-old seedlings, cultured on Murashinge and Skoog basal media containing 0.1 mg/L 1-naphthaleneacetic acid and 2.5 mg/L 6-benzylaminopurine, and incubated in Agrobacterium suspension (OD600 = 0.5) for 3 to 5 min, followed by 2 days of co-cultivation. Integration of T-DNA into the plant genome was confirmed by polymerase chain reaction (PCR), b-glucuronidase histochemical staining, and quantitative real-time PCR. The protocols developed for regeneration, transformation, and rooting described in this study could help to accelerate the development of transgenic spring rape varieties with novel features. PMID:26681030

  16. Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

    PubMed

    Bull, S E; Owiti, J A; Niklaus, M; Beeching, J R; Gruissem, W; Vanderschuren, H

    2009-01-01

    Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop. PMID:20010938

  17. Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration.

    PubMed

    Subramanyam, Kondeti; Subramanyam, Koona; Sailaja, K V; Srinivasulu, M; Lakshmidevi, K

    2011-03-01

    A reproducible and efficient transformation method was developed for the banana cv. Rasthali (AAB) via Agrobacterium-mediated genetic transformation of suckers. Three-month-old banana suckers were used as explant and three Agrobacterium tumefaciens strains (EHA105, EHA101, and LBA4404) harboring the binary vector pCAMBIA1301 were used in the co-cultivation. The banana suckers were sonicated and vacuum infiltered with each of the three A. tumefaciens strains and co-cultivated in the medium containing different concentrations of acetosyringone for 3 days. The transformed shoots were selected in 30 mg/l hygromycin-containing selection medium and rooted in rooting medium containing 1 mg/l IBA and 30 mg/l hygromycin. The presence and integration of the hpt II and gus genes into the banana genome were confirmed by GUS histochemical assay, polymerase chain reaction, and southern hybridization. Among the different combinations tested, high transformation efficiency (39.4 ± 0.5% GUS positive shoots) was obtained when suckers were sonicated and vacuum infiltered for 6 min with A. tumefaciens EHA105 in presence of 50 μM acetosyringone followed by co-cultivation in 50 μM acetosyringone-containing medium for 3 days. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into banana has been developed and that this transformation system could be useful for future studies on transferring economically important genes into banana. PMID:21212957

  18. Agrobacterium-mediated transformation of two Serbian potato cultivars (Solanum tuberosum L. cv. Dragacevka and cv. Jelica)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An efficient protocol for Agrobacterium-mediated transformation of Serbian potato cultivars Dragacevka and Jelica, enabling the introduction of oryzacystatin genes OCI and OCII, was established. Starting with leaf explants a two-stage transformation protocol combining procedures of Webb and Wenzler...

  19. Exploration of new perspectives and limitations in Agrobacterium mediated gene transfer technology. Progress report, [June 1, 1992-- May 31, 1994

    SciTech Connect

    Marton, L.

    1994-12-31

    This report describes progress aimed at constructing gene-transfer technology for Nicotiana plumbaginifolia. Most actual effort as described herein has so far been directed at exploring new perspectives and limitations in Agrobacterium mediated gene transfer. Accomplishments are described using a core homologous gene targeting vector.

  20. Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer.

    PubMed Central

    Schlappi, M; Hohn, B

    1992-01-01

    Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation. PMID:12297627

  1. Development of Agrobacterium-mediated transformation of highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease.

    PubMed

    Elayabalan, Sivalingam; Kalaiponmani, Kalaimughilan; Subramaniam, Sreeramanan; Selvarajan, Ramasamy; Panchanathan, Radha; Muthuvelayoutham, Ramlatha; Kumar, Krish K; Balasubramanian, Ponnuswami

    2013-04-01

    One of the most severe viral diseases of hill banana is caused by banana bunchy top virus (BBTV), a nanovirus transmitted by the aphid Pentalonia nigronervosa. In this study, we reported the Agrobacterium-mediated transformation on a highly valued hill banana cultivar Virupakshi (AAB) for resistance to BBTV disease. The target of the RNA interference (RNAi) is the rep gene, encoded by the BBTV-DNA1. In order to develop RNAi construct targeting the BBTV rep gene, the full-length rep gene of 870 bp was polymerase chain reaction amplified from BBTV infected hill banana sample DNA, cloned and confirmed by DNA sequencing. The partial rep gene fragment was cloned in sense and anti sense orientation in the RNAi intermediate vector, pSTARLING-A. After cloning in pSTARLING-A, the cloned RNAi gene cassette was released by NotI enzyme digestion and cloned into the NotI site of binary vector, pART27. Two different explants, embryogenic cells and embryogenic cell suspension derived microcalli were used for co-cultivation. Selection was done in presence of 100 mg/L kanamycin. In total, 143 putative transgenic hill banana lines were generated and established in green house condition. The presence of the transgenes was confirmed in the selected putative transgenic hill banana lines by PCR and reverse transcription PCR analyses. Transgenic hill banana plants expressing RNAi-BBTV rep were obtained and shown to resist infection by BBTV. The transformed plants are symptomless, and the replication of challenge BBTV almost completely suppressed. Hence, the RNAi mediating resistances were shown to be effective management of BBTV in hill banana. PMID:23184576

  2. Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

    PubMed Central

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

    2013-01-01

    Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

  3. Origin of somatic embryos from repetitively embryogenic cultures of walnut (Juglans regia L.): Implications forAgrobacterium-mediated transformation.

    PubMed

    Polito, V S; McGranahan, G; Pinney, K; Leslie, C

    1989-04-01

    Early stages of somatic embryo development from embryogenic cultures ofJuglans regia (Persian or English walnut) are described. Histological examination reveals that secondary somatic embryos arise from cotyledons and hypocotyls of primary embryos cultured in the dark. The embryos originate by transverse to oblique divisions of surface cells. Single-cell origin of the secondary embryos confirms the potential of the repetitive embryogenesis system forAgrobacterium-mediated transformation and regeneration of non-chimeric, transgenic walnut plants. PMID:24233141

  4. Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation.

    PubMed

    Saika, Hiroaki; Toki, Seiichi

    2010-12-01

    We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning--a common problem with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied our efficient transformation system to Kasalath--a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting. PMID:20853107

  5. Establishment of an Agrobacterium-mediated Inoculation System for Cucumber Green Mottle Mosaic Virus

    PubMed Central

    Kang, Minji; Seo, Jang-Kyun; Song, Dami; Choi, Hong-Soo; Kim, Kook-Hyung

    2015-01-01

    The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor. PMID:26674677

  6. Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation.

    PubMed

    Lu, Chaofu; Kang, Jinling

    2008-02-01

    Camelina sativa is an alternative oilseed crop that can be used as a potential low-cost biofuel crop or a source of health promoting omega-3 fatty acids. Currently, the fatty acid composition of camelina does not uniquely fit any particular uses, thus limit its commercial value and large-scale production. In order to improve oil quality and other agronomic characters, we have developed an efficient and simple in planta method to generate transgenic camelina plants. The method included Agrobacterium-mediated inoculation of plants at early flowering stage along with a vacuum infiltration procedure. We used a fluorescent protein (DsRed) as a visual selection marker, which allowed us to conveniently screen mature transgenic seeds from a large number of untransformed seeds. Using this method, over 1% of transgenic seeds can be obtained. Genetic analysis revealed that most of transgenic plants contain a single copy of transgene. In addition, we also demonstrated that transgenic camelina seeds produced novel hydroxy fatty acids by transforming a castor fatty acid hydroxylase. In conclusion, our results provide a rapid means to genetically improve agronomic characters of camelina, including fatty acid profiles of its seed oils. Camelina may serve as a potential industrial crop to produce novel biotechnology products. PMID:17899095

  7. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

    PubMed

    Zhang, Wan-Jun; Dewey, Ralph E; Boss, Wendy; Phillippy, Brian Q; Qu, Rongda

    2013-02-01

    Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species. PMID:23242917

  8. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    PubMed

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars. PMID:20711728

  9. Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

    PubMed Central

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

  10. Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

    PubMed

    Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

    2014-01-01

    The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

  11. pSiM24 is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants.

    PubMed

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3'rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses. PMID:24897541

  12. pSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

    PubMed Central

    Sahoo, Dipak Kumar; Dey, Nrisingha; Maiti, Indu Bhushan

    2014-01-01

    We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3′rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses. PMID:24897541

  13. Agrobacterium-mediated genetic transformation of commercially elite rice restorer line using nptII gene as a plant selection marker.

    PubMed

    Chakraborty, M; Sairam Reddy, P; Laxmi Narasu, M; Krishna, Gaurav; Rana, Debashis

    2016-01-01

    Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l(-1)) and Paramomycin (70 mg l(-1)) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe. PMID:27186018

  14. A simple shoot multiplication procedure using internode explants, and its application for particle bombardment and Agrobacterium-mediated transformation in watercress.

    PubMed

    Ogita, Shinjiro; Usui, Miki; Shibutani, Nanae; Kato, Yasuo

    2009-07-01

    A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 muM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1-2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 muM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene. PMID:19308313

  15. Agrobacterium-mediated genetic transformation using cotyledons in Japanese pear (Pyrus pyrifolia)

    PubMed Central

    Nakajima, Ikuko; Sato, Yoshihiko; Saito, Toshihiro; Moriguchi, Takaya; Yamamoto, Toshiya

    2013-01-01

    Genetic transformation was successfully established producing both transformed adventitious shoots and calli in Japanese pear (Pyrus pyrifolia Nakai) by using cotyledons as explants. Cotyledons of five cultivars were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying the pBIN19-sgfp, which contained a green fluorescent protein gene and the neomycin phosphotransferase gene. In order to increase transformation efficiency, sonication and ethylenedioxybis (ethylamine)-N,N,N′,N′-tetraacetic acid (EGTA) treatments were applied, which could produce physical wounds across the tissue and prevent plant defense reaction, respectively. Green fluorescent protein (GFP) fluorescence was evaluated two weeks and five months after Agrobacterium inoculation as measures of transient and stable transformations, respectively. As a result, sonication significantly increased both transient and stable expression of GFP fluorescence, whereas EGTA treatment did not show a positive effect on either. Out of 18 regenerated plantlets obtained, one plant regenerated from ‘Agenosho Shinanashi’ showed stable GFP fluorescence. This plant was confirmed as a transformant by PCR and genomic Southern blotting. Three other transformed regenerated shoots by myb gene showed red color, which were derived from ‘Imamuraaki’ by the same transformation method. Transformation system in this study was shown to be reproducible since plural transformants were obtained. PMID:24273422

  16. Enhanced Agrobacterium-mediated transformation of embryogenic calli of upland cotton.

    PubMed

    Zhang, Tianzhen; Wu, Shen-Jie

    2012-01-01

    Agrobacterium tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. The binary vector pBI121 (containing a neomycin phosphotransferase II gene npt-II as a selection marker and a uidA gene as a reporter gene) was used to research transformation efficiency. After 48 h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse granule EC. It indicated that the efficiency of transient transformation was affected by EC morphology. Transient transformation efficiency also was improved by cocultivation on the medium by adding 50 mg/L acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density increased the production of kanamycin-resistant (Km-R) calli lines. From an original 0.3 g EC, an average of 20 Km-R calli lines were obtained from a selection dish, and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments, and the GUS-positive rate of regeneration plants was about 72.6%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and gus gene. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, was integrated into the cotton genome. PMID:22351014

  17. Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Method1

    PubMed Central

    Desfeux, Christine; Clough, Steven J.; Bent, Andrew F.

    2000-01-01

    The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture. To facilitate use with other plant species, we investigated the mechanisms that underlie this method. In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48%. Agrobacterium strains with T-DNA carrying gusA (encoding β-glucuronidase [GUS]) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed. Transformants derived from the same seed pod contained independent T-DNA integration events. In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis. In correlation with this fact, we found that the timing of Agrobacterium infection was critical. Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis. A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium. Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. PMID:10889238

  18. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri.

    PubMed

    Rathod, Jayant Pralhad; Prakash, Gunjan; Pandit, Reena; Lali, Arvind M

    2013-11-01

    Parachlorella kessleri is a unicellular alga which grows in fresh as well as marine water and is commercially important as biomass/lipid feedstock and in bioremediation. The present study describes the successful transformation of marine P. kessleri with the help of Agrobacterium tumefaciens. Transformed marine P. kessleri was able to tolerate more than 10 mg l(-1) hygromycin concentration. Co-cultivation conditions were modulated to allow the simultaneous growth of both marine P. kessleri and A. tumefaciens. For co-cultivation, P. kessleri was shifted from Walne's to tris acetate phosphate medium to reduce the antibiotic requirement during selection. In the present study, the transfer of T-DNA was successful without using acetosyringone. Biochemical and genetic analyses were performed for expression of transgenes by GUS assay and PCR in transformants. Establishment of this protocol would be useful in further genetic modification of oil-bearing Parachlorella species. PMID:24097049

  19. Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

    PubMed

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

    2015-01-01

    Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant. PMID:25102992

  20. Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions.

    PubMed

    Tzima, Aliki K; Paplomatas, Epaminondas J; Schoina, Charikleia; Domazakis, Emmanouil; Kang, Seogchan; Goodwin, Paul H

    2014-08-01

    Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens-mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process. PMID:25110130

  1. Hypocotyl-based Agrobacterium-mediated transformation of soybean (Glycine max) and application for RNA interference.

    PubMed

    Wang, Geliang; Xu, Yinong

    2008-07-01

    An efficient system of gene transformation is necessary for soybean [Glycine max (L.) Merrill] functional genomics and gene modification by using RNA interference (RNAi) technology. To establish such system, we improved the conditions of tissue culture and transformation for increasing the frequency of adventitious shoots and decreasing the browning and necrosis of hypocotyls. Adding N(6)-benzylaminopurine (BAP) and silver nitrate in culture medium enhanced the shoot formation on hypocotyls. BAP increased the frequency of the hypocotyls containing adventitious shoots, while silver nitrate increased the number of shoots on the hypocotyls. As a result, the number of adventitious shoots on hypocotyls cultured in medium containing both BAP and silver nitrate was 5-fold higher than the controls. Adding antioxidants in co-cultivation medium resulted in a significant decrease in occurrence of browning and necrosis of hypocotyls and increase in levels of beta-Glucuronidase (GUS) gene expression. Histochemical assays showed that the apical meristem of hypocotyls was the "target tissue" for Agrobacterium tumefaciens transformation of soybean. Gene silencing of functional gene by using RNAi technology was carried out under above conditions. A silencing construct containing an inverted-repeat fragment of the GmFAD2 gene was introduced into soybean by using the A. tumefaciens-mediated transformation. Several lines with high oleic acid were obtained, in which mean oleic acid content ranged from 71.5 to 81.9%. Our study demonstrates that this transgenic approach could be efficiently used to improve soybean quality and productivity through functional genomics. PMID:18347801

  2. Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda.

    PubMed

    Stevens, Micah E; Pijut, Paula M

    2014-06-01

    This transformation and regeneration protocol provides an integral framework for the genetic improvement of Fraxinus profunda (pumpkin ash) for future development of plants resistant to the emerald ash borer. Using mature hypocotyls as the initial explants, an Agrobacterium tumefaciens-mediated genetic transformation system was successfully developed for pumpkin ash (Fraxinus profunda). This transformation protocol is an invaluable tool to combat the highly aggressive, non-native emerald ash borer (EAB), which has the potential to eliminate native Fraxinus spp. from the natural landscape. Hypocotyls were successfully transformed with Agrobacterium strain EHA105 harboring the pq35GR vector, containing an enhanced green fluorescent protein (EGFP) as well as a fusion gene between neomycin phosphotransferase (nptII) and gusA. Hypocotyls were cultured for 7 days on Murashige and Skoog (MS) medium with 22.2 μM 6-benzyladenine (BA), 4.5 μM thidiazuron (TDZ), 50 mg L(-1) adenine hemisulfate (AS), and 10 % coconut water (CW) prior to transformation. Hypocotyls were transformed using 90 s sonication plus 10 min vacuum infiltration after Agrobacterium was exposed to 100 μM acetosyringone for 1 h. Adventitious shoots were regenerated on MS medium with 22.2 μM BA, 4.5 μM TDZ, 50 mg L(-1) AS, 10 % CW, 400 mg L(-1) timentin, and 20 mg L(-1) kanamycin. Timentin at 400 and 20 mg L(-1) kanamycin were most effective at controlling Agrobacterium growth and selecting for transformed cells, respectively. The presence of nptII, GUS (β-glucuronidase), and EGFP in transformed plants was confirmed using polymerase chain reaction (PCR), while the expression of EGFP was also confirmed through fluorescent microscopy and reverse transcription-PCR. This transformation protocol provides an integral foundation for future genetic modifications of F. profunda to provide resistance to EAB. PMID:24493252

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

    PubMed

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

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals. PMID:19156736

  4. Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

    PubMed Central

    Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

    2015-01-01

    In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

  5. Establishment of a transient expression system for Dictyostelium discoideum.

    PubMed Central

    Howard, P K; Ahern, K G; Firtel, R A

    1988-01-01

    We have established a rapid and sensitive transient expression system for Dictyostelium discoideum. We constructed a gene fusion containing the promoter from the Dictyostelium Actin 15 gene fused to the firefly luciferase gene. The enzymatic activity of this gene fusion, expressed at very high levels in stable transformants, was measured to determine optimum conditions for transient expression using electroporation to introduce the DNA into cells. With these conditions, we show that a luciferase gene fusion driven by a prestalk, cell-type specific promoter from the pst-cathepsin gene expresses luciferase at the appropriate developmental stage. In addition, we present results suggesting that the system will be useful for expressing genes in non-axenic cell lines. Finally, we observe that electroporation is more efficient for obtaining stable transformations than the standard calcium phosphate procedure using extrachromosomally replicating shuttle vectors but less efficient for vectors that integrate into the Dictyostelium chromosomes. PMID:3362676

  6. In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant.

    PubMed

    Alok, Anshu; Shukla, Vishnu; Pala, Zarna; Kumar, Jitesh; Kudale, Subhash; Desai, Neetin

    2016-04-01

    Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog's medium containing 2 mg/L BAP and 0.1 mg/L NAA, after 45 days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1 mg/L IBA or 1 mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15 mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83 % transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2 days at 22 °C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200 mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering. PMID:27436917

  7. Transient Protein Expression by Agroinfiltration in Lettuce.

    PubMed

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

    2016-01-01

    Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level. PMID:26614281

  8. Screening promoters for Anthurium transformation using transient expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Different promoters and tissue types were evaluated for transient '-glucoronidase (GUS) expression in Anthurium andreanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35 S promoter fr...

  9. Identifying a Carotenoid Cleavage Dioxygenase 4a Gene and Its Efficient Agrobacterium-Mediated Genetic Transformation in Bixa orellana L.

    PubMed

    Sankari, Mohan; Hemachandran, Hridya; Anantharaman, Amirtha; Babu, Subramanian; Madrid, Renata Rivera; C, George Priya Doss; Fulzele, Devanand P; Siva, Ramamoorthy

    2016-07-01

    Carotenoids are metabolized to apocarotenoids through the pathway catalysed by carotenoid cleavage oxygenases (CCOs). The apocarotenoids are economically important as it is known to have therapeutic as well as industrial applications. For instance, bixin from Bixa orellana and crocin from Crocus sativus are commercially used as a food colourant and cosmetics since prehistoric time. In our present study, CCD4a gene has been identified and isolated from leaves of B. orellana for the first time and named as BoCCD4a; phylogenetic analysis was carried out using CLUSTAL W. From sequence analysis, BoCCD4a contains two exons and one intron, which was compared with the selected AtCCD4, RdCCD4, GmCCD4 and CmCCD4a gene. Further, the BoCCD4a gene was cloned into pCAMBIA 1301, transformed into Agrobacterium tumefaciens EHA105 strain and subsequently transferred into hypocotyledons and callus of B. orellana by agro-infection. Selection of stable transformation was screened on the basis of PCR detection by using GUS and hptII specific primer, which was followed by histochemical characterization. The percent transient GUS expression in hypocotyledons and callus was 84.4 and 80 %, respectively. The expression of BoCCD4a gene in B. orellana was confirmed through RT-PCR analysis. From our results, the sequence analysis of BoCCD4a gene of B. orellana was closely related to the CsCCD4 gene of C. sativus, which suggests this gene may have a role in various processes such as fragrance, insect attractant and pollination. PMID:26922728

  10. The Physcomitrella patens System for Transient Gene Expression Assays.

    PubMed

    Thévenin, Johanne; Xu, Wenjia; Vaisman, Louise; Lepiniec, Loïc; Dubreucq, Bertrand; Dubos, Christian

    2016-01-01

    Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells. PMID:27557766

  11. DsRed2 transient expression in Culex quinquefasciatus mosquitoes

    PubMed Central

    Wilke, André Barretto Bruno; Scaife, Sarah; Alphey, Luke; Marrelli, Mauro Toledo

    2013-01-01

    Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions. PMID:23828005

  12. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

    PubMed Central

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  13. Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora.

    PubMed

    Khunjan, Uraiwan; Ekchaweng, Kitiya; Panrat, Tanate; Tian, Miaoying; Churngchow, Nunta

    2016-01-01

    This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora. PMID:27337148

  14. Transient Expression Systems in Plants: Potentialities and Constraints.

    PubMed

    Canto, Tomas

    2016-01-01

    Plants have been used from old to extract and isolate by different means the products of interest that they store. In recent years new techniques have emerged that allow the use of plants as factories to overexpress transiently and often efficiently, specific genes of interest, either endogenous or foreign, in their native form or modified. These techniques allow and facilitate the targeted purification of gene products for research and commercial purposes without resorting to lengthy, time-consuming and sometimes challenging plant stable transformations, while avoiding some of their associated regulatory constraints. In this chapter we describe the main strategies available for the transient expression of gene sequences and their encoded products in plants. We discuss biological issues affecting transient expression, including resistance responses elicited by the plant against sequences that it recognizes naturally as foreign, and ways to neutralize them. We also discuss the relative advantages of each expression strategy as well as their inherent drawbacks and technical limitations, and how to partially prevent or overcome them, whenever possible. PMID:27165332

  15. TauG-guidance of transients in expressive musical performance.

    PubMed

    Schogler, Benjaman; Pepping, Gert-Jan; Lee, David N

    2008-08-01

    The sounds in expressive musical performance, and the movements that produce them, offer insight into temporal patterns in the brain that generate expression. To gain understanding of these brain patterns, we analyzed two types of transient sounds, and the movements that produced them, during a vocal duet and a bass solo. The transient sounds studied were inter-tone f (0)(t)-glides (the continuous change in fundamental frequency, f (0)(t), when gliding from one tone to the next), and attack intensity-glides (the continuous rise in sound intensity when attacking, or initiating, a tone). The temporal patterns of the inter-tone f (0)(t)-glides and attack intensity-glides, and of the movements producing them, all conformed to the mathematical function, tau (G)(t) (called tauG), predicted by General Tau Theory, and assumed to be generated in the brain. The values of the parameters of the tau (G)(t) function were modulated by the performers when they modulated musical expression. Thus the tau (G)(t) function appears to be a fundamental of brain activity entailed in the generation of expressive temporal patterns of movement and sound. PMID:18560815

  16. Human Neural Cells Transiently Express Reelin during Olfactory Placode Development

    PubMed Central

    Antal, M. Cristina; Samama, Brigitte; Ghandour, M. Said; Boehm, Nelly

    2015-01-01

    Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed. PMID:26270645

  17. Human Neural Cells Transiently Express Reelin during Olfactory Placode Development.

    PubMed

    Antal, M Cristina; Samama, Brigitte; Ghandour, M Said; Boehm, Nelly

    2015-01-01

    Reelin, an extracellular glycoprotein is essential for migration and correct positioning of neurons during development. Since the olfactory system is known as a source of various migrating neuronal cells, we studied Reelin expression in the two chemosensory olfactory systems, main and accessory, during early developmental stages of human foetuses/embryos from Carnegie Stage (CS) 15 to gestational week (GW) 14. From CS 15 to CS 18, but not at later stages, a transient expression of Reelin was detected first in the presumptive olfactory and then in the presumptive vomeronasal epithelium. During the same period, Reelin-positive cells detach from the olfactory/vomeronasal epithelium and migrate through the mesenchyme beneath the telencephalon. Dab 1, an adaptor protein of the Reelin pathway, was simultaneously expressed in the migratory mass from CS16 to CS17 and, at later stages, in the presumptive olfactory ensheathing cells. Possible involvements of Reelin and Dab 1 in the peripheral migrating stream are discussed. PMID:26270645

  18. Optimizing transient recombinant protein expression in mammalian cells.

    PubMed

    Hopkins, Ralph F; Wall, Vanessa E; Esposito, Dominic

    2012-01-01

    Transient gene expression (TGE) in mammalian cells has become a routine process for expressing recombinant proteins in cell lines such as human embryonic kidney 293 and Chinese hamster ovary cells. The rapidly increasing need for recombinant proteins requires further improvements in TGE technology. While a great deal of focus has been directed toward optimizing the secretion of antibodies and other naturally secreted targets, much less work has been done on ways to improve cytoplasmic expression in mammalian cells. The benefits to protein production in mammalian cells, particularly for eukaryotic proteins, should be very significant - glycosylation and other posttranslational modifications will likely be native or near-native, solubility and protein folding would likely improve overexpression in heterologous hosts, and expression of proteins in their proper intracellular compartments is much more likely to occur. Improvements in this area have been slow, however, due to limited development of the cell culture processes needed for low-cost, higher-throughput expression in mammalian cells, and the relatively low diversity of DNA vectors for protein production in these systems. Here, we describe how the use of recombinational cloning, coupled with improvements in transfection protocols which increase speed and lower cost, can be combined to make mammalian cells much more amenable for routine recombinant protein expression. PMID:21987258

  19. Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

    PubMed

    Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

    2007-10-01

    A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants. PMID:17551731

  20. Agrobacterium-mediated transformation of Eucalyptus globulus using explants with shoot apex with introduction of bacterial choline oxidase gene to enhance salt tolerance.

    PubMed

    Matsunaga, Etsuko; Nanto, Kazuya; Oishi, Masatoshi; Ebinuma, Hiroyasu; Morishita, Yoshihiko; Sakurai, Nozomu; Suzuki, Hideyuki; Shibata, Daisuke; Shimada, Teruhisa

    2012-01-01

    Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants. PMID:22009051

  1. Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

    PubMed

    Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

    2015-02-01

    Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. PMID:25499296

  2. Agrobacterium-mediated transformation of the β-subunit gene in 7S globulin protein in soybean using RNAi technology.

    PubMed

    Qu, J; Liu, S Y; Wang, P W; Guan, S Y; Fan, Y G; Yao, D; Zhang, L; Dai, J L

    2016-01-01

    The objective of this study was to use RNA interference (RNAi) to improve protein quality and decrease anti-nutritional effects in soybean. Agrobacterium tumefaciens-mediated transformation was conducted using RNAi and an expression vector containing the 7S globulin β-subunit gene. The BAR gene was used as the selective marker and cotyledonary nodes of soybean genotype Jinong 27 were chosen as explant material. Regenerated plants were detected by molecular biology techniques. Transformation of the β-subunit gene in the 7S protein was detected by PCR, Southern blot, and q-PCR. Positive plants (10 T0, and 6 T1, and 13 T2) were tested by PCR. Hybridization bands were detected by Southern blot analysis in two of the T1 transgenic plants. RNAi expression vectors containing the soybean 7S protein β-subunit gene were successfully integrated into the genome of transgenic plants. qRT-PCR analysis in soybean seeds showed a clear decrease in expression of the soybean β-subunit gene. The level of 7S protein β-subunit expression in transgenic plants decreased by 77.5% as compared to that of the wild-type plants. This study has established a basis for the application of RNAi to improve the anti-nutritional effects of soybean. PMID:27173254

  3. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

    PubMed

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L; Arzola, Lucas; Lebrilla, Carlito B; Dandekar, Abhaya M; Falk, Bryce W; Nandi, Somen; Rodriguez, Raymond L; McDonald, Karen A

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  4. Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana

    PubMed Central

    Alkanaimsh, Salem; Karuppanan, Kalimuthu; Guerrero, Andrés; Tu, Aye M.; Hashimoto, Bryce; Hwang, Min Sook; Phu, My L.; Arzola, Lucas; Lebrilla, Carlito B.; Dandekar, Abhaya M.; Falk, Bryce W.; Nandi, Somen; Rodriguez, Raymond L.; McDonald, Karen A.

    2016-01-01

    To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed. PMID:27379103

  5. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14.

    PubMed

    Nyaboga, Evans N; Njiru, Joshua M; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1-2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70-80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  6. Factors influencing somatic embryogenesis, regeneration, and Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) cultivar TME14

    PubMed Central

    Nyaboga, Evans N.; Njiru, Joshua M.; Tripathi, Leena

    2015-01-01

    Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 1–2 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 70–80 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

  7. Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

    PubMed Central

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  8. Transient expression of proteins by hydrodynamic gene delivery in mice.

    PubMed

    Kovacsics, Daniella; Raper, Jayne

    2014-01-01

    Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR). PMID:24837006

  9. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  10. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

    NASA Technical Reports Server (NTRS)

    Cardoza, V.; Stewart, C. N.

    2003-01-01

    An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

  11. Transgene expression in pear (Pyrus communis L.) driven by a phloem-specific promoter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A gene expression cassette carrying ß-glucuronidase (uidA) reporter gene under the control of the promoter of the Arabidopsis sucrose-H+ symporter gene (AtSUC2) was introduced to pear plants via an Agrobacterium-mediated leaf-explant transformation procedure. Transgenic shoots were regenerated from...

  12. Transient Expression of cor Gene in Papaver somniferum

    PubMed Central

    Hosseini, Bahman; Shahriari-Ahmadi, Farajollah; Hashemi, Haleh; Marashi, Mohammad-Hasan; Mohseniazar, Mahdi; Farokhzad, Alireza; Sabokbari, Masoume

    2011-01-01

    Introduction Papaver somniferum is the commercial source of morphine and codeine. The isolation of effective genes involved in the morphine biosynthesis of P. somniferum is very important in the production of specific metabolites achieved using metabolic engi-neering techniques. In this pathway, the key enzyme COR is involved in the conversion of codeinone to codeine and morphinone to morphine. Methods the gene encoding of this enzyme was isolated using primers designed on the base of gene sequence available on (NCBI) for P. somniferum. This gene correct size around (960 bp) was first subcloned into pTZ57RIT vector then cloned into expression vectors (pBI121) between BamHI and SacI sites to allow the expression of cor gene driven by the cauliflower mosaic virus 35S pro-moter. The result was confirmed through different molecular methods e.g. PCR and en-zyme digestion by BamHI and SacI. The recombinant plasmid was transformed into the E. coli strain DH5α using a freeze-thaw method. Having selected positive colones on selection medium, plasmid was extracted by miniprep method and recombinant plasmids were selected based on PCR and digestion. The construct was then mobilized in Agrobacterium tumefaciens C58/pGV3850 (KmR RifR). After gene transformation to P. somniferum plants, the agroinfiltration method was also used for transient expression of COR enzyme. Results evaluation results showed that morphine and codeine were detectable in the leaves of transgenic plants containing cor transgene and there was significant difference in the final production. After completing this experiment for three times, results showed that in 11 sets from 15 sets of leaves experiment tested, main alkaloids (codeine, morphine, papaverin, noscapine and thebaine) were detectable. Conclusion Whereas no signal was detected in non-infiltrated control leaves or in leaves infiltrated with non-recombinant bacteria for morphine and codeine, others such as thebaine and papaverine were detectable

  13. Uptake and transient expression of chimeric genes in seed-derived embryos.

    PubMed Central

    Töpfer, R; Gronenborn, B; Schell, J; Steinbiss, H H

    1989-01-01

    Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric "neo" genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly when the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms. PMID:2562504

  14. Transient Stability Simulation of Wind Generator Expressed by Two-Mass Model

    NASA Astrophysics Data System (ADS)

    Shima, Yoshikazu; Takahashi, Rion; Murata, Toshiaki; Tamura, Junji; Tomaki, Yuichi; Tominaga, Shinobu; Sakahara, Atsushi

    Recently, wind power generation is increasing in the world. In the wind power stations, induction machines are mostly used as generators. Since induction generators have a stability problem similar to the transient stability of synchronous machines, it is important to analyze the transient stability of power systems including wind generators. Although there have been some reports analyzing the transient stability problem, wind turbine and wind generator are, in most cases, modeled as one mass shaft system having total inertia constant. This paper presents simulation analyses of transient stability of power system including induction generator which is expressed by a two-mass shaft model and analyzes an effect of shaft system modeling on the transient stability characteristics. Simulations are performed by PSCAD/EMTDC in this study.

  15. [Regulatory potential of S/MAR elements in transient expression].

    PubMed

    Sass, A V; Ruda, V M; Akopov, S B; Snezhkov, E V; Nikolaev, L G; Sverdlov, E D

    2005-01-01

    S/MARs (scaffold/matrix attachment regions) are the DNA regions that are involved in the interaction with the nuclear matrix and are identified by in vitro methods. According to the available information, S/MARs possess an insulating activity, i.e., the ability to block the interaction between the enhancer and promoter in vivo, and are, probably, intact insulators or their fragments. Nevertheless, there is still no direct proof for this correspondence. To obtain additional information on the insulator activity of S/MARs, we selected five DNA fragments of different lengths and affinities for the nuclear matrix from the previously constructed library of S/MARs and tested their ability to serve as insulators. Two of five elements exhibited an insulator (enhancer-blocking) activity upon the transient transfection of CHO cells. None of the S/MARs displayed either promoter or enhancer/silencer activities in these cells. PMID:15787217

  16. Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability

    PubMed Central

    Gopinath, K.; Dragnea, B.; Kao, C.

    2005-01-01

    Brome mosaic virus (BMV) RNA replication has been examined in a number of systems, including Saccharomyces cerevisiae. We developed an efficient T-DNA-based gene delivery system using Agrobacterium tumefaciens to transiently express BMV RNAs in Nicotiana benthamiana. The expressed RNAs can systemically infect plants and provide material to extract BMV replicase that can perform template-dependent RNA-dependent RNA synthesis in vitro. We also expressed the four BMV-encoded proteins from nonreplicating RNAs and analyzed their effects on BMV RNA accumulation. The capsid protein that coinfiltrated with constructs expressing RNA1 and RNA2 suppressed minus-strand levels but increased plus-strand RNA accumulation. The replication proteins 1a and 2a could function in trans to replicate and transcribe the BMV RNAs. None of the BMV proteins or RNA could efficiently suppress posttranscriptional silencing. However, 1a expressed in trans will suppress the production of a recombinant green fluorescent protein expressed from the nontranslated portions of BMV RNA1 and RNA2, suggesting that 1a may regulate translation from BMV RNAs. BMV replicase proteins 1a did not affect the accumulation of the BMV RNAs in the absence of RNA replication, unlike the situation reported for S. cerevisiae. This work demonstrates that the Agrobacterium-mediated gene delivery system can be used to study the cis- and trans-acting requirements for BMV RNA replication in plants and that significant differences can exist for BMV RNA replication in different hosts. PMID:16254357

  17. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    PubMed Central

    Guo, Jianjun; Morrell-Falvey, Jennifer L.; Labbé, Jessy L.; Muchero, Wellington; Kalluri, Udaya C.; Tuskan, Gerald A.; Chen, Jin-Gui

    2012-01-01

    Background Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways. PMID:23028673

  18. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    SciTech Connect

    Guo, Jianjun; Morrell-Falvey, Jennifer L; Labbe, Jessy L; Muchero, Wellington; Kalluri, Udaya C; Tuskan, Gerald A; Chen, Jay

    2012-01-01

    Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  19. Study on transient expression of gus gene in Chlorelia ellipsoidea (Chlorophyta) by using biolistic particle delivery system

    NASA Astrophysics Data System (ADS)

    Chen, Ying; Li, Wen-Bin; Bai, Qin-Hua; Sun, Yong-Ru

    1998-03-01

    Study on the transient expression of GUS gene at different growing stage of Chlorella ellipsoidea using high velocity microprojectiles, the effects of osmosis, the distance between microprojectile and target cell, bombardment times, are reported in this paper. The results showed that C. ellipsoidea in exponential phase has higer level of transient expression and that treatment with osmosis can improve the GUS transient expression notably. The effect of distance or bombardment times was not observed.

  20. An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression

    PubMed Central

    Doherty, Joseph E.; Woodard, Lauren E.; Bear, Adham S.; Foster, Aaron E.; Wilson, Matthew H.

    2013-01-01

    Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.—Doherty, J. E., Woodard, L. E., Bear, A. S., Foster, A. E., Wilson, M. H. An adaptable system for improving transposon-based gene expression in vivo via transient transgene repression. PMID:23752206

  1. Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus 1

    PubMed Central

    Oard, James H.; Paige, David F.; Simmonds, John A.; Gradziel, Thomas M.

    1990-01-01

    An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The β-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 × 10−3. In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals. Images Figure 7 Figure 8 Figure 9 PMID:16667278

  2. Transient Expression of P-type ATPases in Tobacco Epidermal Cells.

    PubMed

    Poulsen, Lisbeth R; Palmgren, Michael G; López-Marqués, Rosa L

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellular space between leaf epidermal cells, which results in DNA transfer from the bacteria to the plant and expression of the corresponding proteins. By injecting mixes of Agrobacterium strains, this system offers the possibility to co-express a number of target proteins simultaneously, thus allowing for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits. PMID:26695049

  3. Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum).

    PubMed

    Fang, Z W; Xu, X Y; Gao, J F; Wang, P K; Liu, Z X; Feng, B L

    2015-01-01

    C-repeat-binding factor (CBF)/dehydration-responsive element (DREB) transcription factors play key roles in plant stress responses. However, little information is available on the regulation of CBF/DREB expression. In this study, we isolated and characterized the FeDREB1 promoter sequence from the common buckwheat accession Xinong 9976. To identify the upstream region of the FeDREB1 gene required for promoter activity, we constructed a series of FeDREB1 promoter deletion derivatives. Each deletion construct was analyzed through Agrobacterium-mediated transient transformation in tobacco leaves treated with 4°C cold or drought stress. Promoter-beta-glucuronidase fusion assays revealed that the pCD1 (-270 bp) deletion in the upstream region of FeDREB1 could activate expression of the GUS gene at 4°C. The pCD1 (-270 bp), pCD2 (-530 bp), and pCD3 (-904 bp) deletion induced low-level GUS expression under drought stress. However, the pCD4 (-1278 bp) deletion clearly activated GUS gene expression. Our results suggest that sections pCD1 (-270 bp) and pCD4 (-1278 bp) in the FeDREB1 gene promoter are new sources of induced promoters for adversity-resistance breeding in plant genetic engineering. PMID:26214481

  4. Polycomb Controls Gliogenesis by Regulating the Transient Expression of the Gcm/Glide Fate Determinant

    PubMed Central

    Diebold, Celine; Van de Bor, Véronique; Schuettengruber, Bernd; González, Inma; Busturia, Ana; Cavalli, Giacomo; Giangrande, Angela

    2012-01-01

    The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system. PMID:23300465

  5. Functional analysis of Cobra Venom Factor/human C3 chimeras transiently expressed in mammalian cells.

    PubMed

    Kölln, Johanna; Matzas, Mark; Jänner, Nathalie; Mix, Thorsten; Klensang, Katrin; Bredehorst, Reinhard; Spillner, Edzard

    2004-05-01

    The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule. PMID:15140572

  6. Expression and function of transient receptor potential channels in the female bovine reproductive tract.

    PubMed

    Ghavideldarestani, Maryam; Atkin, Stephen L; Leese, Henry J; Sturmey, Roger G

    2016-07-15

    The epithelium lining the oviduct is critical for early reproductive events, many of which are mediated via intracellular calcium ions. Despite this, little is known about the regulation of calcium homeostasis in the oviductal epithelium. Epithelial transient receptor potential channels (TRPCs) modulate calcium flux in other tissues, and their expression and functional regulation have therefore been examined using the bovine oviduct as a model for the human. The effects of FSH, LH, 17β-estradiol, and progesterone on TRPCs expression and intracellular calcium flux were determined. Transient receptor potential channels 1, 2, 3, 4, and 6 were expressed in the bovine reproductive tract, and their gene expression varied throughout the estrous cycle. In more detailed studies undertaken on TRPC1 and 6, we show that protein expression varied through the estrus cycle; specifically, 17β-estradiol, FSH, and LH individually and in combination upregulated TRPC1 and 6 expression in cultured bovine oviduct epithelial cells although progesterone antagonized these effects. Functional studies showed changes in calcium mobilization in bovine oviduct epithelial cells were dependent on TRPCs. In conclusion, TRPC1, 2, 3, 4, and 6 are present in the epithelium lining the bovine oviduct, and TRPC1 and 6 vary through the estrous cycle suggesting an important role in early reproductive function. PMID:27001231

  7. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    PubMed Central

    2012-01-01

    Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that

  8. High-level transient expression of ER-targeted human interleukin 6 in Nicotiana benthamiana.

    PubMed

    Nausch, Henrik; Mikschofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    Tobacco plants can be used to express recombinant proteins that cannot be produced in a soluble and active form using traditional platforms such as Escherichia coli. We therefore expressed the human glycoprotein interleukin 6 (IL6) in two commercial tobacco cultivars (Nicotiana tabacum cv. Virginia and cv. Geudertheimer) as well as the model host N. benthamiana to compare different transformation strategies (stable vs. transient expression) and subcellular targeting (apoplast, endoplasmic reticulum (ER) and vacuole). In T(0) transgenic plants, the highest expression levels were achieved by ER targeting but the overall yields of IL6 were still low in the leaves (0.005% TSP in the ER, 0.0008% in the vacuole and 0.0005% in the apoplast). The apoplast variant accumulated to similar levels in leaves and seeds, whereas the ER-targeted variant was 1.2-fold more abundant in seeds and the vacuolar variant was 6-fold more abundant in seeds. The yields improved in subsequent generations, with the best-performing T(2) plants producing the ER-targeted IL6 at 0.14% TSP in both leaves and seeds. Transient expression of ER-targeted IL6 in leaves using the MagnICON system resulted in yields of up to 7% TSP in N. benthamiana, but only 1% in N. tabacum cv. Virginia and 0.5% in cv. Geudertheimer. Although the commercial tobacco cultivars produced up to threefold more biomass than N. benthamiana, this was not enough to compensate for the lower overall yields. The recombinant IL6 produced by transient and stable expression in plants was biologically active and presented as two alternative bands matching the corresponding native protein. PMID:23152824

  9. Transient HEXA expression in a transformed human fetal Tay-Sachs disease neuroglial cell line

    SciTech Connect

    Fernandes, M.J.; Hechtman, P.; Kaplan, F.

    1994-09-01

    Tay-Sachs disease (TSD) is a severe neurodegenerative disorder characterized by the accumulation of GM{sub 2} ganglioside in the neurons of the central cortex. The recessively inherited disorder results from deficiency of hexosaminidase A (Hex A), a heterodimer of an {alpha} and {beta} subunit encoded by the HEXA and HEXB genes. Expression of HEXA mutations in COS cells has several disadvantages including high endogenous hexosaminidase activity. We report a new transient expression system with very low endogenous Hex A activity. An SV40-transformed fetal TSD neuroglial cell line was assessed for transient expression of the HEXA gene. pCMV{alpha}, a vector incorporating the cytomegalovirus promoter with the human {alpha}-subunit cDNA insert, proved to be the most efficient expression vector. Transfection of 4x10{sup 6} cells with 5-20 {mu}g of plasmid resulted in 100 to 500-fold Hex A activity (4MUGS hydrolysis) relative to mock-transfected cells. Use of pCMV{beta}-Gal as a control for transfection efficiency indicated that 10-20% of cells were transfected. Hex A specific activity increased for at least 72 h post-transfection. This new transient expression system should greatly improve the characterization of mutations in which low levels of HEXA expression result in milder clinical phenotypes and permit studies on enzymatic properties of mutant forms of Hex A. Since the cells used are of CNS origin and synthesize gangliosides, it should also be possible to study, in culture, the metabolic phenotype associated with TSD.

  10. Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia.

    PubMed

    Hasegawa, S; Morioka, M; Goto, S; Korematsu, K; Okamura, A; Yano, S; Kai, Y; Hamada, J I; Ushio, Y

    2000-02-15

    We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration. PMID:10652442

  11. Transient Transfection and Expression in the Obligate Intracellular Parasite Toxoplasma gondii

    NASA Astrophysics Data System (ADS)

    Soldati, Dominique; Boothroyd, John C.

    1993-04-01

    Toxoplasma gondii is a protozoan pathogen that produces severe disease in humans and animals. This obligate intracellular parasite provides an excellent model for the study of how such pathogens are able to invade, survive, and replicate intracellularly. DNA encoding chloramphenicol acetyltransferase was introduced into T. gondii and transiently expressed with the use of three vectors based on different Toxoplasma genes. The ability to introduce genes and have them efficiently and faithfully expressed is an essential tool for understanding the structure-function relation of genes and their products.

  12. An oligochaete homologue of the Brachyury gene is expressed transiently in the third quartette of micromeres.

    PubMed

    Kitakoshi, Tomohito; Shimizu, Takashi

    2010-09-01

    We have isolated a Brachyury homologue (Ttu-Bra) from the oligochaete annelid Tubifex tubifex which displays a direct mode of development. Developmental RT-PCR analysis showed that Ttu-Bra transcripts are present in embryos at stages 9-11, 16 and 17, but undetectable at the remaining embryonic stages. Whole-mount in situ hybridization demonstrated that Ttu-Bra is expressed transiently in the third quartette of micromeres, which are located at the prospective stomodaeum (at stages 9-11). The second burst of Ttu-Bra expression occurs at the posterior end of stage 16 embryo that undergoes body elongation. Ttu-Bra-expressing cells, which are organized in a circle at stage 16, become aggregated at the proctodaeum at stage 17. Consistent with the results of the RT-PCR analysis, there is no sign of Ttu-Bra-expressing cells in embryos that undergo gastrulation during stages 12-15. PMID:20603182

  13. Complementary DNA cloning of the pear 1-aminocyclopropane-1-carboxylic acid oxidase gene and agrobacterium-mediated anti-sense genetic transformation.

    PubMed

    Qi, Jing; Dong, Zhen; Zhang, Yu-Xing

    2015-12-01

    The aim of the present study was to genetically modify plantlets of the Chinese yali pear to reduce their expression of ripening-associated 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and therefore increase the shelf-life of the fruit. Primers were designed with selectivity for the conserved regions of published ACO gene sequences, and yali complementary DNA (cDNA) cloning was performed by reverse transcription quantitative polymerase chain reaction (PCR). The obtained cDNA fragment contained 831 base pairs, encoding 276 amino acid residues, and shared no less than 94% nucleotide sequence identity with other published ACO genes. The cDNA fragment was inversely inserted into a pBI121 expression vector, between the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator, in order to construct the anti‑sense expression vector of the ACO gene; it was transfected into cultured yali plants using Agrobacterium LBA4404. Four independent transgenic lines of pear plantlets were obtained and validated by PCR analysis. A Southern blot assay revealed that there were three transgenic lines containing a single copy of exogenous gene and one line with double copies. The present study provided germplasm resources for the cultivation of novel storage varieties of pears, therefore providing a reference for further applications of anti‑sense RNA technology in the genetic improvement of pears and other fruit. PMID:26460204

  14. Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

    PubMed Central

    Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

    2013-01-01

    In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

  15. Transient expression of somatostatin messenger RNA and peptide in the hypoglossal nucleus of the neonatal rat.

    PubMed

    Seroogy, K B; Bayliss, D A; Szymeczek, C L; Hökfelt, T; Millhorn, D E

    1991-06-21

    The postnatal developmental expression of somatostatin mRNA and peptide in the rat hypoglossal nucleus was analyzed using immunocytochemical and in situ hybridization techniques. Both the neuropeptide and its cognate mRNA were found to be transiently present within a subpopulation of hypoglossal motoneurons during the neonatal period. At the day of birth, a large population of perikarya situated in caudal, ventral regions of the hypoglossal nucleus expressed somatostatin. By postnatal day 7, the number of hypoglossal somata which expressed somatostatin had diminished considerably, and by 2 weeks postnatal, only few such cell bodies were found. By 3-4 weeks postnatal, somatostatin peptide- and mRNA-containing hypoglossal motoneurons were rarely observed, and in the adult, they were never detected, despite the use of colchicine. A double-labeling co-localization technique was used to demonstrate that somatostatin, when present perinatally, always coexisted with calcitonin gene-related peptide in hypoglossal motoneurons. The latter peptide, in contrast to somatostatin, was expressed in large numbers of somata throughout the entire hypoglossal nucleus and persisted within the motoneurons throughout development into adulthood. These results demonstrate that somatostatin is transiently expressed in motoneurons of the caudal, ventral tier of the hypoglossal nucleus in the neonatal rat. The developmental disappearance of somatostatin is most likely not due to cell death; hypoglossal somata continue to express calcitonin gene-related peptide, with which somatostatin coexisted perinatally, a high levels throughout development. Thus, it appears that the regulation of somatostatin expression in hypoglossal neurons occurs at the level of gene transcription or mRNA stability/degradation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1680035

  16. A transient three-plasmid expression system for the production of high titer retroviral vectors.

    PubMed

    Soneoka, Y; Cannon, P M; Ramsdale, E E; Griffiths, J C; Romano, G; Kingsman, S M; Kingsman, A J

    1995-02-25

    We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants. PMID:7899083

  17. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    PubMed Central

    Ooi, Amanda; Wong, Aloysius; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Chris

    2016-01-01

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins. PMID:27486406

  18. A simple plasmid-based transient gene expression method using High Five cells.

    PubMed

    Shen, Xiao; Pitol, Ana K; Bachmann, Virginie; Hacker, David L; Baldi, Lucia; Wurm, Florian M

    2015-12-20

    The High Five (H5) cell line, derived from the lepidopteran Trichoplusia ni, is one of the major insect cell hosts for the production of recombinant proteins using the baculovirus expression vector system (BEVS). Here, we describe a simple polyethylenimine (PEI)-based transient gene expression (TGE) process for the rapid production of recombinant proteins from suspension-adapted H5 cells. The method was optimized using two model proteins, enhanced green fluorescent protein (EGFP) and human tumor necrosis factor receptor-Fc fusion protein (TNFR-Fc). After screening several promoter and enhancer combinations for high levels of TNFR:Fc production, an expression vector containing the Autographa californica multicapsid nucleopolyhedrovirus immediate early 1 (ie1) promoter and homologous region 5 (hr5) enhancer was selected. Cells were transfected at a density of 2×10(6) cells/mL by direct addition of DNA and PEI. Under optimized conditions, a 90% transfection efficiency (percentage of EGFP-positive cells) was obtained. In addition, we observed volumetric TNFR-Fc yields over 150μg/mL within 4 days of transfection. The method was found to be reproducible and scalable to 300mL. This plasmid-based transient transfection process is a simple and efficient alternative to the BEVS for recombinant protein production in H5 cells. PMID:26476358

  19. Poinsettia protoplasts - a simple, robust and efficient system for transient gene expression studies

    PubMed Central

    2012-01-01

    Background Transient gene expression systems are indispensable tools in molecular biology. Yet, their routine application is limited to few plant species often requiring substantial equipment and facilities. High chloroplast and chlorophyll content may further impede downstream applications of transformed cells from green plant tissue. Results Here, we describe a fast and simple technique for the high-yield isolation and efficient transformation (>70%) of mesophyll-derived protoplasts from red leaves of the perennial plant Poinsettia (Euphorbia pulccherrima). In this method no particular growth facilities or expensive equipments are needed. Poinsettia protoplasts display an astonishing robustness and can be employed in a variety of commonly-used downstream applications, such as subcellular localisation (multi-colour fluorescence) or promoter activity studies. Due to low abundance of chloroplasts or chromoplasts, problems encountered in other mesophyll-derived protoplast systems (particularly autofluorescence) are alleviated. Furthermore, the transgene expression is detectable within 90 minutes of transformation and lasts for several days. Conclusions The simplicity of the isolation and transformation procedure renders Poinsettia protoplasts an attractive system for transient gene expression experiments, including multi-colour fluorescence, subcellular localisation and promoter activity studies. In addition, they offer hitherto unknown possibilities for anthocyan research and industrial applications. PMID:22559320

  20. Transient SNAIL1 Expression is Necessary for Metastatic Competence in Breast Cancer

    PubMed Central

    Tran, Hung D.; Luitel, Krishna; Kim, Michael; Zhang, Kun; Longmore, Gregory D.; Tran, David D.

    2016-01-01

    SNAIL1 has been suggested to regulate breast cancer metastasis based on analyses of human breast tumor transcriptomes and experiments using cancer cell lines and xenografts. However, in vivo genetic experimental support for a role for SNAIL1 in breast cancer metastasis that develops in an immunocompetent tumor microenvironment has not been determined. To address this question, we created a genetic SNAIL1 model by coupling an endogenous SNAIL1 reporter with an inducible SNAIL1 transgene. Using multiple genetic models of breast cancer, we demonstrated that endogenous SNAIL1 expression was restricted to primary tumors that ultimately disseminate. SNAIL1 gene deletion either during the premalignant phase or after primary tumors have reached a palpable size blunted metastasis, indicating that late metastasis was the main driver of metastasis and that this was dependent on SNAIL1. Importantly, SNAIL1 expression during breast cancer metastasis was transient and forced transient, but not continuous, SNAIL1 expression in breast tumors was sufficient to increase metastasis. PMID:25164016

  1. RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

    PubMed Central

    Diener, Yvonne; Jurk, Marion; Kandil, Britta; Choi, Yeong-Hoon; Wild, Stefan; Bissels, Ute; Bosio, Andreas

    2015-01-01

    Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5′- and 3′-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs. PMID:26599627

  2. Transient functional expression of alphaVbeta 3 on vascular cells during wound repair.

    PubMed Central

    Clark, R. A.; Tonnesen, M. G.; Gailit, J.; Cheresh, D. A.

    1996-01-01

    During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 Figure 9 Figure 10 PMID:8623913

  3. Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor▿ †

    PubMed Central

    Moon, Yong-Sun; Donzelli, Bruno G. G.; Krasnoff, Stuart B.; McLane, Heather; Griggs, Mike H.; Cooke, Peter; Vandenberg, John D.; Gibson, Donna M.; Churchill, Alice C. L.

    2008-01-01

    Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor. PMID:18502925

  4. Emergence delirium with transient associative agnosia and expressive aphasia reversed by flumazenil in a pediatric patient.

    PubMed

    Drobish, Julie K; Kelz, Max B; DiPuppo, Patricia M; Cook-Sather, Scott D

    2015-06-01

    Multiple factors may contribute to the development of emergence delirium in a child. We present the case of a healthy 12-year-old girl who received preoperative midazolam with the desired anxiolytic effect, underwent a brief general anesthetic, and then exhibited postoperative delirium, consisting of a transient associative agnosia and expressive aphasia. Administration of flumazenil led to immediate and lasting resolution of her symptoms. We hypothesize that γ-aminobutyric acid type A receptor-mediated effects, most likely related to an atypical offset of midazolam, are an important subset of emergence delirium that is amenable to pharmacologic therapy with flumazenil. PMID:26035220

  5. Emergence Delirium with Transient Associative Agnosia and Expressive Aphasia Reversed by Flumazenil in a Pediatric Patient

    PubMed Central

    Drobish, Julie K.; Kelz, Max B.; DiPuppo, Patricia M.; Cook-Sather, Scott D.

    2014-01-01

    Multiple factors may contribute to the development of emergence delirium in a child. We present the case of a healthy 12-year-old girl who received preoperative midazolam with the desired anxiolytic effect, underwent a brief general anesthetic, and then exhibited postoperative delirium, consisting of a transient associative agnosia and expressive aphasia. Administration of flumazenil led to immediate and lasting resolution of her symptoms. We hypothesize that γ-aminobutyric acid type A receptor-mediated effects, most likely related to an atypical offset of midazolam, are an important subset of emergence delirium that is amenable to pharmacologic therapy with flumazenil. PMID:26035220

  6. Production of the Main Celiac Disease Autoantigen by Transient Expression in Nicotiana benthamiana

    PubMed Central

    Marín Viegas, Vanesa S.; Acevedo, Gonzalo R.; Bayardo, Mariela P.; Chirdo, Fernando G.; Petruccelli, Silvana

    2015-01-01

    Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to

  7. Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development

    PubMed Central

    Spiegel, Holger; Boes, Alexander; Voepel, Nadja; Beiss, Veronique; Edgue, Gueven; Rademacher, Thomas; Sack, Markus; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-01-01

    Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route toward the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification, and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs, and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here, we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility, and stability using fluorescent fusion proteins. Our

  8. Application of a Scalable Plant Transient Gene Expression Platform for Malaria Vaccine Development.

    PubMed

    Spiegel, Holger; Boes, Alexander; Voepel, Nadja; Beiss, Veronique; Edgue, Gueven; Rademacher, Thomas; Sack, Markus; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-01-01

    Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route toward the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification, and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs, and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here, we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility, and stability using fluorescent fusion proteins. Our

  9. Increases of Catalase and Glutathione Peroxidase Expressions by Lacosamide Pretreatment Contributes to Neuroprotection Against Experimentally Induced Transient Cerebral Ischemia.

    PubMed

    Choi, Hyun Young; Park, Joon Ha; Chen, Bai Hui; Shin, Bich Na; Lee, Yun Lyul; Kim, In Hye; Cho, Jeong-Hwi; Lee, Tae-Kyeong; Lee, Jae-Chul; Won, Moo-Ho; Ahn, Ji Hyeon; Tae, Hyun-Jin; Yan, Bing Chun; Hwang, In Koo; Cho, Jun Hwi; Kim, Young-Myeong; Kim, Sung Koo

    2016-09-01

    Lacosamide is a new antiepileptic drug which is widely used to treat partial-onset seizures. In this study, we examined the neuroprotective effect of lacosamide against transient ischemic damage and expressions of antioxidant enzymes such as Zn-superoxide dismutase (SOD1), Mn-superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPX) in the hippocampal cornu ammonis 1 (CA1) region following 5 min of transient global cerebral ischemia in gerbils. We found that pre-treatment with 25 mg/kg lacosamide protected CA1 pyramidal neurons from transient global cerebral ischemic insult using hematoxylin-eosin staining and neuronal nuclear antigen immunohistochemistry. Transient ischemia dramatically changed expressions of SOD1, SOD2 and GPX, not CAT, in the CA1 pyramidal neurons. Lacosamide pre-treatment increased expressions of CAT and GPX, not SOD1 and 2, in the CA1 pyramidal neurons compared with controls, and their expressions induced by lacosamide pre-treatment were maintained after transient cerebral ischemia. In brief, pre-treatment with lacosamide protected hippocampal CA1 pyramidal neurons from ischemic damage induced by transient global cerebral ischemia, and the lacosamide-mediated neuroprotection may be closely related to increases of CAT and GPX expressions by lacosamide pre-treatment. PMID:27209305

  10. Canonical transient receptor potential channels expression is elevated in a porcine model of metabolic syndrome.

    PubMed

    Hu, Guoqing; Oboukhova, Elena A; Kumar, Sanjay; Sturek, Michael; Obukhov, Alexander G

    2009-05-01

    Plasma epinephrine and heart rate are elevated in metabolic syndrome, suggesting enhanced catecholamine secretion from the adrenal medulla. Canonical transient receptor potential (TRPC) channels are implicated in mediating hormone-induced Ca(2+) influx and catecholamine secretion in adrenomedullary chromaffin cells. We studied the pattern of TRPC expression in the pig adrenal medulla and investigated whether adrenal TRPC expression is altered in prediabetic metabolic syndrome Ossabaw miniature pigs. We used a combination of molecular biological, biochemical, and fluorescence imaging techniques. We determined the sequence of pig TRPC1 and TRPC3-7 channels. We found that the pig adrenal medulla expressed predominantly TRPC1, TRPC5, and TRPC6 transcripts. The expression level of these TRPCs was significantly elevated in the adrenal medulla from pigs with metabolic syndrome. Interestingly, aldosterone, which is endogenously secreted in the adjacent adrenal cortex, increased TRPC1, TRPC5, and TRPC6 expression in adrenal chromaffin cells isolated from metabolic syndrome but not control pigs. Spironolactone, a blocker of mineralocorticoid receptors, inhibited the aldosterone effect. Dexamethasone also increased TRPC5 expression in metabolic syndrome chromaffin cells. The amplitude of hormone-induced divalent cation influx correlated with the level of TRPC expression in adrenal chromaffin cells. Orai1/Stim1 protein expression was not significantly altered in the metabolic syndrome adrenal medulla when compared with the control. We propose that in metabolic syndrome, abnormally elevated adrenal TRPC expression may underlie increased plasma epinephrine and heart rate. The excess of plasma catecholamines and increased heart rate are risk factors for cardiovascular disease. Thus, TRPCs are potential therapeutic targets in the fight against cardiovascular disease. PMID:19221052

  11. A Plant-Based Transient Expression System for the Rapid Production of Malaria Vaccine Candidates.

    PubMed

    Boes, Alexander; Reimann, Andreas; Twyman, Richard M; Fischer, Rainer; Schillberg, Stefan; Spiegel, Holger

    2016-01-01

    There are currently no vaccines that provide sterile immunity against malaria. Various proteins from different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates, but none of them have fulfilled expectations. Therefore, combinations of key antigens from different stages of the parasites life cycle may be essential for the development of efficacious malaria vaccines. Following the identification of promising antigens using bioinformatics, proteomics, and/or immunological approaches, it is necessary to express, purify, and characterize these proteins and explore the potential of fusion constructs combining different antigens or antigen domains before committing to expensive and time-consuming clinical development. Here, using malaria vaccine candidates as an example, we describe how Agrobacterium tumefaciens-based transient expression in plants can be combined with a modular and flexible cloning strategy as a robust and versatile tool for the rapid production of candidate antigens during research and development. PMID:27076325

  12. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana.

    PubMed

    Pitino, Marco; Armstrong, Cheryl M; Cano, Liliana M; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus "Las" is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  13. Transient Expression of Candidatus Liberibacter Asiaticus Effector Induces Cell Death in Nicotiana benthamiana

    PubMed Central

    Pitino, Marco; Armstrong, Cheryl M.; Cano, Liliana M.; Duan, Yongping

    2016-01-01

    Candidatus Liberibacter asiaticus “Las” is a phloem-limited bacterial plant pathogen, and the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB), a devastating disease of citrus worldwide. Although, the complete sequence of the Las genome provides the basis for studying functional genomics of Las and molecular mechanisms of Las-plant interactions, the functional characterization of Las effectors remains a slow process since remains to be cultured. Like other plant pathogens, Las may deliver effector proteins into host cells and modulate a variety of host cellular functions for their infection progression. In this study, we identified 16 putative Las effectors via bioinformatics, and transiently expressed them in Nicotiana benthamiana. Diverse subcellular localization with different shapes and aggregation patterns of the effector candidates were revealed by UV- microscopy after transient expression in leaf tissue. Intriguingly, one of the 16 candidates, Las5315mp (mature protein), was localized in the chloroplast and induced cell death at 3 days post inoculation (dpi) in N. benthamiana. Moreover, Las5315mp induced strong callose deposition in plant cells. This study provides new insights into the localizations and potential roles of these Las effectors in planta. PMID:27458468

  14. [Efficient transient expression to analyze miRNA targets in rice protoplasts].

    PubMed

    Guo, Ping; Wu, Yao; Li, Jia; Fang, Rongxiang; Jia, Yantao

    2014-11-01

    Compared with the transgenic approach, transient assays provide a convenient alternative to analyze gene expression. To analyze the relationship between miRNAs and their target genes, a rice protoplast system to detect target gene activity was established. The MIRNA and GFP-fused target sequence (or GFP-fused mutated sequence as a non-target control) were constructed into the same plasmid, and then delivered into rice protoplasts. The GFP expression level decreased significantly when the protoplasts were transfected with the plasmid containing GFP-fused target compared to that of the plasmid with non-target sequence either by fluorescence microscopy or qRT-PCR method. Two microRNA genes, osaMIR156 and osaMIR397, and their target sequences were used to prove the feasibility of the rice protoplast transient assay system. This method will facilitate large-scale screening of rice miRNA target in vivo, and may be suitable for functional analysis of miRNAs of other monocot plants that might share the evolutionarily conserved small RNA processing system with rice. PMID:25985526

  15. Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis.

    PubMed

    Garcia-Fabiani, Maria B; Montanaro, Mauro A; Lacunza, Ezequiel; Cattaneo, Elizabeth R; Coleman, Rosalind A; Pellon-Maison, Magali; Gonzalez-Baro, Maria R

    2015-10-15

    Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications. PMID:26268560

  16. Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis

    PubMed Central

    Garcia-Fabiani, Maria B.; Montanaro, Mauro A.; Lacunza, Ezequiel; Cattaneo, Elizabeth R.; Coleman, Rosalind A.; Pellon-Maison, Magali; Gonzalez-Baro, Maria R.

    2015-01-01

    Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on–off expression pattern responds predominantly to epigenetic modifications. PMID:26268560

  17. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    PubMed Central

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells. PMID:25206849

  18. Transient lung-specific expression of the chemokine KC improves outcome in invasive aspergillosis.

    PubMed

    Mehrad, Borna; Wiekowski, Maria; Morrison, Brad E; Chen, Shu-Cheng; Coronel, Elizabeth C; Manfra, Denise J; Lira, Sergio A

    2002-11-01

    Invasive aspergillosis is a common and devastating pneumonia in immunocompromised hosts. Neutrophils are critical for defense against this infection, and ELR+ CXC chemokines are potent neutrophil chemoattractants. We hypothesized that transient lung-specific overexpression of one such ligand, KC, in mice with invasive aspergillosis improves the outcome of disease. We generated mice in which transgenic expression of KC was limited to the lungs and occurred only upon exposure to tetracycline analogues, and we exposed them to doxycycline after the onset of invasive aspergillosis. Transgenic mice had a threefold greater survival, a 74% lower lung fungal burden, a greater magnitude of lung KC induction, and an earlier and higher peak of lung neutrophil influx compared with wild-type mice. In addition to a higher number of neutrophils, we found a 1.8-fold higher number of monocytes-macrophages in the lungs of transgenic mice as compared with wild-type mice. Furthermore, transgenic mice had greater lung expression of interferon-gamma and interleukin-12 in response to infection, suggesting that transgenic expression of KC indirectly regulated the expression of other cytokines associated with improved host defense against this pathogen. Taken together, these data suggest that overexpression of KC in the lung in the setting of established invasive aspergillosis results in improved host defense and outcome of disease. PMID:12403697

  19. Bcmimp1, a Botrytis cinerea Gene Transiently Expressed in planta, Encodes a Mitochondrial Protein

    PubMed Central

    Benito-Pescador, David; Santander, Daniela; Arranz, M.; Díaz-Mínguez, José M.; Eslava, Arturo P.; van Kan, Jan A. L.; Benito, Ernesto P.

    2016-01-01

    Botrytis cinerea is a widespread necrotrophic fungus which infects more than 200 plant species. In an attempt to characterize the physiological status of the fungus in planta and to identify genetic factors contributing to its ability to infect the host cells, a differential gene expression analysis during the interaction B. cinerea-tomato was carried out. Gene Bcmimp1 codes for a mRNA detected by differential display in the course of this analysis. During the interaction with the host, it shows a transient expression pattern with maximal expression levels during the colonization and maceration of the infected tissues. Bioinformatic analysis suggested that BCMIMP1 is an integral membrane protein located in the mitochondrial inner membrane. Co-localization experiments with a BCMIMP1-GFP fusion protein confirmed that the protein is targeted to the mitochondria. ΔBcmimp1 mutants do not show obvious phenotypic differences during saprophytic growth and their infection ability was unaltered as compared to the wild-type. Interestingly, the mutants produced increased levels of reactive oxygen species, likely as a consequence of disturbed mitochondrial function. Although Bcmimp1 expression is enhanced in planta it cannot be considered a pathogenicity factor. PMID:26952144

  20. Molecular Determinants of Cardiac Transient Outward Potassium Current (Ito) Expression and Regulation

    PubMed Central

    Niwa, Noriko; Nerbonne, Jeanne M.

    2009-01-01

    Rapidly activating and inactivating cardiac transient outward K+ currents, Ito, are expressed in most mammalian cardiomyocytes, and contribute importantly to the early phase of action potential repolarization and to plateau potentials. The rapidly recovering (Ito,f) and slowly recovering (Ito,s) components are differentially expressed in the myocardium, contributing to regional heterogeneities in action potential waveforms. Consistent with the marked differences in biophysical properties, distinct pore-forming (α) subunits underlie the two Ito components: Kv4.3/Kv4.2 subunits encode Ito,f, whereas Kv1.4 encodes Ito,s, channels. It has also become increasingly clear that cardiac Ito channels function as components of macromolecular protein complexes, comprising (four) Kv α subunits and a variety of accessory subunits and regulatory proteins that influence channel expression, biophysical properties and interactions with the actin cytoskeleton, and contribute to the generation of normal cardiac rhythms. Derangements in the expression or the regulation of Ito channels in inherited or acquired cardiac diseases would be expected to increase the risk of potentially life-threatening cardiac arrhythmias. Indeed, a recently identified Brugada syndrome mutation in KCNE3 (MiRP2) has been suggested to result in increased Ito,f densities. Continued focus in this area seems certain to provide new and fundamentally important insights into the molecular determinants of functional Ito channels and into the molecular mechanisms involved in the dynamic regulation of Ito channel functioning in the normal and diseased myocardium. PMID:19619557

  1. Comparative study of polyethylenimines for transient gene expression in mammalian HEK293 and CHO cells.

    PubMed

    Delafosse, Laurence; Xu, Ping; Durocher, Yves

    2016-06-10

    Three commercially available linear polyethylenimines (25kDa LPEI, 40kDa PEI"Max" and PEIpro™) were compared regarding their potency to transfect serum-free growing and suspension-adapted HEK293 and CHO cells. We determined the optimal DNA:PEI ratios for maximal expression of the reporter gene SEAP while monitoring cytotoxicity following transfection. PEIs acylation was determined by (1)H NMR and their apparent size and polydispersity assessed by size-exclusion chromatography. The propensity of PEIs to condense plasmid DNA was evaluated by agarose-gel electrophoresis. The zeta potentials and particle sizes at optimal DNA:PEI ratio were analyzed. Polyplex attachment to the cells and internalization kinetics were monitored. The quantity of PEIpro™ needed to efficiently transfect the cells was significantly lower than with LPEI and PEI"Max" and, interestingly, the maximal amount of internalized PEIpro™-based polyplexes was approximately half of that observed with its counterparts. PEIpro™ was the largest and least polydisperse polymer, but also the most cytotoxic. The optimal transfection conditions were subsequently used to express three monoclonal antibodies at larger-scale. The use of the deacylated PEI"Max" and PEIpro™ resulted in a significant increase of recombinant protein expression compared to LPEI. These findings demonstrate the importance of properly choosing the most suitable polymers to obtain optimal recombinant protein transient expression. PMID:27085888

  2. Singular subsets of locus coeruleus neurons may recover tyrosine hydroxylase phenotype transiently expressed during development.

    PubMed

    Bezin, L; Marcel, D; Desgeorges, S; Pujol, J F; Weissmann, D

    2000-03-29

    The number of tyrosine hydroxylase (TH)-expressing neurons appears to be precisely determined in basal conditions within the noradrenergic pontine nucleus locus coeruleus (LC). However, additional neurons exhibiting TH phenotype have been observed in the adult rat LC following a single administration of RU 24722, a potent inducer of TH expression specific to the LC. The neurons acquiring TH phenotype following treatment had a topographical localization similar to that of the neurons, which transiently expressed TH during postnatal development and lost TH phenotype during the third postnatal week. The idea that the fluctuation of TH phenotype in singular subsets of LC neurons during development may be selectively restored in adults is of particular interest. The present study attempted to determine whether the cells in which TH expression was repressed during the third postnatal week could correspond to those which exhibited TH phenotype in response to RU 24722 treatment in adults. We first verified that no massive cell death occurred in the LC during the period ranging from days 13 to 30. Then, we observed that both cell populations exhibited the same altered steady-state concentration of TH-mRNA as compared to cells that permanently expressed TH. Finally, we demonstrated the presence of TH-negative neurons expressing the homeodomain transcription factor Phox2a, specific for the determination of noradrenergic phenotype, providing further evidence that "resting-noradrenergic" neurons exist in the adult rat LC under basal conditions. These neurons provide interesting prospective for gain of noradrenergic function when classical noradrenergic LC neurons are impaired. PMID:10762702

  3. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  4. Transient Expression of WNT2 Promotes Somatic Cell Reprogramming by Inducing β-Catenin Nuclear Accumulation.

    PubMed

    Kimura, Mizuki; Nakajima-Koyama, May; Lee, Joonseong; Nishida, Eisuke

    2016-06-14

    Treatment with several Wnt/β-catenin signaling pathway regulators can change the cellular reprogramming efficiency; however, the dynamics and role of endogenous Wnt/β-catenin signaling in reprogramming remain largely unanswered. Here we identify the upregulation of WNT2 and subsequent β-catenin nuclear accumulation as key events in reprogramming. Transient nuclear accumulation of β-catenin occurs early in MEF reprogramming. Wnt2 is strongly expressed in the early stage of reprogramming. Wnt2 knockdown suppresses the nuclear accumulation of β-catenin and reduces the reprogramming efficiency. WNT2 overexpression promotes β-catenin nuclear accumulation and enhances the reprogramming efficiency. WNT2 contributes to the promotion of cell proliferation. Experiments with several drugs that control the Wnt pathway also indicate the importance of β-catenin nuclear accumulation in reprogramming. Our findings reveal the role of WNT2/β-catenin signaling in reprogramming. PMID:27211212

  5. Protocols for Studying Protein Stability in an Arabidopsis Protoplast Transient Expression System.

    PubMed

    Planchais, Séverine; Camborde, Laurent; Jupin, Isabelle

    2016-01-01

    Protein stability influences many aspects of biology, and measuring their stability in vivo can provide important insights into biological systems.This chapter describes in details two methods to assess the stability of a specific protein based on its transient expression in Arabidopsis protoplasts. First, a pulse-chase assay based on radioactive metabolic labeling of cellular proteins, followed by immunoprecipitation of the protein of interest. The decrease in radioactive signal is monitored over time and can be used to determine the protein's half-life.Alternatively, we also present a nonradioactive assay based on the use of reporter proteins, whose ratio can be quantified. This assay can be used to determine the relative stability of a protein of interest under specific conditions. PMID:27424754

  6. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

    PubMed

    Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

    2016-01-01

    Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression. PMID:27386255

  7. ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle*

    PubMed Central

    Buvinic, Sonja; Almarza, Gonzalo; Bustamante, Mario; Casas, Mariana; López, Javiera; Riquelme, Manuel; Sáez, Juan Carlos; Huidobro-Toro, Juan Pablo; Jaimovich, Enrique

    2009-01-01

    ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca2+ concentration, with an EC50 value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y2 receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca2+ homeostasis and muscle physiology. PMID:19822518

  8. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in spaceflight.

    PubMed

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-06-01

    Microgravity, or an altered gravity environment different from the 1 g of the Earth, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies that have been conducted in space or by using simulated microgravity on the ground have focused on the growth or differentiation of these cells. It has not been specifically addressed whether nonproliferating cultured cells will sense the presence of microgravity in space. In an experiment conducted onboard the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 d, respectively, to investigate changes in gene and microRNA (miRNA) expression profiles in these cells. Results of the experiment showed that on d 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67(+) cells. Gene and miRNA expression data indicated activation of NF-κB and other growth-related pathways that involve hepatocyte growth factor and VEGF as well as the down-regulation of the Let-7 miRNA family. On d 14, when the cells were mostly nonproliferating, the gene and miRNA expression profile of the flight sample was indistinguishable from that of the ground sample. Comparison of gene and miRNA expressions in the d 3 samples, with respect to d 14, revealed that most of the changes observed on d 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for α-tubulin and fibronectin showed no difference between the flown and ground samples. Taken together, our study suggests that in true nondividing human fibroblast cells in culture, microgravity experienced in space has little effect on gene and miRNA expression profiles.-Zhang, Y., Lu, T., Wong, M., Wang, X., Stodieck, L., Karouia, F., Story, M., Wu, H. Transient gene and microRNA expression profile changes of

  9. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research. PMID:19255730

  10. Biochemical and functional characterization of transiently expressed in neural precursor (TENP) protein in emu egg white.

    PubMed

    Maehashi, Kenji; Ueda, Megumi; Matano, Mami; Takeuchi, Junko; Uchino, Masataka; Kashiwagi, Yutaka; Watanabe, Toshihiro

    2014-06-01

    A protein transiently expressed in the neural precursors of developing tissues (TENP) was found to be present in emu (Dromaius novaehollandiae) egg white as one of the major proteins. Nucleotide analysis of its encoding cDNA revealed a sequence of 452 amino acids including a 19 amino acid peptide signal. Phylogenetic analysis determined that emu TENP was clustered within the bactericidal/permeability-increasing protein (BPI) superfamily together with other avian TENPs. RT-PCR analysis revealed that the emu TENP gene was highly expressed in the magnum of the oviduct, indicating that TENP is a major egg white component. Emu TENP was purified by anion exchange chromatography and ammonium sulfate fractionation. Unlike BPI, emu TENP exhibited antibacterial activity against Gram-positive bacteria, including Micrococcus luteus and Bacillus subtilis, but not against Gram-negative bacteria such as Escherichia coli and Salmonella Typhimurium. The results suggest that emu TENP is a potent novel antibacterial protein with a spectrum distinct from that of BPI. PMID:24820544

  11. Olfactory neurons expressing transient receptor potential channel M5 (TRPM5) are involved in sensing semiochemicals

    PubMed Central

    Lin, Weihong; Margolskee, Robert; Donnert, Gerald; Hell, Stefan W.; Restrepo, Diego

    2007-01-01

    Olfactory sensory neurons (OSNs) in the main olfactory epithelium respond to environmental odorants. Recent studies reveal that these OSNs also respond to semiochemicals such as pheromones and that main olfactory input modulates animal reproduction, but the transduction mechanism for these chemosignals is not fully understood. Previously, we determined that responses to putative pheromones in the main olfactory system were reduced but not eliminated in mice defective for the canonical cAMP transduction pathway, and we suggested, on the basis of pharmacology, an involvement of phospholipase C. In the present study, we find that a downstream signaling component of the phospholipase C pathway, the transient receptor potential channel M5 (TRPM5), is coexpressed with the cyclic nucleotide-gated channel subunit A2 in a subset of mature OSNs. These neurons project axons primarily to the ventral olfactory bulb, where information from urine and other socially relevant signals is processed. We find that these chemosignals activate a subset of glomeruli targeted by TRPM5-expressing OSNs. Our data indicate that TRPM5-expressing OSNs that project axons to glomeruli in the ventral area of the main olfactory bulb are involved in processing of information from semiochemicals. PMID:17267604

  12. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  13. Determinants of rat albumin promoter tissue specificity analyzed by an improved transient expression system.

    PubMed Central

    Heard, J M; Herbomel, P; Ott, M O; Mottura-Rollier, A; Weiss, M; Yaniv, M

    1987-01-01

    The 150-base-pairs region located upstream of the transcriptional start site of the rat albumin gene contains all of the critical sequences necessary for this gene's tissue-specific expression in rat hepatoma cells. In transient expression assays using an improved CAT system or direct mRNA analysis we were able to detect a faithful transcription from the albumin promoter in albumin-negative dedifferentiated H5 hepatoma cells which was 250-fold weaker than in differentiated H4II hepatoma cells producing albumin. This strong tissue specificity could be completely overcome through the cis action of a non-tissue-specific enhancer. Two upstream regions from nucleotides -151 to -119 and from -118 to -94, were required for efficient transcription in H4II cells. Each region contained a sequence motif highly conserved among different species. The effect of the -151/-119 region was strictly tissue specific, while the -118/-94 region was also involved in the low level of transcription observed in H5 cells. Finally, sequences between the CCAAT box and the TATA box also contributed to the overall tissue specificity of rat albumin gene transcription. Images PMID:3475566

  14. High-throughput mAb expression and purification platform based on transient CHO.

    PubMed

    Barnard, Gavin C; Hougland, Maria D; Rajendra, Yashas

    2015-01-01

    A high-cell-density transient transfection system was recently developed in our laboratory based on a CHO-GS-KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7-day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24-deep-well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high-throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX(p) liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer-exchange steps. We optimized both the batch-binding and the batch elution steps. The length of the batch-binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early-stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high-yielding transient transfection and semiautomated high-throughput protein A purification represents a valuable mAb drug discovery tool. PMID:25403790

  15. Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia.

    PubMed

    de Oliveira, R M Weffort; Martin, S; de Oliveira, C Lino; Milani, H; Schiavon, A P; Joca, S; Pardo, L A; Stühmer, W; Del Bel, E A

    2012-03-01

    Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system. PMID:22006722

  16. Expression of canonical transient receptor potential (TRPC) proteins in human glomerular mesangial cells.

    PubMed

    Sours, Sherry; Du, Juan; Chu, Shaoyou; Ding, Min; Zhou, Xin J; Ma, Rong

    2006-06-01

    Mesangial cells are located within glomerular capillary loops and contribute to the physiological regulation of glomerular hemodynamics. The function of mesangial cells is controlled by a variety of ion channels in the plasma membrane, including nonselective cation channels, receptor-operated Ca2+ channels, and recently identified store-operated Ca2+ channels. Although the significance of these channels has been widely acknowledged, their molecular identities are still unknown. Recently, the members of the canonical transient receptor potential (TRPC) protein family have been demonstrated to behave as cation channels. The present study was performed to identify the isoforms of endogenous TRPC proteins in human mesangial cells (HMCs) and their interactions. Western blotting showed that TRPC1, 3, 4, and 6 were expressed in cultured HMCs. Consistently, immunofluorescent confocal microscopy revealed specific stainings for TRPC1, 3, 4, and 6 with predominant intracellular localization. However, TRPC5 and 7 were not detectable at protein level by either Western blotting or immunofluorescent staining. The expression of TRPC1, 3, 4, and 6 was also observed in rat and human glomeruli using fluorescent immunohistochemistry. Furthermore, coimmunoprecipitation experiments and immunofluorescent double staining displayed that TRPC1 had physical interaction with TRPC4 and 6, while no interactions were detected among other isoforms of TRPCs. Ca2+ fluorescent ratiometry measurement showed that store-operated Ca2+ entry in HMCs was significantly reduced by knocking down TRPC1, but enhanced by overexpressing TRPC1. These results suggest that HMCs specifically express isoforms of TRPC1, 3, 4, and 6 proteins. These isoforms of TRPCs might selectively assemble to form functional complexes. PMID:16418302

  17. Transient Expression of Transgenic IL-12 in Mouse Liver Triggers Unremitting Inflammation Mimicking Human Autoimmune Hepatitis.

    PubMed

    Gil-Farina, Irene; Di Scala, Marianna; Salido, Eduardo; López-Franco, Esperanza; Rodríguez-García, Estefania; Blasi, Mercedes; Merino, Juana; Aldabe, Rafael; Prieto, Jesús; Gonzalez-Aseguinolaza, Gloria

    2016-09-15

    The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encoding murine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, anti-nuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-γ remained elevated during the entire study period. IFN-γ was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies. PMID:27511737

  18. Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system.

    PubMed

    Estes, Bram; Hsu, Yueh-Rong; Tam, Lei-Ting; Sheng, Jackie; Stevens, Jennitte; Haldankar, Raj

    2015-01-01

    Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems. PMID:25395220

  19. Transient Receptor Potential Vanilloid 1 Expression and Functionality in MCF-7 Cells: A Preliminary Investigation

    PubMed Central

    Barbero, Raffaella; Cuniberti, Barbara; Racca, Silvia; Abbadessa, Giuliana; Piccione, Francesca; Re, Giovanni

    2014-01-01

    Purpose Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel belonging to the transient receptor potential family, and it is expressed in different neoplastic tissues. Its activation is associated with regulation of cancer growth and progression. The aim of this research was to study the expression and pharmacological characteristics of TRPV1 in cells derived from human breast cancer MCF-7 cells. Methods TRPV1 presence was assessed by binding studies and Western blotting. Receptor binding characteristics were evaluated through competition assays, while 3-(4,5-dimethylthiazol-2-yl)-2,5,-dipheyltetrazolium bromide reduction assays were performed to confirm an early hypothesis regarding the modulation of cancer cell proliferation. The functionality of TRPV1 was evaluated by measuring Ca2+ uptake in the presence of increasing concentrations of TRPV1 agonists and antagonists. Results Binding studies identified a single class of TRPV1 (Bmax 1,492±192 fmol/mg protein), and Western blot showed a signal at 100 kDa corresponding to the molecular weight of human TRPV1. Among the different tested agonists and antagonists, anandamide (Ki: 2.8×10-11 M) and 5-iodoresiniferatoxin (5-I-RTX) (Ki: 5.6×10-11 M) showed the highest degrees of affinity for TRPV1, respectively. All tested TRPV1 agonists and antagonists caused a significant (p<0.05) decrease in cell growth rate in MCF-7 cells. For agonists and antagonists, the efficacy of tested compounds displayed the following rank order: resiniferatoxin>anandamide>capsaicin and 5-I-RTX=capsazepine, respectively. Conclusion These data indicate that both TRPV1 agonists and antagonists induce significant inhibition of MCF-7 cell growth. Even though the mechanisms involved in the antiproliferative effects of TRPV1 agonists and antagonists should be further investigated, it has been suggested that agonists cause desensitization of the receptor, leading to alteration in Ca2+-influx regulation. By contrast

  20. Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture.

    PubMed

    Rozkov, Aleksei; Larsson, Bert; Gillström, Stefan; Björnestedt, Robert; Schmidt, Stefan R

    2008-02-15

    Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less. PMID:17680665

  1. Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts.

    PubMed

    Nicolia, Alessandro; Proux-Wéra, Estelle; Åhman, Inger; Onkokesung, Nawaporn; Andersson, Mariette; Andreasson, Erik; Zhu, Li-Hua

    2015-06-20

    Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production. PMID:25848989

  2. Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins.

    PubMed

    Solé-Magdalena, Antonio; Revuelta, Enrique G; Menénez-Díaz, Ivan; Calavia, Marta G; Cobo, Teresa; García-Suárez, Olivia; Pérez-Piñera, Pablo; De Carlos, Felix; Cobo, Juan; Vega, Jose A

    2011-05-01

    Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (α, β, and γ) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except α-ENa(+) C. The intensity of immunostaining was high for β-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, γ-ENa(+) C, and TRPV4, being absent for α-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission. PMID:20836083

  3. High-yield expression of recombinant soybean agglutinin in plants using transient and stable systems.

    PubMed

    Tremblay, Reynald; Feng, Mary; Menassa, Rima; Huner, Norman P A; Jevnikar, Anthony M; Ma, Shengwu

    2011-04-01

    Soybean agglutinin (SBA) is a specific N-acetylgalactosamine-binding plant lectin that can agglutinate a wide variety of cells. SBA has great potential for medical and biotechnology-focused applications, including screening and treatment of breast cancer, isolation of fetal cells from maternal blood for genetic screening, the possibility as a carrier system for oral drug delivery, and utilization as an affinity tag for high-quality purification of tagged proteins. The success of these applications, to a large degree, critically depends on the development of a highly efficient expression system for a source of recombinant SBA (rSBA). Here, we demonstrate the utility of transient and stable expression systems in Nicotiana benthamiana and potato, respectively, for the production of rSBA, with the transgenic protein accumulated to 4% of total soluble protein (TSP) in Nicotiana benthamiana leaves and 0.3% of TSP in potato tubers. Furthermore, we show that both plant-derived rSBAs retain their ability to induce the agglutination of red blood cells, are similarly glycosylated when compared with native SBA, retained their binding specificity for N-acetylgalactosamine, and were highly resistant to degradation in simulated gastric and intestinal fluids. Affinity column purification using N-acetylgalactosamine as a specific ligand resulted in high recovery and purity of rSBA. This work is the first step toward use of rSBA for various new applications, including the development of rSBA as a novel affinity tag for simplified purification of tagged proteins and as a new carrier molecule for delivery of oral drugs. PMID:20559869

  4. Galectin-3 expression in hippocampal CA2 following transient forebrain ischemia and its inhibition by hypothermia or antiapoptotic agents

    PubMed Central

    Hisamatsu, Kenji; Kobayashi, Kazuhiro; Miyazaki, Tatsuhiko; Hirata, Akihiro; Hatano, Yuichiro; Tomita, Hiroyuki; Hara, Akira

    2016-01-01

    Recent evidence has suggested that the hippocampal CA2 region plays an important role in the recognition process. We have reported that ischemic damage in the hippocampal CA2 region following transient ischemia is caused by apoptosis, but the underlying mechanisms are still not clear. Galectin-3 is a β-galactosidase-binding lectin that is important in cell proliferation and apoptotic regulation. We have also reported that galectin-3 was expressed in activated microglia in the CA1 region 96 h after transient ischemia. The aim of this study is to determine the localization and time course of galectin-3 expression in the CA2 region following transient forebrain ischemia. Galectin-3 immunostaining was observed in both interior side of CA1 region and CA2 region in hippocampus 60 h after ischemic insult. At 66 h, galectin-3 was observed in the whole CA1 region adjacent to the CA2 region in the hippocampus. Both galectin-3 expression and neuronal cell death in the CA2 region were significantly inhibited by hypothermia and by apoptosis-inhibiting reagents. These results suggest that galectin-3 in the CA2 region is expressed independent of that in the CA1 region. Protection of the expression of galectin-3 in the CA2 region might contribute toward the survival of CA2 pyramidal neurons. PMID:26848998

  5. Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.

    PubMed Central

    Scott, R W; Tilghman, S M

    1983-01-01

    The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a

  6. A transient assay to evaluate the expression of polyhydroxybutyrate genes regulated by oil palm mesocarp-specific promoter.

    PubMed

    Omidvar, V; Siti Nor Akmar, A; Marziah, M; Maheran, A A

    2008-09-01

    The promoter of the oil palm metallothionein-like gene (MT3-A) demonstrated mesocarp-specific activity in functional analysis using transient expression assay of reporter gene in bombarded oil palm tissue slices. In order to investigate the tissue-specific expression of polyhydroxybutyrate (PHB) biosynthetic pathway genes, a multi-gene construct carrying PHB genes fused to the oil palm MT3-A promoter was co-transferred with a construct carrying GFP reporter gene using microprojectile bombardment targeting the mesocarp and leaf tissues of the oil palm. Transcriptional analysis using RT-PCR revealed successful transcription of all the three phbA, phbB, and phbC genes in transiently transformed mesocarp but not in transiently transformed leaf tissues. Furthermore, all the three expected sizes of PHB-encoded protein products were only detected in transiently transformed mesocarp tissues on a silver stained polyacrylamide gel. Western blot analysis using polyclonal antibody specific for phbB product confirmed successful translation of phbB mRNA transcript into protein product. This study provided valuable information, supporting the future engineering of PHB-producing transgenic palms. PMID:18563415

  7. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

    PubMed Central

    Zhang, Yi; Liang, Zhen; Zong, Yuan; Wang, Yanpeng; Liu, Jinxing; Chen, Kunling; Qiu, Jin-Long; Gao, Caixia

    2016-01-01

    Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. PMID:27558837

  8. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.

    PubMed

    Zhang, Yi; Liang, Zhen; Zong, Yuan; Wang, Yanpeng; Liu, Jinxing; Chen, Kunling; Qiu, Jin-Long; Gao, Caixia

    2016-01-01

    Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. PMID:27558837

  9. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells.

    PubMed

    Krishna, B A; Lau, B; Jackson, S E; Wills, M R; Sinclair, J H; Poole, E

    2016-01-01

    Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens. PMID:27091512

  10. Transient activation of human cytomegalovirus lytic gene expression during latency allows cytotoxic T cell killing of latently infected cells

    PubMed Central

    Krishna, B. A.; Lau, B.; Jackson, S. E.; Wills, M. R.; Sinclair, J. H.; Poole, E.

    2016-01-01

    Human cytomegalovirus (HCMV) latency in the myeloid lineage is maintained by repressive histone modifications around the major immediate early promoter (MIEP), which results in inhibition of the lytic viral life cycle. We now show that pharmacological inhibition of histone deacetylases (HDACs) relieves this repression of the MIEP and induces transient expression of the viral lytic immediate early (IE) antigens but, importantly, not full virus reactivation. In turn, these latently infected cells now become targets for IE-specific cytotoxic T cells (CTLs) which are present at high frequency in all normal healthy HCMV positive carriers but would normally be unable to target latent (lytic antigen-negative) cells. This approach of transiently inducing viral lytic gene expression by HDAC inhibition, in otherwise latently infected cells, offers a window of opportunity to target and purge the latent myeloid cell reservoir by making these normally immunologically undetectable cells visible to pre-existing host immune responses to viral lytic antigens. PMID:27091512

  11. Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and to drought and salt stresses.

    PubMed

    Liu, Hongxia; Zhou, Xianyao; Dong, Na; Liu, Xin; Zhang, Huaiyu; Zhang, Zengyan

    2011-09-01

    MYB transcription factors play diverse roles in plant growth, developmental processes and stress responses. A full-length cDNA sequence of a MYB gene, namely TaPIMP1, was isolated from wheat (Triticum aestivum L.). The TaPIMP1 transcript level was significantly up-regulated by inoculation with a fungal pathogen Bipolaris sorokiniana and by drought treatment. TaPIMP1 encodes the MYB protein TaPIMP1 consisting of 323 amino acids. TaPIMP1 contains two MYB DNA binding domains (R2, R3), two putative nuclear localization sites and two putative transcription activation domains. TaPIMP1 is a new member of the R2R3-MYB transcription factor subfamily. Transient expression in onion epidermal cells of GFP fused with TaPIMP1 proved that subcellular localization of TaPIMP1 occurred in the nucleus. The TaPIMP1 gene was transferred into tobacco (Nicotiana tabacum L.) cultivar W38 by Agrobacterium-mediated transformation. After screening through PCR and RT-PCR analyses, transgenic tobacco lines expressing TaPIMP1 were identified and evaluated for pathogen resistance, and drought and salt tolerance. Compared to untransformed tobacco host plants, TaPIMP1 expressing plants displayed significantly enhanced resistance to Ralstonia solanacearum and exhibited improved tolerances to drought and salt stresses. In these transgenic lines, the activities of phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were significantly increased relative to wild-type tobacco plants. The results suggested that the wheat R2R3-MYB transcription factor plays an important role in modulating responses to biotic and abiotic stresses. PMID:21597961

  12. A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters*

    PubMed Central

    Shibasaki, Koji; Ikenaka, Kazuhiro; Tamalu, Fuminobu; Tominaga, Makoto; Ishizaki, Yasuki

    2014-01-01

    Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca2+ transients in astrocytes, and these Ca2+ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4+ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4+ astrocytes. After activation, both TRPV4+ and TRPV4− astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4+ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4+ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands. PMID:24737318

  13. Transient Co-Expression of Post-Transcriptional Gene Silencing Suppressors for Increased in Planta Expression of a Recombinant Anthrax Receptor Fusion Protein

    PubMed Central

    Arzola, Lucas; Chen, Junxing; Rattanaporn, Kittipong; Maclean, James M.; McDonald, Karen A.

    2011-01-01

    Potential epidemics of infectious diseases and the constant threat of bioterrorism demand rapid, scalable, and cost-efficient manufacturing of therapeutic proteins. Molecular farming of tobacco plants provides an alternative for the recombinant production of therapeutics. We have developed a transient production platform that uses Agrobacterium infiltration of Nicotiana benthamiana plants to express a novel anthrax receptor decoy protein (immunoadhesin), CMG2-Fc. This chimeric fusion protein, designed to protect against the deadly anthrax toxins, is composed of the von Willebrand factor A (VWA) domain of human capillary morphogenesis 2 (CMG2), an effective anthrax toxin receptor, and the Fc region of human immunoglobulin G (IgG). We evaluated, in N. benthamiana intact plants and detached leaves, the expression of CMG2-Fc under the control of the constitutive CaMV 35S promoter, and the co-expression of CMG2-Fc with nine different viral suppressors of post-transcriptional gene silencing (PTGS): p1, p10, p19, p21, p24, p25, p38, 2b, and HCPro. Overall, transient CMG2-Fc expression was higher on intact plants than detached leaves. Maximum expression was observed with p1 co-expression at 3.5 days post-infiltration (DPI), with a level of 0.56 g CMG2-Fc per kg of leaf fresh weight and 1.5% of the total soluble protein, a ten-fold increase in expression when compared to absence of suppression. Co-expression with the p25 PTGS suppressor also significantly increased the CMG2-Fc expression level after just 3.5 DPI. PMID:21954339

  14. Co-expression of non-selective cation channels of the transient receptor potential canonical family in central aminergic neurones.

    PubMed

    Sergeeva, Olga A; Korotkova, Tatiana M; Scherer, Annette; Brown, Ritchie E; Haas, Helmut L

    2003-06-01

    The mammalian transient receptor potential canonical (TRPC) group of channels is a family of Ca2+-permeable cation channels that are activated following receptor-mediated stimulation of different isoforms of phospholipase C. In vitro TRPC proteins can form hetero- or homo-oligomeric channels. We performed single-cell RT-PCR analysis to reveal the co-expression of seven TRPC channels in identified rat aminergic neurones. All serotonergic neurones of the dorsal raphe (DR), the majority of histaminergic (tuberomamillary nucleus; TMN) and dopaminergic cells of the ventral tegmental area (VTA), as well as some GABAergic neurones from the VTA, expressed at least one variant of TRPC channels. No TRPC channel expression was found in the locus coeruleus. In raphe neurones TRPC6 and TRPC5 mRNAs occurred most frequently. In VTA and TMN co-expression of TRPC4 with TRPC5 and TRPC6 with TRPC7 was not found in individual neurones (in contrast to the whole-brain regions). Their co-expression in non-neuronal cells could not be excluded. The neonatal TRPC3 subunit was rarely seen. In DR, but not in the other nuclei studied, the expression of orexin receptors correlated with the expression of TRPC channels. We conclude that several TRPC channel populations exist in individual neurones and that their subunit co-expression pattern is region and cell-type specific. PMID:12787073

  15. Proteomic analysis reveals protein expression differences in Escherichia coli strains associated with persistent versus transient mastitis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli is a leading cause of bacterial mastitis in dairy cattle. Typically this infection is transient in nature, causing an infection that lasts 2-3 days. However, in a minority of cases, E. coli has been shown to cause a persistent intramammary infection. The mechanisms that allow for...

  16. Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs.

    PubMed Central

    Xu, G.; Redard, M.; Gabbiani, G.; Neuville, P.

    1997-01-01

    We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 PMID:9403724

  17. Transient and persistent expression of NT-3/HDNF mRNA in the rat brain during postnatal development.

    PubMed

    Friedman, W J; Ernfors, P; Persson, H

    1991-06-01

    Neurotrophin-3 (NT-3) is closely related to two known neurotrophic agents, NGF and brain-derived neurotrophic factor (BDNF), and acts upon overlapping, yet distinct, populations of peripheral ganglia. NT-3 mRNA expression in the adult rat brain is largely confined to the hippocampus. In this study, we have used in situ hybridization to examine expression of this novel neurotrophic factor during postnatal development. The striking observation was made that NT-3 mRNA was transiently expressed at high levels in the cingulate cortex during the first 2 weeks of age. In the hippocampus, the adult pattern of expression, in the CA2, medial CA1, and granule layer of the dentate gyrus, was detected at all ages examined. However, there were two major differences in NT-3 mRNA expression in the developing hippocampus: Labeled cells were detected in the hilar region of the dentate gyrus at postnatal day 1 (P1) and 1 week that were absent by 2 weeks of age. Further, the caudal hippocampus, which has a lower intensity of labeling than the rostral region in the adult, was devoid of NT-3-expressing cells in the P1 and 1-week-old rat brain. These data indicate a substantial plasticity in NT-3 mRNA expression and suggest that the spectrum of neurons supported by NT-3 during development is partially different from that in the mature rat brain. PMID:2045877

  18. Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

    PubMed Central

    Buyel, Johannes Felix; Fischer, Rainer

    2014-01-01

    Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems. PMID:24514765

  19. Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor alpha1 subunits (R528H)

    PubMed

    Jurkat-Rott, K; Uetz, U; Pika-Hartlaub, U; Powell, J; Fontaine, B; Melzer, W; Lehmann-Horn, F

    1998-02-20

    Rabbit cDNA of the alpha1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L-type calcium currents and transients were recorded for the wild type and a mutant alpha1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L-type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L-type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura-2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L-type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T-type current and changes in the expression level of the third-type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis. PMID:9512357

  20. Delay-Induced Transient Increase and Heterogeneity in Gene Expression in Negatively Auto-Regulated Gene Circuits

    PubMed Central

    Maithreye, R.; Sarkar, Ram Rup; Parnaik, Veena K.; Sinha, Somdatta

    2008-01-01

    A generic feature in all intracellular biochemical processes is the time required to complete the whole sequence of reactions to yield any observable quantity-from gene expression to circadian rhythms. This widespread phenomenon points towards the importance of time delay in biological functions. Theoretically time delay is known to be the source of instability, and has been attributed to lead to oscillations or transient dynamics in several biological functions. Negative feedback loops, common in biochemical pathways, have been shown to provide stability and withstand considerable variations and random perturbations of biochemical parameters. The interaction of these two opposing factors-of instability and homeostasis-are features that are widespread in intracellular processes. To test the effect of these divergent forces in the dynamics of gene expression, we have designed and constructed simple negatively auto-regulated gene circuits consisting of a basic regulator and transcriptional repressor module, and compared it with one, which has delayed repression. We show, both theoretically and experimentally, that delayed repression induces transient increase and heterogeneity in gene expression before the gain of stability effected by the negative feedback. This design, therefore, seems to be suitable for conferring both stability and variability in cells required for adaptive response to a noisy environment. PMID:18698420

  1. Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells.

    PubMed

    Hester, Ian; McKee, Sarah; Pelletier, Phillip; Thompson, Charles; Storbeck, Christopher; Mears, Alan; Schulz, Jörg B; Hakim, Antoine A; Sabourin, Luc A

    2007-03-01

    Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNAs induced by potassium depolarization in vivo. Using this approach, we have isolated a cDNA encoding the SIM2-related bHLH-PAS protein Nxf. Our results confirm that Nxf mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD. Reporter assays show that Nxf is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged Nxf results in cell death and the direct activation of the Bax gene in cultured cells. However, RNA interference studies show that endogenous Nxf is required for optimal neuroprotection by preconditioning in cultured F-11 cells. Together, our data indicate that Nxf is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of Nxf may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury. PMID:17214977

  2. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed

    Alwine, J C

    1985-05-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. PMID:2987671

  3. Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

    PubMed Central

    Alwine, J C

    1985-01-01

    The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed. Images PMID:2987671

  4. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

    PubMed Central

    Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

    1990-01-01

    Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

  5. Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes.

    PubMed

    Lundberg, U; Vinatzer, U; Berdnik, D; von Gabain, A; Baccarini, M

    1999-06-01

    Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date. PMID:10348855

  6. Growth Phase-Regulated Induction of Salmonella-Induced Macrophage Apoptosis Correlates with Transient Expression of SPI-1 Genes

    PubMed Central

    Lundberg, Urban; Vinatzer, Ursula; Berdnik, Daniela; von Gabain, Alexander; Baccarini, Manuela

    1999-01-01

    Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date. PMID:10348855

  7. Decreased expression of transient receptor potential channels in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage.

    PubMed

    Thilo, Florian; Suess, Olaf; Liu, Ying; Tepel, Martin

    2011-01-01

    Recent data indicate that transient receptor potential (TRP) cation channels play an important role in hypertension. Now, we tested the hypothesis that TRP expression is altered in human cerebral vascular tissue in patients who had experienced hypertensive intracerebral hemorrhage. TRPC1, TRPC3, TRPC5, TRPC6, TRPM4, TRPM6, and TRPM7 channels were detected in cerebral vascular tissue by quantitative real-time RT-PCR. Control cerebral vascular tissue was obtained from normotensive patients who underwent neurosurgical operation because of brain tumor. To examine a possible relation between the expression of TRP expression and hypoxic conditions caused by the intracerebral bleeding, we examined the expression of hypoxia inducible factor 1a (HIF1a). Transcripts of TRPC3, TRPC5, TRPM6, and HIF1a were significantly reduced in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage compared to controls. TRPC3 mRNA correlated well with the expression of HIF1a mRNA (r(2) = 0.59; p = 0.01). TRPC3 expression is associated with hypertension and hypoxic conditions in human cerebral vascular tissue. PMID:21957871

  8. High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

    PubMed

    Subedi, Ganesh P; Johnson, Roy W; Moniz, Heather A; Moremen, Kelley W; Barb, Adam

    2015-01-01

    The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield. PMID:26779721

  9. Optimatization of transient transformation methods to study gene expression in Musa acuminata (AAA group) cultivar Ambon Lumut

    NASA Astrophysics Data System (ADS)

    Prayuni, Kinasih; Dwivany, Fenny M.

    2015-09-01

    Banana is classified as a climateric fruit, whose ripening is regulated by ethylene. Ethylene is synthesized from ACC (1-aminocyclopropane-1-carboxylic acid) by ACC oxidase enzyme which is encoded by ACO gene. Controling an important gene expression in ethylene biosynthesis pathway has became a target to delay the ripening process. Therefore in the previous study we have designed a MaACO-RNAi construct to control MaACO gene expression. In this research, we study the effectiveness of different transient transformation methods to deliver the construct. Direct injection, with or no vaccum infiltration methods were used to deliver MaACO-RNAi construct. All of the methods succesfully deliver the construct into banana fruits based on RT-PCR result.

  10. Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy.

    PubMed

    Liu, Yiting; Liao, Sifang; Veenstra, Jan A; Nässel, Dick R

    2016-01-01

    The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0-10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles. PMID:27197757

  11. Drosophila insulin-like peptide 1 (DILP1) is transiently expressed during non-feeding stages and reproductive dormancy

    PubMed Central

    Liu, Yiting; Liao, Sifang; Veenstra, Jan A.; Nässel, Dick R.

    2016-01-01

    The insulin/insulin-like growth factor signaling pathway is evolutionarily conserved in animals, and is part of nutrient-sensing mechanisms that control growth, metabolism, reproduction, stress responses, and lifespan. In Drosophila, eight insulin-like peptides (DILP1-8) are known, six of which have been investigated in some detail, whereas expression and functions of DILP1 and DILP4 remain enigmatic. Here we demonstrate that dilp1/DILP1 is transiently expressed in brain insulin producing cells (IPCs) from early pupa until a few days of adult life. However, in adult female flies where diapause is triggered by low temperature and short days, within a time window 0–10h post-eclosion, the dilp1/DILP1 expression remains high for at least 9 weeks. The dilp1 mRNA level is increased in dilp2, 3, 5 and dilp6 mutant flies, indicating feedback regulation. Furthermore, the DILP1 expression in IPCs is regulated by short neuropeptide F, juvenile hormone and presence of larval adipocytes. Male dilp1 mutant flies display increased lifespan and reduced starvation resistance, whereas in female dilp1 mutants oviposition is reduced. Thus, DILP1 is expressed in non-feeding stages and in diapausing flies, is under feedback regulation and appears to play sex-specific functional roles. PMID:27197757

  12. Do fasudil and Y-27632 affect the level of transient receptor potential (TRP) gene expressions in breast cancer cell lines?

    PubMed

    Gogebakan, Bulent; Bayraktar, Recep; Suner, Ali; Balakan, Ozan; Ulasli, Mustafa; Izmirli, Muzeyyen; Oztuzcu, Serdar; Camci, Celaletdin

    2014-08-01

    Breast cancer (BC) is the most frequent cancer type in women, and the mortality rate is high especially in metastatic disease. Ion channels such as the transient receptor potential (TRP) channels correlate with malignant growth and cancer progression. Hence, some authors have suggested that the expression levels of TRP channels may be used as a marker in the diagnosis and predicting the prognosis of BC. Also, in some recent studies, targeting TRP channels are suggested as a novel treatment strategy in BC. The aim of this study was to investigate the effect of two Rho-kinase (ROCK) inhibitors, fasudil and Y-27632, on the expression levels of TRP channel genes in breast cancer cell lines (ZR-75-1, MCF7, and MDA-MB-231) and breast epithelial cell line (hTERT-HME1). The expression levels of TRP genes were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We found that fasudil had reduced the TRPC1, TRPV2 expression levels in the ZR-75-1, MCF7, and MDA-MB-231 cell lines. On the other hand, fasudil and Y-27632 had reduced TRPM6 expression levels in all cell lines. Y-27632 increased the expression levels of TRPC7 in all cell lines. In conclusion, this is the first study demonstrating that the inhibition of ROCK pathway changes the expression levels of some TRP genes. Also, our study has firstly shown that the expression levels of the TRP genes which are suggested as a diagnostic and prognostic biomarker in BC, were changed with the treatment of fasudil and Y-27632. PMID:24839003

  13. Hypoxia drives transient site-specific copy gain and drug-resistant gene expression

    PubMed Central

    Black, Joshua C.; Atabakhsh, Elnaz; Kim, Jaegil; Biette, Kelly M.; Van Rechem, Capucine; Ladd, Brendon; Burrowes, Paul d.; Donado, Carlos; Mattoo, Hamid; Kleinstiver, Benjamin P.; Song, Bing; Andriani, Grasiella; Joung, J. Keith; Iliopoulos, Othon; Montagna, Cristina; Pillai, Shiv; Getz, Gad

    2015-01-01

    Copy number heterogeneity is a prominent feature within tumors. The molecular basis for this heterogeneity remains poorly characterized. Here, we demonstrate that hypoxia induces transient site-specific copy gains (TSSGs) in primary, nontransformed, and transformed human cells. Hypoxia-driven copy gains are not dependent on HIF1α or HIF2α; however, they are dependent on the KDM4A histone demethylase and are blocked by inhibition of KDM4A with a small molecule or the natural metabolite succinate. Furthermore, this response is conserved at a syntenic region in zebrafish cells. Regions with site-specific copy gain are also enriched for amplifications in hypoxic primary tumors. These tumors exhibited amplification and overexpression of the drug resistance gene CKS1B, which we recapitulated in hypoxic breast cancer cells. Our results demonstrate that hypoxia provides a biological stimulus to create transient site-specific copy alterations that could result in heterogeneity within tumors and cell populations. These findings have major implications in our understanding of copy number heterogeneity and the emergence of drug resistance genes in cancer. PMID:25995187

  14. Enhanced expression of transient receptor potential channel 3 in uterine smooth muscle tissues of lipopolysaccharide-induced preterm delivery mice

    PubMed Central

    Zheng, Dongming; Zhang, Lijuan; Na, Quan; Liu, Sishi; Zhuang, Yanyan; Lv, Yuan; Liu, Caixia

    2016-01-01

    Objective(s): We aimed to investigate the influence of transient receptor potential channel 3 (TRPC3) on lipopolysaccharide-induced (LPS) preterm delivery mice. Materials and Methods: Mice were randomly assigned to the four groups: an unpregnant group, a mid-pregnancy group (E15), a term delivery group, and an LPS-induced preterm delivery group (intraperitoneal injection LPS at 15 days). Uterine smooth muscles were obtained through caesarean section; TRPC3 expression was measured by real-time PCR, western blotting, and immunohistochemistry. A specific inhibitor of TRPC3 (SKF96365) was injected into the LPS-induced preterm delivery group to determine whether the delivery interval was prolonged. Results: TRPC3 was primarily expressed in the uterine smooth muscle layer. In addition, the LPS-induced preterm delivery group had an obviously higher expression level of TRPC3 mRNA and protein compared with the unpregnant and E15 groups, which were close to term delivery. More importantly, SKF96365 prolongs the delivery interval of LPS-induced preterm delivery mice. Conclusion: Enhanced expression of TRPC3 may be associated with LPS-induced preterm delivery in mice. The specific inhibitor of TRPC3 (SKF96365) may be helpful for clinical treatment of preterm delivery. PMID:27403264

  15. Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

    NASA Astrophysics Data System (ADS)

    Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

    2007-01-01

    Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

  16. Functional characterization and transient expression manipulation of a new sesquiterpene synthase involved in β-caryophyllene accumulation in Ocimum.

    PubMed

    Jayaramaiah, Ramesha H; Anand, Atul; Beedkar, Supriya D; Dholakia, Bhushan B; Punekar, Sachin A; Kalunke, Raviraj M; Gade, Wasudeo N; Thulasiram, Hirekodathakallu V; Giri, Ashok P

    2016-04-22

    The genus Ocimum has a unique blend of diverse secondary metabolites, with major proportion of terpenoids including mono- and sesquiterpenes. Although, β-Caryophyllene, bicyclic sesquiterpene, is one of the major terpene found in Ocimum species and known to possess several biological activities, not much is known about its biosynthesis in Ocimum. Here, we describe isolation and characterization of β-caryophyllene synthase gene from Ocimum kilimandscharicum Gürke (OkBCS- GenBank accession no. KP226502). The open reading frame of 1629 bp encoded a protein of 542 amino acids with molecular mass of 63.6 kDa and pI value of 5.66. The deduced amino acid sequence revealed 50-70% similarity with known sesquiterpene synthases from angiosperms. Recombinant OkBCS converted farnesyl diphosphate to β-caryophyllene as a major product (94%) and 6% α-humulene. Expression variation of OkBCS well corroborated with β-caryophyllene levels in different tissues from five Ocimum species. OkBCS transcript revealed higher expression in leaves and flowers. Further, agro-infiltration based transient expression manipulation with OkBCS over-expression and silencing confirmed its role in β-caryophyllene biosynthesis. These findings may potentially be further utilized to improve plant defense against insect pests. PMID:27005818

  17. Protooncogene expression identifies a transient columnar organization of the forebrain within the late embryonic ventricular zone

    SciTech Connect

    Johnston, J.G.; Van Der Kooy, D. )

    1989-02-01

    Immunocytochemical studies using monoclonal antibodies directed against oncogenic peptides revealed a heterogeneous distribution of the peptides within the ventricular zone of the embryonic day 18 rat forebrain. This sis-, src-, ras-, and myc-encoded peptides were concentrated in the same isolated clusters of 5-25 radial glial cells (also identified by vimentin staining), providing a transient columnar compartmentalization to the ventricular zone. An increased number of ({sup 3}H)thymidine-labeled ventricular zone cells were observed within the protooncogene stained radial glial cell columns as compared to noncolumn areas. The columnar heterogeneity of radial glial cells reveals the mosaicism of the embryonic ventricular zone and the differential proliferation of its cells.

  18. Transient Gene and miRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Wang, Xiaoyu; Wu, Honglu

    2015-01-01

    Microgravity or an altered gravity environment from the static 1 gravitational constant has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of the cells. Whether non-dividing cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted on the International Space Station, confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days for investigations of gene and miRNA (microRNA) expression profile changes in these cells. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly even though they were confluent, as measured by the expression of the protein Ki-67 positive cells, and the cells in space grew slightly faster. Gene and miRNA expression data indicated activation of NF(sub kappa)B (nuclear factor kappa-light-chain-enhancer of activated B cells) and other growth related pathways involving HGF and VEGF in the flown cells. On Day 14 when the cells were mostly non-dividing, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples in respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeleton changes by immunohistochemistry staining of the cells with antibodies for alpha-tubulin showed no difference between the flight and ground samples. Results of our study suggest that in true non-dividing human fibroblast cells, microgravity in

  19. Transient gene and microRNA expression profile changes of confluent human fibroblast cells in space

    NASA Astrophysics Data System (ADS)

    Wu, Honglu; Story, Michael; Karouia, Fathi; Stodieck, Louis; Zhang, Ye; Lu, Tao

    2016-07-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NFkB and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for αa-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  20. Transient Gene and MicroRNA Expression Profile Changes of Confluent Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Zhang, Ye; Lu, Tao; Wong, Michael; Wang, Xiaoyu; Stodieck, Louis; Karouia, Fathi; Story, Michael; Wu, Honglu

    2016-01-01

    Microgravity, or an altered gravity environment from the Earth1g, has been shown to influence global gene expression patterns and protein levels in cultured cells. However, most of the reported studies conducted in space or using simulated microgravity on the ground have focused on the growth or differentiation of these cells. Whether non-proliferating cultured cells will sense the presence of microgravity in space has not been specifically addressed. In an experiment conducted onboard the International Space Station (ISS), confluent human fibroblast cells were fixed after being cultured in space for 3 and 14 days, respectively, for investigations of gene and miRNA expression profile changes in these cells. Results of the experiment showed that on Day 3, both the flown and ground cells were still proliferating slowly, as measured by the percentage of Ki-67 positive cells. Gene and miRNA expression data indicated activation of NF(kappa)B and other growth related pathways involving HGF and Vegf along with down regulation of the Let-7 miRNA family. On Day 14 when the cells were mostly non-proliferating, the gene and miRNA expression profiles between the flight and ground samples were indistinguishable. Comparison of gene and miRNA expressions in the Day 3 samples with respect to Day 14 revealed that most of the changes observed on Day 3 were related to cell growth for both the flown and ground cells. Analysis of cytoskeletal changes via immunohistochemistry staining of the cells with antibodies for alpha-tubulin and fibronectin showed no difference between flown and ground samples. Taken together, our study suggests that in true non-dividing human fibroblast cells in culture, microgravity experienced in space has little effect on the gene and miRNA expression profiles.

  1. Transient and Stable Expression of the Neurotensin Receptor NTS1: A Comparison of the Baculovirus-Insect Cell and the T-REx-293 Expression Systems

    PubMed Central

    Xiao, Su; White, Jim F.; Betenbaugh, Michael J.; Grisshammer, Reinhard; Shiloach, Joseph

    2013-01-01

    Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [3H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs. PMID:23696845

  2. Transient expression of Bcl6 is sufficient for oncogenic function and induction of mature B-cell lymphoma

    PubMed Central

    Green, Michael R; Vicente-Dueñas, Carolina; Romero-Camarero, Isabel; Liu, Chih Long; Dai, Bo; González-Herrero, Inés; García-Ramírez, Idoia; Alonso-Escudero, Esther; Iqbal, Javeed; Chan, Wing C; Campos-Sanchez, Elena; Orfao, Alberto; Pintado, Belén; Flores, Teresa; Blanco, Oscar; Jiménez, Rafael; Martínez-Climent, Jose Angel; Criado, Francisco Javier García; Cenador, María Begoña García; Zhao, Shuchun; Natkunam, Yasodha; Lossos, Izidore S; Majeti, Ravindra; Melnick, Ari; Cobaleda, César; Alizadeh, Ash A.; Sánchez-García, Isidro

    2015-01-01

    Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma and can be separated into two subtypes based upon molecular features with similarities to germinal center B-cells (GCB-like) or activated B-cells (ABC-like). Here we identify gain of 3q27.2 as being significantly associated with adverse outcome in DLBCL and linked with the ABC-like subtype. This lesion includes the BCL6 oncogene, but does not alter BCL6 transcript levels or target-gene repression. Separately, we identify expression of BCL6 in a subset of human hematopoietic stem/progenitor cells (HSPCs). We therefore hypothesize that BCL6 may act by hit-and-run oncogenesis. We model this by transiently expressing Bcl6 within murine HSPCs, and find it causes mature B-cell lymphomas that lack Bcl6 expression and target-gene repression, are transcriptionally similar to post-GCB cells, and show epigenetic changes that are conserved from HSPCs to mature B-cells. Together these results suggest that Bcl6 may function in a hit-and-run role in lymphomagenesis. PMID:24887457

  3. Expression of vesicular glutamate transporters in transient receptor potential melastatin 8 (TRPM8)-positive dental afferents in the mouse.

    PubMed

    Kim, Y S; Kim, T H; McKemy, D D; Bae, Y C

    2015-09-10

    Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cool and noxious cold and plays a crucial role in cold-induced acute pain and pain hypersensitivity. To help understand the mechanism of TRPM8-mediated cold perception under normal and pathologic conditions, we used light microscopic immunohistochemistry and Western blot analysis in mice expressing a genetically encoded axonal tracer in TRPM8-positive (+) neurons. We investigated the coexpression of TRPM8 and vesicular glutamate transporter 1 (VGLUT1) and VGLUT2 in the trigeminal ganglion (TG) and the dental pulp before and after inducing pulpal inflammation. Many TRPM8+ neurons in the TG and axons in the dental pulp expressed VGLUT2, while none expressed VGLUT1. TRPM8+ axons were dense in the pulp horn and peripheral pulp and also frequently observed in the dentinal tubules. Following pulpal inflammation, the proportion of VGLUT2+ and of VGLUT2+/TRPM8+ neurons increased significantly, whereas that of TRPM8+ neurons remained unchanged. Our findings suggest the existence of VGLUT2 (but not VGLUT1)-mediated glutamate signaling in TRPM8+ neurons possibly underlying the cold-induced acute pain and hypersensitivity to cold following pulpal inflammation. PMID:26166724

  4. Studies of Anthocyanin Regulatory Genes in Phalaenopsis Using a Transient Expression System

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anthocyanins are one of the primary color-producing compounds in plants, and their expression is controlled by a complex pathway of regulatory genes and transcription factors. Plant pigmentation, especially in leaves and flowers, is especially important in ornamental plants, where novel colors often...

  5. Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells.

    PubMed

    Mergler, Stefan; Mertens, Charlotte; Valtink, Monika; Reinach, Peter S; Székely, Violeta Castelo; Slavi, Nefeli; Garreis, Fabian; Abdelmessih, Suzette; Türker, Ersal; Fels, Gabriele; Pleyer, Uwe

    2013-11-01

    Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca(2+) transients induced by either menthol (500 μmol/l), eucalyptol (3 mmol/l), or icilin (2-60 μmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La(3+), 100 μmol/l) as well as the TRPM8 blockers BCTC (10 μmol/l) and capsazepine (CPZ, 10 μmol/l) suppressed icilin-induced Ca(2+) increases. In and outward currents induced by application of menthol (500 μmol/l) or icilin (50 μmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca(2+) increases that were inhibited by a TRPM8 blocker BCTC (10 μmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca(2+) response patterns are suggestive of other Ca(2+) handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype. PMID:24135298

  6. Transient gene expression in tobacco using Gibson assembly and the Gene Gun.

    PubMed

    Mattozzi, Matthew D; Voges, Mathias J; Silver, Pamela A; Way, Jeffrey C

    2014-01-01

    In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5' mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work(11), and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy. PMID:24796418

  7. Production of bioactive wheat puroindoline proteins in Nicotiana benthamiana using a virus-based transient expression system.

    PubMed

    Niknejad, Azadeh; Webster, Diane; Bhave, Mrinal

    2016-09-01

    The emergence of antibiotic resistant pathogenic strains of bacteria has necessitated the development of novel antimicrobial agents. The puroindoline A and B (PINA and PINB) proteins of wheat, well-known for their roles in determining the important phenotype of grain texture, are also antimicrobial, making them attractive as natural bio-control agents. However, the biochemical basis of PIN functionality remains unclear due to limitations in expressing them at the required yield and purity and lack of accurate tertiary structure. This study focussed on rapid transient expression of PINs targeted to different subcellular compartments (chloroplast, apoplast, endoplasmic reticulum and cytosol) of Nicotiana benthamiana leaf cells using the deconstructed tobacco mosaic virus-based 'magnICON®' system. The expressed recombinant PINs were characterised by Western blot using the Durotest anti-friabilin antibody, enzyme-linked immunosorbent assays (ELISA) and antimicrobial activity tests. Maximum yield of the His-tagged PINs occurred when targeted to the chloroplast. Both PINs exhibited oligomeric and monomeric forms on gels, but Western blots with the widely used Durotest anti-friabilin antibody identified only oligomeric forms. Only the PINs purified by a hydrophobic interaction method exhibited monomeric forms with the anti-His tag antibody, indicating correct folding. Interestingly, the Durotest antibody did not bind to monomers, suggesting their epitope may be obscured. PINs purified by His-tag affinity purification under native conditions or by the hydrophobic method exhibited antimicrobial activities. The successful in planta expression and optimisation of purification will enable future studies to examine the detailed structure of the PINs and explore novel bio-control applications in health, food and/or agriculture. PMID:26363114

  8. Transient expression of Fc-fused human glycoprotein 130 in Expi293F suspension cells.

    PubMed

    Zhao, Xiaozhi; Chen, Wei; Ge, Liyuan; Jiang, Wei; Tang, Bo; Zhang, Qing; Xu, Xiaoyu; Wang, Chong; Cao, Lin; Guo, Hongqian

    2016-08-01

    Human glycoprotein 130 (gp130) is a signal-transducing receptor for interleukin 6 (IL-6), whose signaling plays a critical role in chronic inflammation and cancer. The soluble form of gp130 specifically inhibits IL-6 trans-signaling. However, achieving high-level expression of a large glycoprotein such as gp130 is difficult. Here, we designed and constructed one Fc-gp130-pcDNA mammalian expression vector, with the mouse IgG2a Fc fragment added to the N-terminus of human gp130, which greatly increased the secretion of recombinant gp130 protein from Expi293F suspension cells. Recombinant fusion Fc-gp130 was easily and efficiently purified from the supernatant of transfected cells by one-step affinity chromatography. Moreover, Fc-gp130 could automatically form dimers by the disulfide bond. Fc-gp130 was confirmed as a more efficient IL-6 trans-signaling blocker by its higher biological activity against signal transducer and activator of transcription 3 (STAT3). This purified active Fc-gp130 could be used to develop valuable therapeutic agents against inflammatory diseases and cancers. PMID:27113713

  9. Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts.

    PubMed

    Hayashi, Shimpei; Wakasa, Yuhya; Ozawa, Kenjirou; Takaiwa, Fumio

    2016-06-01

    In some eukaryotes, endoplasmic reticulum (ER) stress induces regulated inositol-requiring enzyme 1 (IRE1)-dependent decay (RIDD) of mRNAs. Recently, the expression levels of the mRNAs encoding some secretory proteins were reported to be downregulated by RIDD in the vegetative tissues of plants. However, the characteristics of plant RIDD have been insufficiently investigated due to difficulty of in planta analyses. Here, the RIDD susceptibilities of various mRNAs that are difficult to analyze in planta were examined using transient expression analyses of rice protoplasts. In this system, the mRNAs encoding three rice seed storage proteins (SSPs) - namely α-globulin, 16-kDa prolamin and 10-kDa prolamin - were downregulated in response to ER stress. The rapid ER stress-induced degradation of these mRNAs was repressed in cells in which the ribonuclease activity of IRE1 was specifically abolished by genome editing, suggesting that the mRNAs encoding certain SSPs are strong targets of RIDD. Furthermore, we investigated whether these RIDD targets are substrates of the IRE1 ribonuclease using a recombinant IRE1 protein, and identified candidate IRE1-mediated cleavage sites. Overall, the results demonstrate the existence of a post-transcriptional mechanism of regulation of SSPs, and illustrate the basic and multifaceted characteristics of RIDD in higher plants. PMID:26831622

  10. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

    PubMed Central

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G.; Sunde, Margaret; Whan, Renee; Graham, Robert M.; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  11. Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer.

    PubMed

    Constantine, Maryrose; Liew, Chu Kong; Lo, Victor; Macmillan, Alex; Cranfield, Charles G; Sunde, Margaret; Whan, Renee; Graham, Robert M; Martinac, Boris

    2016-01-01

    Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel. PMID:26785754

  12. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies.

    PubMed

    Grefen, Christopher; Donald, Naomi; Hashimoto, Kenji; Kudla, Jörg; Schumacher, Karin; Blatt, Michael R

    2010-10-01

    Fluorescent tagging of proteins and confocal imaging techniques have become methods of choice in analysing the distributions and dynamic characteristics of proteins at the subcellular level. In common use are a number of strategies for transient expression that greatly reduce the preparation time in advance of imaging, but their applications are limited in success outside a few tractable species and tissues. We previously developed a simple method to transiently express fluorescently-tagged proteins in Arabidopsis root epidermis and root hairs. We describe here a set of Gateway-compatable vectors with fluorescent tags incorporating the ubiqutin-10 gene promoter (P(UBQ10) ) of Arabidopsis that gives prolonged expression of the fluorescently-tagged proteins, both in tobacco and Arabidopsis tissues, after transient transformation, and is equally useful in generating stably transformed lines. As a proof of principle, we carried out transformations with fluorescent markers for the integral plasma membrane protein SYP121, a member of the SNARE family of vesicle-trafficking proteins, and for DHAR1, a cytosolic protein that facilitates the scavenging of reactive oxygen species. We also carried out transformations with SYP121 and its interacting partner, the KC1 K(+) channel, to demonstrate the utility of the methods in bimolecular fluorescence complementation (BiFC). Transient transformations of Arabidopsis using Agrobacterium co-cultivation methods yielded expression in all epidermal cells, including root hairs and guard cells. Comparative studies showed that the P(UBQ10) promoter gives similar levels of expression to that driven by the native SYP121 promoter, faithfully reproducing the characteristics of protein distributions at the subcellular level. Unlike the 35S-driven construct, expression under the P(UBQ10) promoter remained elevated for periods in excess of 2 weeks after transient transformation. This toolbox of vectors and fluorescent tags promises significant

  13. Effects of chronic exposure to cigarette smoke on canonical transient receptor potential expression in rat pulmonary arterial smooth muscle

    PubMed Central

    Wang, Jian; Chen, Yuqin; Lin, Chunyi; Jia, Jing; Tian, Lichun; Yang, Kai; Zhao, Lei; Lai, Ning; Jiang, Qian; Sun, Yueqian; Zhong, Nanshan; Ran, Pixin

    2013-01-01

    To clarify the possible mechanism of cigarette smoke (CS)-induced pulmonary hypertension and furthermore provide effective targets for prevention and treatment, the effects of chronic CS on rat pulmonary arterial smooth muscle in vivo and nicotine treatment on rat pulmonary arterial smooth muscle cells (PASMCs) in vitro were investigated. In this study, we demonstrated that chronic CS exposure led to rat weight loss, right ventricular hypertrophy, and pulmonary arterial remodeling. A fluorescence microscope was used to measure intracellular calcium concentration ([Ca2+]i) in rat distal PASMCs. Results showed that basal [Ca2+]i and store-operated calcium entry (SOCE) levels in PASMCs from 3- and 6-mo CS-exposed rats were markedly higher than those in cells from the unexposed control animals (the increases in 6-mo CS group were more significant than that in 3-mo group), accompanied with increased canonical transient receptor potential 1 (TRPC1) and TRPC6 expression at both mRNA and protein levels in isolated distal PA. Simultaneously, in vitro study showed that nicotine treatment (10 nM) significantly increased basal [Ca2+]i and SOCE and upregulated TRPC1 and TRPC6 expression in cultured rat distal PASMCs. TRPC siRNA knockdown strategies revealed that the elevations of basal [Ca2+]i and SOCE induced by nicotine in PASMCs were TRPC1 and TRPC6 dependent. These results suggested that chronic CS-induced changes in vascular tone and structure in PA and the development of pulmonary hypertension might be largely due to upregulation of TRPC1 and TRPC6 expression in PASMCs, in which nicotine played an important role. PMID:24336649

  14. Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice

    PubMed Central

    Anziliero, D.; Weiblen, R.; Kreutz, L.C.; Spilki, F.; Flores, E.F.

    2014-01-01

    The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines. PMID:24519126

  15. Stress induced gene expression drives transient DNA methylation changes at adjacent repetitive elements

    PubMed Central

    Secco, David; Wang, Chuang; Shou, Huixia; Schultz, Matthew D; Chiarenza, Serge; Nussaume, Laurent; Ecker, Joseph R; Whelan, James; Lister, Ryan

    2015-01-01

    Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress. DOI: http://dx.doi.org/10.7554/eLife.09343.001 PMID:26196146

  16. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress

    PubMed Central

    Malcolm, Tim I. M.; Villarese, Patrick; Fairbairn, Camilla J.; Lamant, Laurence; Trinquand, Amélie; Hook, C. Elizabeth; Burke, G. A. Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D.

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM–ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  17. Transient Expression of Functional Glucocerebrosidase for Treatment of Gaucher's Disease in the Goat Mammary Gland.

    PubMed

    Tavares, Kaio Cesar Simiano; Dias, Ana Christina de Oliveira; Lazzarotto, Cícera Regina; Gaudencio Neto, Saul; de Sá Carneiro, Igor; Ongaratto, Felipe Ledur; Pinto, Antônio Frederico Michel; de Aguiar, Luís Henrique; Calderón, Carlos Enrique Mendez; Toledo, Jorge Roberto; Castro, Fidel Ovidio; Santos, Diogenes Santiago; Chies, Jocelei Maria; Bertolini, Marcelo; Bertolini, Luciana Relly

    2016-01-01

    Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk. PMID:26589705

  18. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress.

    PubMed

    Malcolm, Tim I M; Villarese, Patrick; Fairbairn, Camilla J; Lamant, Laurence; Trinquand, Amélie; Hook, C Elizabeth; Burke, G A Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM-ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  19. Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

    PubMed

    Robert, Stéphanie; Goulet, Marie-Claire; D'Aoust, Marc-André; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 μg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 μg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins. PMID:26286859

  20. Expression and cellular distribution of transient receptor potential vanilloid 4 in cortical tubers of the tuberous sclerosis complex.

    PubMed

    Chen, Xin; Yang, Meihua; Sun, Feiji; Liang, Chao; Wei, Yujia; Wang, Lukang; Yue, Jiong; Chen, Bing; Li, Song; Liu, Shiyong; Yang, Hui

    2016-04-01

    Cortical tubers in patients with tuberous sclerosis complex (TSC) are highly associated with intractable epilepsy. Recent evidence has shown that transient receptor potential vanilloid 4 (TRPV4) has direct effects on both neurons and glial cells. To understand the role of TRPV4 in pathogenesis of cortical tubers, we investigated the expression patterns of TRPV4 in cortical tubers of TSC compared with normal control cortex (CTX). We found that TRPV4 was clearly up-regulated in cortical tubers at the protein levels. Immunostaining indicated that TRPV4 was specially distributed in abnormal cells, including dysplastic neurons (DNs) and giant cells (GCs). In addition, double immunofluorescent staining revealed that TRPV4 was localized on neurofilament proteins (NF200) positive neurons and glial fibrillary acidic portein (GFAP) positive reactive astrocytes. Moreover, TRPV4 co-localized with both glutamatergic and GABAergic neurons. Furthermore, protein levels of protein kinase C (PKC), but not protein kinase A (PKA), the important upstream factors of the TRPV4, were significantly increased in cortical tubers. Taken together, the overexpression and distribution patterns of TRPV4 may be linked with the intractable epilepsy caused by TSC. PMID:26874068

  1. Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.

    PubMed

    Tsuji, Akihiko; Tsukamoto, Kana; Iwamoto, Keiko; Ito, Yuka; Yuasa, Keizo

    2013-01-01

    Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination. PMID:23112094

  2. Optimization of transient gene expression in mammalian cells and potential for scale-up using flow electroporation.

    PubMed

    Parham, J H; Iannone, M A; Overton, L K; Hutchins, J T

    1998-11-01

    The goals of this study were to identify mammalian cell lines which could be efficiently transiently-transfected and scaled-up for protein production. The transfection efficiencies of eight cell lines (NSO, NSO-TAg, CV-1, COS-7, CHO, CHO-TAg, HEK 293, and 293-EBNA) were measured using electroporation for DNA delivery and green fluorescent protein (Evans, 1996) as the reporter gene. In addition, we have evaluated the effects of stable expression of viral proteins, cell cycle manipulation, and butyrate post-treatment in small scale experiments. The cell lines varied widely in their GFP transfection efficiencies. Stable expression of simian virus 40 large T-antigen or Epstein Barr nuclear antigen failed to significantly increase transfection efficiency above that seen in the parental lines. Aphidicolin (a DNA polymerase inhibitor), which blocked cells from S or G2/M, brought about an increase in transfection efficiency in two cell lines. The primary effect of butyrate (a histone deacetylase inhibitor) post-treatment was an increased intensity of the fluorescent signal of green fluorescent protein, as measured by flow cytometry (1.0 to 4.2-fold, depending on the cell line). The combined use of aphidicolin pretreatment followed by butyrate treatment post- electroporation yielded increases in fluorescence intensities ranging from 0.9 to 6.8-fold. Based on their high transfection efficiencies in small scale experiments, rapid growth, and ability to grow in suspension culture, CHO, CHO-TAg, and 293-EBNA were selected to assess the feasibility of using flow electroporation for large-scale transfections. Using secreted placental alkaline phosphatase as a reporter, 293-EBNA cells produced the highest protein levels in both the presence and absence of butyrate. These data indicate that flow electroporation provides an efficient method of DNA delivery into large numbers of cells for mammalian protein production. PMID:19003416

  3. An efficient Agrobacterium-mediated transformation and regeneration system for leaf explants of two elite aspen hybrid clones Populus alba × P. berolinensis and Populus davidiana × P. bolleana.

    PubMed

    Wang, Haihai; Wang, Cuiting; Liu, Hua; Tang, Renjie; Zhang, Hongxia

    2011-11-01

    Transgenic technology has been successfully used for gene function analyses and trait improvement in cereal plants. However, its usage is limited in woody plants, especially in the difficult-to-transform but commercially viable hybrid poplar. In this work, an efficient regeneration and transformation system was established for the production of two hybrid aspen clones: Populus alba × P. berolinensis and Populus davidiana × P. bolleana. A plant transformation vector designed to express the reporter gene uidA, encoding β-glucuronidase (GUS), driven by the cauliflower mosaic virus 35S promoter, was used to detect transformation event at early stages of plant regeneration, and to optimize the parameters that may affect poplar transformation efficiency. Bacterium strain and age of leaf explant are two major factors that affect transformation efficiency. Addition of thidiazuron (TDZ) improved both regeneration and transformation efficiency. The transformation efficiency is approximately 9.3% for P. alba × P. berolinensis and 16.4% for P. davidiana × P. bolleana. Using this system, transgenic plants were usually produced in less than 1 month after co-cultivation. The growth characteristics and morphology of transgenic plants were identical to the untransformed wild type plants, and the transgenes could be inherited by vegetative propagation, as confirmed by PCR, Southern blotting, RT-PCR and β-glucuronidase staining analyses. The establishment of this system will help to facilitate the studies of gene functions in tree growth and development at a genome level, and as well as the introduction of some valuable traits in aspen breeding. PMID:21717184

  4. Transient Mesp1 expression

    PubMed Central

    Liu, Yu; Schwartz, Robert J

    2013-01-01

    Mesp1 sits on the tip of the cardiac regulatory hierarchy, recent evidences support that it is regulated by stem cell factor Oct4, early gastrulation signal canonical Wnts and a couple of T-box factors, T and Eomes. With other transcription factors, Mesp1 programs/reprograms human cells toward cardiomyocytes. PMID:23584093

  5. Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea.

    PubMed

    Wang, Jie; Guo, Liqiong; Zhang, Kai; Wu, Qi; Lin, Junfang

    2008-11-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to the edible straw mushroom, Volvariella volvacea. Mycelium pellets were transformed to cold stress resistance using the afp gene as both a selective marker and a reporter gene, under the control of a heterologous Lentinula edodes gpd promoter. The efficiency of transformation is over 100 times higher than that previously reported in V. volvacea. Stable integration of the afp gene with 1-4 copy numbers was confirmed in all 10 randomly selected transgenic events by Southern blot analysis. The mitotic stability of the transformants was demonstrated after five successive transfers on PDA medium without selection pressure and the PCR analysis of basidiospores harvested from transformants. PMID:18434137

  6. Genetic transformation of wheat via Agrobacterium-mediated DNA delivery.

    PubMed

    Sparks, Caroline A; Doherty, Angela; Jones, Huw D

    2014-01-01

    The method described involves an initial incubation of wheat immature embryos in a liquid culture of Agrobacterium tumefaciens. The Agrobacterium strain is engineered to contain a binary vector with a gene of interest and a selectable marker gene placed between the T-DNA borders; the T-DNA is the region transferred to the plant cells, thus harnessing the bacterium's natural ability to deliver specific DNA into host cells. Following the initial inoculation with the Agrobacterium, the embryos are co-cultivated for several days after which the Agrobacterium is selectively destroyed using an antibiotic. Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants. This protocol has been used successfully to generate transformed plants of a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.). PMID:24243208

  7. Agrobacterium-mediated genetic transformation of Prunus salicina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report Agrobacterium tumefaciens-mediated transformation from hypocotyls slices of two Prunus salicina varieties, 'Angeleno' and 'Larry Anne', using a modification of the technique previously described for P. domestica. Regeneration rates on thidiazuron (TDZ) and indole-3-butyric acid (IBA) supp...

  8. The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein

    PubMed Central

    Saunders, Keith; Lomonossoff, George P.

    2015-01-01

    Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation. PMID:26734041

  9. A Cre-lox approach for transient transgene expression in neural precursor cells and long-term tracking of their progeny in vitro and in vivo

    PubMed Central

    Geoffroy, Cédric G; Raineteau, Olivier

    2007-01-01

    Background Neural precursor cells (NPCs) can be isolated from various regions of the postnatal central nervous system (CNS). Manipulation of gene expression in these cells offers a promising strategy to manipulate their fate both in vitro and in vivo. In this study, we developed a technique that allows the transient manipulation of single/multiple gene expression in NPCs in vitro, and the long-term tracking of their progeny both in vitro and in vivo. Results In order to combine the advantages of transient transfection with the long-term tracking of the transfected cells progeny, we developed a new approach based on the cre-lox technology. We first established a fast and reliable protocol to isolate and culture NPCs as monolayer, from the spinal cord of neonatal transgenic Rosa26-YFP cre-reporter mice. These cells could be reliably transfected with single/multiple plasmids by nucleofection. Nucleofection with mono- or bicistronic plasmids containing the Cre recombinase gene resulted in efficient recombination and the long-term expression of the YFP-reporter gene. The transient cre-expression was non-toxic for the transfected cells and did not alter their intrinsic properties. Finally, we demonstrated that cre-transfected cells could be transplanted into the adult brain, where they maintained YFP expression permitting long-term tracking of their migration and differentiation. Conclusion This approach allows single/multiple genes to be manipulated in NPCs, while at the same time allowing long-term tracking of the transfected cells progeny to be analyzed both in vitro and in vivo. PMID:17504531

  10. Ontogeny of the serotonergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas

    SciTech Connect

    D'Amato, R.J.; Blue, M.E.; Largent, B.L.; Lynch, D.R.; Ledbetter, D.J.; Molliver, M.E.; Snyder, S.H.

    1987-06-01

    The development of serotonergic innervation to rat cerebral cortex was characterized by immunohistochemical localization of serotonin combined with autoradiographic imaging of serotonin-uptake sites. In neonatal rat, a transient, dense, serotonergic innervation appears in all primary sensory areas of cortex. In somatosensory cortex, dense patches of serotonergic innervation are aligned with specialized cellular aggregates called barrels. The dense patches are not apparent after 3 weeks of age, and the serotonergic innervation becomes more uniform in adult neocortex. This precocious neonatal serotonergic innervation may play a transient physiologic role in sensory areas of cortex or may exert a trophic influence on the development of cortical circuitry and thalamocortical connections.

  11. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics

    PubMed Central

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-01-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3′-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  12. Differential regulation of mRNA stability controls the transient expression of genes encoding Drosophila antimicrobial peptide with distinct immune response characteristics.

    PubMed

    Wei, Youheng; Xiao, Qianghai; Zhang, Ting; Mou, Zongchun; You, Jia; Ma, Wei-Jun

    2009-10-01

    The tight regulation of transiently expressed antimicrobial peptides (AMPs) with a distinct antimicrobial spectrum and different expression kinetics contributes greatly to the properly regulated immune response for resistance to pathogens and for the maintenance of mutualistic microbiota in Drosophila. The important role of differential regulation of AMP expression at the posttranscriptional level needs to be elucidated. It was observed that the highly expressed Cecropin A1 (CecA1) mRNA encoding a broad antimicrobial spectrum AMP against both bacteria and fungi decayed more quickly than did the moderately expressed Diptericin mRNA encoding AMP against Gram negative bacteria. The mRNA stability of AMPs is differentially regulated and is attributed to the specific interaction between cis-acting ARE in 3'-UTR of AMP mRNA and the RNA destabilizing protein transactor Tis11 as shown in co-immunoprecipitation of the Tis11 RNP complex with CecA1 mRNA but not other AMP mRNA. The p38MAPK was further demonstrated to play a crucial role in stabilizing ARE-bearing mRNAs by inhibiting Tis11-mediated degradation in LPS induced AMP expression. This evidence suggests an evolutionarily conserved and functionally important molecular basis for and effective approach to exact control of AMP gene expression. These mechanisms thereby orchestrate a well balanced and dynamic antimicrobial spectrum of innate immunity to resist infection and maintain resident microbiota properly. PMID:19726583

  13. Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

    PubMed Central

    Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

    2013-01-01

    The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

  14. Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells

    PubMed Central

    Sadofsky, Laura R.; Sreekrishna, Koti T.; Lin, Yakang; Schinaman, Renee; Gorka, Kate; Mantri, Yogita; Haught, John Christian; Huggins, Thomas G.; Isfort, Robert J.; Bascom, Charles C.; Morice, Alyn H.

    2014-01-01

    Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1. PMID:24921186

  15. Cxcr4 is transiently expressed in both epithelial and mesenchymal compartments of nascent hair follicles but is not required for follicle formation.

    PubMed

    Sennett, Rachel; Rezza, Amélie; Dauber, Katherine L; Clavel, Carlos; Rendl, Michael

    2014-10-01

    Hair follicle (HF) morphogenesis relies on the coordinated exchange of signals between mesenchymal and epithelial compartments of embryonic skin. Chemokine receptor Cxcr4 expression was recently identified in dermal condensates (DCs) of nascent HFs, but its role in promoting HF morphogenesis remains unknown. Our analyses confirmed Cxcr4 expression in condensate cells, and additionally revealed transient Cxcr4 expression in incipient epithelial hair placodes. Placodal Cxcr4 appeared prior to detection in DCs, representing a switch of expression between epithelial and mesenchymal compartments. To explore the functional role of this receptor in both compartments for early HF formation, we conditionally ablated Cxcr4 with condensate-targeting Tbx18(cre) knock-in and epidermis-targeting Krt14-cre transgenic mice. Conditional knockouts for both crosses were viable throughout embryogenesis and into adulthood. Morphological and biochemical marker analyses revealed comparable numbers of HFs forming in knockout embryos compared to wild-type littermate controls in both cases, suggesting that neither dermal nor epithelial Cxcr4 expression is required for early HF morphogenesis. We conclude that Cxcr4 expression and chemokine signaling through this receptor in embryonic mouse skin is dispensable for HF formation. PMID:25066162

  16. Acute but not chronic exposure to abiotic stress results in transient reduction of expression levels of the transgene driven by the 35S promoter.

    PubMed

    Boyko, Alex; Molinier, Jean; Chatter, Waine; Laroche, André; Kovalchuk, Igor

    2010-02-28

    The transgenic plant performance depends on the stable expression of the integrated transgene. In this paper, we have analyzed the stability of the most frequently used constitutive promoter, the cauliflower mosaic virus (CaMV) 35S promoter. We used several independent Nicotiana tabacum lines transgenic for the luciferase (LUC) or green fluorescence protein (GFP) coding genes driven by the same 35S promoter. As an indication of the expression level, we measured the steady state RNA level, protein level and protein activity. Exposure of plants to an acute single dose of UVC, UVB or X-ray radiation resulted in a decrease of the transgene expression level, whereas exposure to high temperature increased it. In most of the cases, the expression changed at one to two hours post exposure and returned to normal at four hours. By contrast, plants germinated and grown in the presence of a low dose of either UVB radiation or CuSO(4) for two weeks did not show any changes in expression level. We conclude that although the expression level of the transgenes driven by the 35S promoter can be transiently altered by the acute exposure, no substantial changes occur upon constant low exposure. PMID:19800040

  17. Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

    PubMed Central

    Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

    2013-01-01

    The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

  18. Changes in synaptic plasticity and expression of glutamate receptor subunits in the CA1 and CA3 areas of the hippocampus after transient global ischemia.

    PubMed

    Han, Xin-Jia; Shi, Zhong-Shan; Xia, Luo-Xing; Zhu, Li-Hui; Zeng, Ling; Nie, Jun-Hua; Xu, Zao-Cheng; Ruan, Yi-Wen

    2016-07-01

    Excess glutamate release from the presynaptic membrane has been thought to be the major cause of ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while CA3 neurons remain relatively intact. This suggests that changes in the properties of pyramidal cells may be the main cause related to ischemic neuronal death. Our previous studies have shown that the densities of dendritic spines and asymmetric synapses in the CA1 area are increased at 12h and 24h after ischemia. In the present study, we investigated changes in synaptic structures in the CA3 area and compared the expression of glutamate receptors in the CA1 and CA3 hippocampal regions of rats after ischemia. Our results demonstrated that the NR2B/NR2A ratio became larger after ischemia although the expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival pathway) decreased in the CA1 area from 6h to 48h after reperfusion. Furthermore, expression of the GluR2 subunit (calcium impermeable) of the AMPA receptor class significantly decreased while the GluR1 subunit (calcium permeable) remained unchanged at the same examined reperfusion times, which subsequently caused an increase in the GluR1/GluR2 ratio. Despite these notable differences in subunit expression, there were no obvious changes in the density of synapses or expression of NMDAR and AMPAR subunits in the CA3 area after ischemia. These results suggest that delayed CA1 neuronal death may be related to the dramatic fluctuation in the synaptic structure and relative upregulation of NR2B and GluR1 subunits induced by transient global ischemia. PMID:27090818

  19. Transient Expression of Secretory IgA In Planta is Optimal Using a Multi-Gene Vector and may be Further Enhanced by Improving Joining Chain Incorporation

    PubMed Central

    Westerhof, Lotte B.; Wilbers, Ruud H. P.; van Raaij, Debbie R.; van Wijk, Christina Z.; Goverse, Aska; Bakker, Jaap; Schots, Arjen

    2016-01-01

    Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA. PMID:26793201

  20. Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines

    PubMed Central

    Ho, Tsui-Ting; He, Xiaolong; Mo, Yin-Yuan; Beck, William T

    2016-01-01

    The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance. PMID:27570640

  1. Transient resistance to DNA damaging agents is associated with expression of microRNAs-135b and -196b in human leukemia cell lines.

    PubMed

    Ho, Tsui-Ting; He, Xiaolong; Mo, Yin-Yuan; Beck, William T

    2016-01-01

    The acquisition of resistance to anticancer drugs is widely viewed as a key obstacle to successful cancer therapy. However, detailed knowledge of the initial molecular events in the response of cancer cells to these chemotherapeutic and stress responses, and how these lead to the development of chemoresistance, remains incompletely understood. Using microRNA array and washout and rechallenge experiments, we found that short term treatment of leukemia cells with etoposide led a few days later to transient resistance that was associated with a corresponding transient increase in expression of ABCB1 mRNA, as well as microRNA (miR)-135b and miR-196b. This phenomenon was associated with short-term exposure to genotoxic agents, such as etoposide, topotecan, doxorubicin and ionizing radiation, but not agents that do not directly damage DNA. Further, this appeared to be histiotype-specific, and was seen in leukemic cells, but not in cell lines derived from solid tumors. Treatment of leukemic cells with either 5-aza-deoxycytidine or tricostatin A produced similar increased expression of ABCB1, miR-135b, and miR-196b, suggesting a role for epigenetic regulation of this phenomenon. Bioinformatics analyses revealed that CACNA1E, ARHGEF2, PTK2, SIAH1, ARHGAP6, and NME4 may be involved in the initial events in the development of drug resistance following the upregulation of ABCB1, miR-135b and miR-196b. In summary, we report herein that short-term exposure of cells to DNA damaging agents leads to transient drug resistance, which is associated with elevations in ABCB1, miR-135b and miR-196b, and suggests novel components that may be involved in the development of anticancer drug resistance. PMID:27570640

  2. A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature-sensitive processes in plant-pathogen interactions.

    PubMed

    Del Toro, Francisco; Tenllado, Francisco; Chung, Bong-Nam; Canto, Tomas

    2014-10-01

    Localized expression of genes in plants from T-DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time-consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host-environment and host-pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T-DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus. PMID:24581046

  3. Highly efficient transient gene expression and gene targeting in primate embryonic stem cells with helper-dependent adenoviral vectors

    PubMed Central

    Suzuki, Keiichiro; Mitsui, Kaoru; Aizawa, Emi; Hasegawa, Kouichi; Kawase, Eihachiro; Yamagishi, Toshiyuki; Shimizu, Yoshihiko; Suemori, Hirofumi; Nakatsuji, Norio; Mitani, Kohnosuke

    2008-01-01

    Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey (Macaca fasicularis) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells. PMID:18768795

  4. Transient expression of the ectodomain of matrix protein 2 (M2e) of Avian Influenza A Virus in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously reported an expression system based on capsid protein gene (CP) of Cucumber mosaic virus (CMV) placed under transcriptional control of Potato virus X (PVX)-based vector. PVX-expressed CMV CP formed virus-like particles, which served as carriers for heterologous antigens of Ne...

  5. Creation of transgenic Brassica napus L. plants expressing human alpha 2b interferon gene.

    PubMed

    Sakhno, L O; Kvasko, O Y; Olevinska, Z M; Spivak, M Y; Kuchuk, M V

    2012-01-01

    Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacterium-mediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system--superoxide dismutase (SOD)--in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins. PMID:23285745

  6. rbcS SRS4 promoter from Glycine max and its expression activity in transgenic tobacco.

    PubMed

    Cui, X Y; Chen, Z Y; Wu, L; Liu, X Q; Dong, Y Y; Wang, F W; Li, H Y

    2015-01-01

    The regulatory region of the ribulose-1,5-bisphosphate carboxylase small subunit gene SRS4 from soybean (Glycine max) was cloned using TAIL-PCR and general PCR, and named the rbcS promoter. The promoter was fused with the GUS gene and introduced into Nicotiana tabacum via Agrobacterium-mediated leaf disk transformation. In 4-week-old transgenic tobacco plants, the highest GUS expression levels were observed in the leaves, GUS activity was 7.13- and 7.40-fold higher in leaves than in stems and roots, respectively. Moreover, GUS activity was stimulated by light. In conclusion, spatial and light regulation of the soybean rbcS promoter was observed in N. tabacum, thus illustrating a leaf-specific and light-induced promoter. PMID:26214418

  7. Engineering herbicide resistance in plants by expression of a detoxifying enzyme

    PubMed Central

    Block, M. De; Botterman, J.; Vandewiele, M.; Dockx, J.; Thoen, C.; Gosselé, V.; Movva, N. Rao; Thompson, C.; Montagu, M. Van; Leemans, J.

    1987-01-01

    Phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase in plants and is used as a non-selective herbicide. The bar gene which confers resistance in Streptomyces hygroscopicus to bialaphos, a tripeptide containing PPT, encodes a phosphinothricin acetyltransferase (PAT) (see accompanying paper). The bar gene was placed under control of the 35S promoter of the cauliflower mosaic virus and transferred to plant cells using Agrobacterium-mediated transformation. PAT was used as a selectable marker in protoplast co-cultivation. The chimeric bar gene was expressed in tobacco, potato and tomato plants. Transgenic plants showed complete resistance towards high doses of the commercial formulations of phosphinothricin and bialaphos. These data present a successful approach to obtain herbicide-resistant plants by detoxification of the herbicide. ImagesFig. 2.Fig. 4.Fig. 5. PMID:16453789

  8. Quinic acids from Aster caucasicus and from transgenic callus expressing a beta-amyrin synthase.

    PubMed

    Pecchia, Paola; Cammareri, Maria; Malafronte, Nicola; Consiglio, M Federica; Gualtieri, Maria Josefina; Conicella, Clara

    2011-11-01

    Several different classes of secondary metabolites, including flavonoids, triterpenoid saponins and quinic acid derivatives, are found in Aster spp. (Fam. Asteraceae). Several Aster compounds revealed biological as well as pharmacological activities. In this work, a phytochemical investigation of A. caucasicus evidenced the presence of quinic acid derivatives, as well as the absence of triterpene saponins. To combine in one species the production of different phytochemicals, including triterpenes, an Agrobacterium-mediated transformation of A. caucasicus was set up to introduce A. sedifolius beta-amyrin synthase (AsOXA1)-encoding gene under the control of the constitutive promoter CaMV35S. The quali-quantitative analysis of transgenic calli with ectopic expression of AsOXA1 showed, in one sample, a negligible amount of triterpene saponins combined with higher amount of quinic acid derivatives as compared with the wild type callus. PMID:22224284

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

    PubMed Central

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

    2013-01-01

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

  10. Expression of genes involved in the salicylic acid pathway in type h1 thioredoxin transiently silenced pepper plants during a begomovirus compatible interaction.

    PubMed

    Luna-Rivero, Marianne S; Hernández-Zepeda, Cecilia; Villanueva-Alonzo, Hernán; Minero-García, Yereni; Castell-González, Salvador E; Moreno-Valenzuela, Oscar A

    2016-04-01

    The type-h thioredoxins (TRXs) play a fundamental role in oxidative stress tolerance and defense responses against pathogens. In pepper plants, type-h TRXs participate in the defense mechanism against Cucumber mosaic virus. The goal of this study was to analyze the role of the CaTRXh1-cicy gene in pepper plants during compatible interaction with a DNA virus, the Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The effects of a transient silencing of the CaTRXh1-cicy gene in pepper plants wëre evaluated by observing the accumulation of viral DNA and the visible symptoms of pepper plants under different treatments. The accumulation of salicylic acid (SA) and the relative expression of the defense genes NPR1 and PR10 were also evaluated. Results showed that viral DNA accumulation was higher in transiently CaTRXh1-cicy silenced plants that were also infected with EuMV-YP. Symptoms in these plants were more severe compared to the non-silenced plants infected with EuMV-YP. The SA levels in the EuMV-YP-infected plants were rapidly induced at 1 h post infection (hpi) in comparison to the non-silenced plants inoculated with EuMV-YP. Additionally, in pepper plants infected with EuMV-YP, the expression of NPR1 decreased by up to 41 and 58 % at 28 days post infection (dpi) compared to the non-silenced pepper plants infected with only EuMV-YP and healthy non-inoculated pepper plants, respectively. PR10 gene expression decreased by up to 70 % at 28 dpi. Overall, the results indicate that the CaTRXh1-cicy gene participates in defense mechanisms during the compatible interaction of pepper plants with the EuMV-YP DNA virus. PMID:26606929

  11. Multiple human papillomavirus type 16 glucocorticoid response elements functional for transformation, transient expression, and DNA-protein interactions.

    PubMed Central

    Mittal, R; Pater, A; Pater, M M

    1993-01-01

    We have previously shown that human papillomavirus type 16 (HPV-16) can efficiently transform primary baby rat kidney cells in the presence of the steroid hormones progesterone and the glucocorticoid dexamethasone. To study this effect of hormone, different combinations of the previously identified glucocorticoid response element (GRE) at nucleotide 7640 of HPV-16 and the other two GREs that we have recently identified, at nucleotides 7385 and 7474, were mutated. The previously described GRE and the other two GREs were shown to be functional for the induction of transformation by dexamethasone. In addition, transient assays in cervical HeLa cells demonstrated the functional importance of the three individual GREs. Assays for in vitro interaction demonstrated the specific binding of a 97-kDa protein, the glucocorticoid receptor, to both recently identified HPV-16 GREs. Images PMID:8394465

  12. A quantitative analysis of the spatiotemporal pattern of transient receptor potential gene expression in the developing mouse cochlea.

    PubMed

    Asai, Yukako; Holt, Jeffrey R; Géléoc, Gwenaëlle S G

    2010-03-01

    TRP genes encode a diverse family of ion channels which have been implicated in many sensory functions. Because several TRP channels have similar properties to the elusive hair cell transduction channel, recent attention has focused on TRP gene expression in the inner ear. At least four TRP genes are known to be expressed in hair cells: TRPC3, TRPV4, TRPA1, and TRPML3. However, there is little evidence supporting any of these as a component of the transduction complex. Other less well-characterized TRP channels are expressed in the inner ear, in particular, within the organ of Corti. Because of their potential role in sensory function, we investigated the developmental expression of RNA that encodes all 33 TRP subunits as well as several splice variants. We designed a quantitative PCR screen using cochlear samples acquired before, during, and after the time when mechanotransduction is acquired in sensory hair cells (embryonic day 17 to postnatal day 8). Cochleas, which included the organ of Corti, stria vascularis, and Reissner's membrane, were subdivided into four equal quadrants which allowed for regional comparison during development. Expression of RNA transcripts that encoded 33 TRP subunits plus several splice forms and beta-actin were quantified in 28 samples for a total of 1,092 individual measurements, each done in triplicate. We detected RNA that encoded all TRP channels except two: TRPC7 and TRPM8. The largest changes in RNA expression were for TRPA1 (>100-fold), TRPP3 (>50-fold), and TRPC5.2 (>20-fold) which suggested that these subunits may contribute to normal cochlear function. Furthermore, the screen revealed TRPP3 and PKD1L3 RNA expression patterns that were correlated with the acquisition of sensory transduction in outer hair cells (Lelli et al., J Neurophysiol. 101:2961-2973, 2009). Numerous spatiotemporal expression gradients were identified many of which may contribute to the normal functional development of the mouse cochlea. PMID:19834762

  13. Identification of differentially expressed genes involved in transient regeneration of the neonatal C57BL/6J mouse heart by digital gene expression profiling.

    PubMed

    Liu, Ming; Zhu, Jin-Gai; Yu, Zhang-Bin; Song, Gui-Xian; Shen, Ya-Hui; Liu, Yao-Qiu; Zhu, Chun; Qian, Ling-Mei

    2014-06-01

    Accumulating evidence has revealed that the mammalian heart possesses a measurable capacity for renewal. Neonatal mice retain a regenerative capacity over a short time-frame (≤6 days), but this capacity is lost by 7 days of age. In the present study, differential gene expression profiling of mouse cardiac tissue was performed to further elucidate the mechanisms underlying this process. The global gene expression patterns of the neonatal C57BL/6J mouse heart were examined at three key time-points (1, 6 and 7 days old) using digital gene expression analysis. In the distribution of total clean tags, high-expression tags (>100 copies) were found to be predominant, whereas low expression tags (<5 copies) occupied the majority of distinct tag distributions. In total, 306 differentially expressed genes (DEGs) were detected in cardiac tissue, with the expression levels of 115 genes upregulated and those of 191 genes downregulated in 7-day-old mice compared with expression levels in 1- and 6-day-old mice, respectively. The expression levels of five DEGs were confirmed using quantitative polymerase chain reaction. Gene ontology analysis revealed a large proportion of DEGs distributed throughout the cell, and these DEGs were associated with binding as well as catalytic, hydrolase, transferase and molecular transducer activities. Furthermore, these genes were involved in cellular, metabolic and developmental processes, as well as biological regulation and signaling pathways. Pathway analysis identified the oxidative phosphorylation pathway to be the process most significantly putatively affected by the differential expression of these genes. These data provide the basis for future analysis of the gene expression patterns that regulate the molecular mechanism of cardiac regeneration. PMID:24699800

  14. Alterations in protein expression and membrane properties during Müller cell gliosis in a murine model of transient retinal ischemia.

    PubMed

    Hirrlinger, Petra G; Ulbricht, Elke; Iandiev, Ianors; Reichenbach, Andreas; Pannicke, Thomas

    2010-03-12

    Retinal Müller glial cells are involved in K+ ion homeostasis of the tissue. Inwardly rectifying K(+) (Kir) channels play a decisive role in the process of spatial K+ buffering. It has been demonstrated that Kir-mediated currents of Müller cells are downregulated in various cases of retinal neurodegeneration. However, this has not yet been verified for any murine animal model. The aim of the present study was to investigate Müller cells after transient retinal ischemia in mice. High intraocular pressure was applied for 1h; the retina was analysed 1 week later. We studied protein expression in the tissue by immunohistochemistry, and membrane currents of isolated cells by patch-clamp experiments. We found the typical indicators of reactive gliosis such as upregulation of glial fibrillary acidic protein. Moreover, the membrane capacitance of isolated Müller cells was increased and the amplitudes of Kir-mediated currents were slightly, but significantly decreased. This murine high intraocular pressure model of transient retinal ischemia is proposed as a versatile tool for further studies on Müller cell functions in retinal degeneration. PMID:20132867

  15. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  16. A Quantitative Analysis of the Spatiotemporal Pattern of Transient Receptor Potential Gene Expression in the Developing Mouse Cochlea

    PubMed Central

    Asai, Yukako; Holt, Jeffrey R.

    2009-01-01

    TRP genes encode a diverse family of ion channels which have been implicated in many sensory functions. Because several TRP channels have similar properties to the elusive hair cell transduction channel, recent attention has focused on TRP gene expression in the inner ear. At least four TRP genes are known to be expressed in hair cells: TRPC3, TRPV4, TRPA1, and TRPML3. However, there is little evidence supporting any of these as a component of the transduction complex. Other less well-characterized TRP channels are expressed in the inner ear, in particular, within the organ of Corti. Because of their potential role in sensory function, we investigated the developmental expression of RNA that encodes all 33 TRP subunits as well as several splice variants. We designed a quantitative PCR screen using cochlear samples acquired before, during, and after the time when mechanotransduction is acquired in sensory hair cells (embryonic day 17 to postnatal day 8). Cochleas, which included the organ of Corti, stria vascularis, and Reissner’s membrane, were subdivided into four equal quadrants which allowed for regional comparison during development. Expression of RNA transcripts that encoded 33 TRP subunits plus several splice forms and beta-actin were quantified in 28 samples for a total of 1,092 individual measurements, each done in triplicate. We detected RNA that encoded all TRP channels except two: TRPC7 and TRPM8. The largest changes in RNA expression were for TRPA1 (>100-fold), TRPP3 (>50-fold), and TRPC5.2 (>20-fold) which suggested that these subunits may contribute to normal cochlear function. Furthermore, the screen revealed TRPP3 and PKD1L3 RNA expression patterns that were correlated with the acquisition of sensory transduction in outer hair cells (Lelli et al., J Neurophysiol. 101:2961–2973, 2009). Numerous spatiotemporal expression gradients were identified many of which may contribute to the normal functional development of the mouse cochlea. Electronic

  17. Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing.

    PubMed

    Merlin, Matilde; Gecchele, Elisa; Arcalis, Elsa; Remelli, Sabrina; Brozzetti, Annalisa; Pezzotti, Mario; Avesani, Linda

    2016-03-01

    Plants have emerged as competitive production platforms for pharmaceutical proteins that are required in large quantities. One example is the 65-kDa isoform of human glutamic acid decarboxylase (GAD65), a major autoimmune diabetes autoantigen that has been developed as a vaccine candidate for the primary prevention of diabetes. The expression of GAD65 in plants has been optimized but large-scale purification is hampered by its tendency to associate with membranes. We investigated the potential for large-scale downstream processing by evaluating different combinations of plant-based expression systems and engineered forms of GAD65 in terms of yield, subcellular localization and solubility in detergent-free buffer. We found that a modified version of GAD65 lacking the first 87 amino acids accumulates to high levels in the cytosol and can be extracted in detergent-free buffer. The highest yields of this variant protein were achieved using the MagnICON transient expression system. This combination of truncated GAD65 and the MagnICON system dramatically boosts the production of the recombinant protein and helps to optimize downstream processing for the establishment of a sustainable plant-based production platform for an autoimmune diabetes vaccine candidate. PMID:26710327

  18. Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2).

    PubMed

    Darwich, L; Segalés, J; Resendes, A; Balasch, M; Plana-Durán, J; Mateu, E

    2008-04-01

    Immunological impairment by porcine circovirus type 2 (PCV2) infection is well documented in pigs suffering from postweaning multisystemic wasting syndrome. Nonetheless, little is known about immune status of pigs that remain PCV2 subclinically infected. Thus, seven pigs successfully infected in an experimental inoculation and without developing disease and nine control non-inoculated pigs were examined. Serological, virological and immunological determinations were done throughout ten weeks post-infection (PI). At week 3 PI, inoculated animals presented the peak of viremia and produced higher levels of IL-10 than the controls; correlation between viral load and IL-10 amounts was observed (p<0.05). Also, the ratio IgM/IgG suffered a shift skewing IgM production towards an IgG response. By 10 weeks PI, levels of IL-10 disappeared and the viremia decreased. In summary, subclinically PCV2-infected pigs developed a transient PCV2-specific IL-10 response during the viremic phase of infection which coincided with the inversion of the IgM/IgG ratio. PMID:17592737

  19. Expression and Activation of STAT3 in the Astrocytes of Optic Nerve in a Rat Model of Transient Intraocular Hypertension

    PubMed Central

    Zhang, Shaodan; Li, Weiyi; Wang, Wenqian; Zhang, Samuel S.; Huang, Ping; Zhang, Chun

    2013-01-01

    Lamina cribosa, an astrocyte-rich region, is the origin of axonal degeneration in glaucomatous neuropathy. Astrocytes are particularly activated during optic nerve (ON) degeneration and are likely to contribute to the pathogenesis of glaucomatous optic neuropathy. Signalling mechanisms that regulate different aspects of astrocyte reactiviation in response to intraocular hypertensive injury are not well defined. Signal transducer and activator of transcription protein-3 (STAT3) is a transcription factor that participates in many biological processes and has been implicated as activator of reactive astrogliosis. In this study, we investigated the role of STAT3 in regulating the activation of astrocytes to transient intraocular hypertension in vivo by using a rat ocular hypertension model. ON astrocytes hypertrophy was observed early after intraocular hypertensive stress. Morphological changes in glial fibrillary acidic protein (GFAP) positive cells coupled with axon loss in the optic nerve was detected at day 7 after the injury. Nestin was significantly upregulated in ON astrocytes as early as day 2 post injury and kept elevated through post injury day 7. Phosphorylated STAT3 (pSTAT3) was markedly upregulated in ON astrocytes at post injury day 1, prior to the reactivation of ON astrocytes. These findings indicate that STAT3 signalling is involved in the initiation of astrocyte reactivation in optic nerve injury. PMID:23383263

  20. Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta.

    PubMed

    Martin, Kathleen; Kopperud, Kristin; Chakrabarty, Romit; Banerjee, Rituparna; Brooks, Robert; Goodin, Michael M

    2009-07-01

    Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community. PMID:19309457

  1. Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

    PubMed Central

    Bae, Eun Joo; Chen, Bai Hui; Yan, Bing Chun; Shin, Bich Na; Cho, Jeong Hwi; Kim, In Hye; Ahn, Ji Hyeon; Lee, Jae Chul; Tae, Hyun-Jin; Hong, Seongkweon; Kim, Dong Won; Cho, Jun Hwi; Lee, Yun Lyul; Won, Moo-Ho; Park, Joon Ha

    2015-01-01

    The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults. PMID:26199612

  2. Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

    PubMed

    Fujiuchi, Naomichi; Matsuda, Ryo; Matoba, Nobuyuki; Fujiwara, Kazuhiro

    2016-04-01

    The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 μg gFW(-1), was comparable to the mean HA yield of 846 μg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory. PMID

  3. BMP4 Increases Canonical Transient Receptor Potential Protein Expression by Activating p38 MAPK and ERK1/2 Signaling Pathways in Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Li, Xiaoyan; Lu, Wenju; Fu, Xin; Zhang, Yi; Yang, Kai; Zhong, Nanshan; Ran, Pixin

    2013-01-01

    Abnormal bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of pulmonary hypertension. We previously found that BMP4 elevated basal intracellular Ca2+ ([Ca2+]i) concentrations in distal pulmonary arterial smooth muscle cells (PASMCs), attributable in large part to enhanced store-operated Ca2+ entry through store-operated Ca2+ channels (SOCCs). Moreover, BMP4 up-regulated the expression of canonical transient receptor potential (TRPC) proteins thought to compose SOCCs. The present study investigated the signaling pathways through which BMP4 regulates TRPC expression and basal [Ca2+]i in distal PASMCs. Real-time quantitative PCR was used for the measurement of mRNA, Western blotting was used for the measurement of protein, and fluorescent microscopic for [Ca2+]i was used to determine the involvement of p38 and extracellular regulated kinase (ERK)–1/2 mitogen-activated protein kinase (MAPK) signaling in BMP4–induced TRPC expression and the elevation of [Ca2+]i in PASMCs. We found that the treatment of BMP4 led to the activation of both p38 MAPK and ERK1/2 in rat distal PASMCs. The induction of TRPC1, TRPC4, and TRPC6 expression, and the increases of [Ca2+]i caused by BMP4 in distal PASMCs, were inhibited by treatment with either SB203580 (10 μM), the selective inhibitor for p38 activation, or the specific p38 small interfering RNA (siRNA). Similarly, those responses induced by BMP4 were also abolished by treatment with PD98059 (5 μM), the selective inhibitor of ERK1/2, or by the knockdown of ERK1/2 using its specific siRNA. These results indicate that BMP4 participates in the regulation of Ca2+ signaling in PASMCs by modulating TRPC channel expression via activating p38 and ERK1/2 MAPK pathways. PMID:23526217

  4. Region-specific changes in transient receptor potential vanilloid channel expression in the vasopressin magnocellular system in hepatic cirrhosis-induced hyponatraemia.

    PubMed

    Nedungadi, T P; Carreño, F R; Walch, J D; Bathina, C S; Cunningham, J T

    2012-04-01

    The present study aimed to measure the expression of transient receptor potential (TRP) channels in the magnocellular neurones of the paraventricular (PVN) and supraoptic nucleus (SON) in an animal model of hepatic cirrhosis associated with inappropriate vasopressin (AVP) release. In these studies, we used chronic bile duct ligation (BDL) in the rat, which is a commonly used model of hepatic cirrhosis, associated with elevated plasma AVP. The present study tested the hypothesis that changes in TRP vanilloid (TRPV) channel expression may be related to inappropriate AVP release in BDL rats. To test our hypothesis, we utilised laser capture microdissection of AVP neurones in the PVN and SON and western blot analysis from brain punches. Laser capture microdissection and quantitative reverse transcriptase-polymerase chain reaction demonstrated elevated TRPV2 mRNA in the PVN and SON of BDL compared to sham-ligated controls. AVP transcription was also increased as determined using intron specific primers to measure heteronuclear RNA. Immunohistochemistry demonstrated increased AVP and TRPV2 positive cells in both the PVN and SON after BDL. Also, there was an increased co-expression of TRPV2 and AVP cells after BDL. However, there was no change in the colocalisation counts of TRPV2 and oxytocin in both the magnocellular regions evaluated. In the SON but not the PVN, the transcription levels of TRPV4 were also significantly increased in BDL rats. Western blot analysis of punches containing the PVN and SON revealed that TRPV2 protein content was significantly increased in these brain regions in BDL rats compared to sham rats. Our data suggest that regionally specific changes in TRPV expression in the magnocellular neurosecretory cell AVP neurones could alter their osmosensing ability. PMID:22188460

  5. Transient performance

    NASA Astrophysics Data System (ADS)

    Curnock, Barry

    Gas turbine engine transient behavior, that which is concerned with the changes in engine parameters during acceleration or decceleration of an engine from one steady state point to a different steady state point, is considered. An engine can also experience cyclic aerodynamic phenomena which occur at a nominally steady condition; examples are compressor rotator stall and intake or afterburner buzz. The following are discussed: certification requirements; mechanism of acceleration; compressor working lines and surge; and some important factors (pressure level, moment of inertia, heat soakage, clearances, measurement of transients, thrust reversal, and transient maneuvers which involve significant changes to the shaft speeds of the engine). A set of graphics illustrating transient performance is presented.

  6. Transient developmental expression of IgY and secretory component like protein in the gut of the axolotl (Ambystoma mexicanum).

    PubMed

    Fellah, J S; Iscaki, S; Vaerman, J P; Charlemagne, J

    1992-01-01

    We previously reported that a primitive vertebrate, the Mexican axolotl (Amphibian, Urodela) synthesizes two classes of immunoglobulins. IgM are present in serum early in the development, and represent the bulk of specific antibody synthesis after an antigenic challenge. IgY occur in the serum later during the development, and are relatively insensitive to immunization. We demonstrate in the present work, using immunofluorescence with specific Mabs, that IgY are expressed in the gut epithelium, as secretory molecules. Secretory IgY are well expressed in the stomach and intestinal mucosae of young animals from 1 month after hatching to the seventh month. Thereafter, IgY progressively disappear from the gut and become readily detectable in the serum of 9-month-old preadult immunologically mature animals. Axolotl IgY are closely associated in the gut to secretory component-like (SC) molecules that are well-recognized by antisera to the SC of different mammalian species. This is the first description, in a primitive tetrapode, of an immunoglobulin class that could be the physiological counterpart of mammalian IgA. PMID:1627950

  7. The transient nature of Bunyamwera orthobunyavirus NSs protein expression: effects of increased stability of NSs protein on virus replication.

    PubMed

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein. PMID:23667701

  8. The Transient Nature of Bunyamwera Orthobunyavirus NSs Protein Expression: Effects of Increased Stability of NSs Protein on Virus Replication

    PubMed Central

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M.

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein. PMID:23667701

  9. Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

    PubMed Central

    Mignaqui, Ana Clara; Ruiz, Vanesa; Perret, Sylvie; St-Laurent, Gilles; Singh Chahal, Parminder; Transfiguracion, Julia; Sammarruco, Ayelén; Gnazzo, Victoria; Durocher, Yves; Wigdorovitz, Andrés

    2013-01-01

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV. PMID:23977353

  10. CHO-S Antibody Titers >1 Gram/Liter Using Flow Electroporation-Mediated Transient Gene Expression followed by Rapid Migration to High-Yield Stable Cell Lines

    PubMed Central

    Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-01-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities. PMID:25520372

  11. Up-Scaled Synthesis and Characterization of Nonviral Gene Delivery Particles for Transient In Vitro and In Vivo Transgene Expression.

    PubMed

    Taschauer, Alexander; Geyer, Antonia; Gehrig, Sebastian; Maier, Julia; Sami, Haider; Ogris, Manfred

    2016-06-01

    Polyethylenimine-based polyplexes are promising nonviral gene delivery systems for preclinical and clinical applications. Pipette-based polyplexing is associated with several disadvantages, such as batch-to-batch variability, restriction to smaller volumes, and variable gene delivery results. The present protocol describes syringe-pump-mediated upscaled synthesis of well-defined gene delivery nanoparticles capable of efficient in vitro and in vivo gene delivery. Syringe-pump-based synthesis ensures controlled mixing, upscaling, and reproducible gene delivery. Nanoparticle tracking analysis of the upscaled formulations involved single nanoparticle tracking, thereby generating highly resolved biophysical characterization. Gene delivery performance was investigated by luciferase gene expression in cells and three-dimensional bioluminescence imaging in mice. PMID:27169568

  12. Differences in the protein expression levels of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils following transient global cerebral ischemia.

    PubMed

    Lee, Choong Hyun; Park, Joon Ha; Cho, Jeong-Hwi; Ahn, Ji Hyeon; Bae, Eun Joo; Won, Moo-Ho

    2015-08-01

    The thioredoxin (Trx) and peroxiredoxin (Prx) redox system is associated with neuronal damage and neuroprotective effects via the regulation of oxidative stress in brain ischemia. In the present study, ischemia-induced changes in the protein expression levels of Trx2 and Prx3 in the stratum pyramidale (SP) of the hippocampal CA1 region were investigated in adult and aged gerbils, subjected to 5 min of transient global cerebral ischemia, using immunohistochemistry and western blot analysis. In the adult ischemia-group, minimal Trx2 immunoreactivity was detected in the SP 2 days after ischemia-reperfusion. In the aged animals, the Trx2 immunoreactivity in the sham-group was marginally lower compared with that in the adult sham-group. In the aged ischemia-group, Trx2 immunoreactivity in the SP was significantly higher 1, 2 and 4 days post-ischemia, compared with that in the adult ischemia-group and, in the 5 days post-ischemia group, Trx2 immunoreactivity was significantly decreased in the SP. Prx3 immunoreactivity in the SP of the adult ischemia-group was significantly decreased from 4 days after ischemia-reperfusion. In the aged animals, Prx3 immunoreactivity in the sham-group was also marginally lower compared with that in the adult sham-group. Prx3 immunoreactivity in the aged ischemia-group was also significantly higher 1, 2 and 4 days post-ischemia, compared with the adult ischemia-group; however, the Prx3 immunoreactivity was significantly decreased 5 days post-ischemia. The western blot analyses revealed that the pattern of changes in the protein levels of Trx2 and Prx3 in the adult and aged hippocampal CA1 region following ischemic damage were similar to the results obtained in the immunohistochemical data. These findings indicated that cerebral ischemia lead to different protein expression levels of Trx2 and Prx3 in the hippocampal CA1 region between adult and aged gerbils, and these differences may be associated with more delayed neuronal death in the aged

  13. Infantile 4-tert-octylphenol exposure transiently inhibits rat ovarian steroidogenesis and steroidogenic acute regulatory protein (StAR) expression

    SciTech Connect

    Myllymaeki, S.A. . E-mail: saanmy@utu.fi; Karjalainen, M.; Haavisto, T.E.; Toppari, J.; Paranko, J.

    2005-08-22

    Phenolic compounds, such as 4-tert-octylphenol (OP), have been shown to interfere with rat ovarian steroidogenesis. However, little is known about steroidogenic effects of infantile OP exposure on immature ovary. The aim of the present study was to investigate the effects of infantile OP exposure on plasma FSH, LH, estradiol, and progesterone levels in 14-day-old female rats. The effect on ovarian steroidogenic acute regulatory protein (StAR) and FSH receptor (FSHr) expression was analyzed by Western blotting. Ex vivo analysis was carried out for follicular estradiol, progesterone, testosterone, and cAMP production. Sprague-Dawley rats were given OP (0, 10, 50, or 100 mg/kg) subcutaneously on postnatal days 6, 8, 10, and 12. On postnatal day 14, plasma FSH was decreased and progesterone increased significantly at a dose of 100 mg OP/kg. In addition, the highest OP dose advanced the time of vaginal opening in puberty. OP had no effect on infantile LH and estradiol levels or ovarian FSHr content. Ovarian StAR protein content and ex vivo hormone and cAMP production were decreased at all OP doses compared to controls. However, hormone levels recovered independent on FSH and even increased above the control level during a prolonged culture. On postnatal day 35, no statistically significant differences were seen between control and OP-exposed animals in plasma FSH, LH, estradiol, and progesterone levels, or in ovarian StAR protein content. The results indicate that the effect of OP on the infantile ovary is reversible, while more permanent effects in the hypothalamus and pituitary, as described earlier, are involved in the reduction of circulating FSH levels and premature vaginal opening.

  14. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene.

    PubMed

    Fujii, N; Uchimiya, H

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5'-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial beta-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in beta-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of beta-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of beta-glucuronidase activities, in concord with embryogenesis, were also noted. PMID:16667958

  15. Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene 1

    PubMed Central

    Fujii, Nobuharu; Uchimiya, Hirofumi

    1991-01-01

    We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted. Images Figure 2 PMID:16667958

  16. CCR5 Gene Editing of Resting CD4+ T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice

    PubMed Central

    Yi, Guohua; Choi, Jang Gi; Bharaj, Preeti; Abraham, Sojan; Dang, Ying; Kafri, Tal; Alozie, Ogechika; Manjunath, Manjunath N; Shankar, Premlata

    2014-01-01

    CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4+ T cells. Both activated and resting primary CD4+ T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4+ T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN–transduced resting CD4+ T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy. PMID:25268698

  17. Co-expression of Foxa.a, Foxd and Fgf9/16/20 defines a transient mesendoderm regulatory state in ascidian embryos

    PubMed Central

    Hudson, Clare; Sirour, Cathy; Yasuo, Hitoyoshi

    2016-01-01

    In many bilaterian embryos, nuclear β-catenin (nβ-catenin) promotes mesendoderm over ectoderm lineages. Although this is likely to represent an evolutionary ancient developmental process, the regulatory architecture of nβ-catenin-induced mesendoderm remains elusive in the majority of animals. Here, we show that, in ascidian embryos, three nβ-catenin transcriptional targets, Foxa.a, Foxd and Fgf9/16/20, are each required for the correct initiation of both the mesoderm and endoderm gene regulatory networks. Conversely, these three factors are sufficient, in combination, to produce a mesendoderm ground state that can be further programmed into mesoderm or endoderm lineages. Importantly, we show that the combinatorial activity of these three factors is sufficient to reprogramme developing ectoderm cells to mesendoderm. We conclude that in ascidian embryos, the transient mesendoderm regulatory state is defined by co-expression of Foxa.a, Foxd and Fgf9/16/20. DOI: http://dx.doi.org/10.7554/eLife.14692.001 PMID:27351101

  18. Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease

    PubMed Central

    Becker, Lore; Hartenstein, Bettina; Schenkel, Johannes; Kuhse, Jochen; Betz, Heinrich; Weiher, Hans

    2000-01-01

    Startle disease or hereditary hyperekplexia has been shown to result from mutations in the α1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR α or β-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR β transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR β-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR β transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at ∼ 3–4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease. PMID:10651857

  19. Mild hypothermia causes differential, time-dependent changes in cytokine expression and gliosis following endothelin-1-induced transient focal cerebral ischemia

    PubMed Central

    2011-01-01

    Background Stroke is an important cause of morbidity and mortality and few therapies exist thus far. Mild hypothermia (33°C) is a promising neuroprotective strategy to improve outcome after ischemic stroke. However, its complete mechanism of action has not yet been fully elaborated. This study is the first to investigate whether this neuroprotection occurs through modulation of the neuroinflammatory response after stroke in a time-dependent manner. Methods The Endothelin-1 (Et-1) model was used to elicit a transient focal cerebral ischemia in male Wistar rats. In this model, the core and penumbra of the insult are represented by the striatum and the cortex respectively. We assessed the effects of 2 hours of hypothermia, started 20 minutes after Et-1 injection on neurological outcome and infarct volume. Furthermore, pro- and anti-inflammatory cytokine expression was determined using ELISA. Microgliosis and astrogliosis were investigated using CD-68 and GFAP staining respectively. All parameters were determined 8, 24, 72 hours and 1 week after the administration of Et-1. Results Et-1 infusion caused neurological deficit and a reproducible infarct size which increased up to 3 days after the insult. Both parameters were significantly reduced by hypothermia. The strongest reduction in infarct volume with hypothermia, at 3 days, corresponded with increased microglial activation. Reducing the brain temperature affected the stroke induced increase in interleukin-1β and tumor necrosis factor α in the striatum, 8 hours after its induction, but not at later time points. Transforming growth factor β increased as a function of time after the Et-1-induced insult and was not influenced by cooling. Hypothermia reduced astrogliosis at 1 and 3 days after stroke onset. Conclusions The beneficial effects of hypothermia after stroke on infarct volume and functional outcome coincide with a time-dependent modulation of the cytokine expression and gliosis. PMID:21627837

  20. Transient inactivation of the ventral tegmental area selectively disrupts the expression of conditioned place preference for pup- but not cocaine-paired contexts

    PubMed Central

    Seip, Katharine M.; Morrell, Joan I.

    2010-01-01

    The ventral tegmental area (VTA) plays a critical role in motivated behavior. However, it remains unclear whether intact VTA function is necessary for motivated behavior to seek contexts repeatedly paired with natural stimuli and/or pharmacological stimuli. In the present study, conditioned place preference (CPP) was induced using highly salient natural or drug stimuli attributed with strong incentive-motivational value in each of two female models: postpartum females were conditioned to associate one unique context in the CPP apparatus with young offspring (pups) and a second context with a neutral stimulus, and virgin females were conditioned to associate unique contexts with cocaine (5mg/kg i.p.) and saline injections. Immediately prior to CPP testing, each female received a microinfusion of bupivacaine bilaterally into the VTA to transiently inactivate the region; subjects were also tested following saline microinfusion into the VTA. Postpartum females’ preference for the pup-paired context was abolished by VTA inactivation but was restored to high control levels following saline microinfusion. In separate tests, VTA inactivation also reduced motivated pup licking and pup retrieval in postpartum females, suggesting that intact VTA function is required for the expression of both pup CPP and motivated pup-directed behaviors. Cocaine CPP remained unaffected by VTA inactivation. Locomotion was not affected by VTA microinfusions but was severely impaired by bupivacaine microinfusions into the substantia nigra. We conclude that the VTA is differentially involved in the expression of conditioned preference for contexts paired with pups, a salient natural stimulus, and contexts paired with cocaine. PMID:20001116

  1. Cell layer-specific distribution of transiently expressed barley ESCRT-III component HvVPS60 in developing barley endosperm.

    PubMed

    Hilscher, Julia; Kapusi, Eszter; Stoger, Eva; Ibl, Verena

    2016-01-01

    The significance of the endosomal sorting complexes required for transport (ESCRT)-III in cereal endosperm has been shown by the identification of the recessive mutant supernumerary aleurone layer1 (SAL1) in maize. ESCRT-III is indispensable in the final membrane fission step during biogenesis of multivesicular bodies (MVBs), responsible for protein sorting to vacuoles and to the cell surface. Here, we annotated barley ESCRT-III members in the (model) crop Hordeum vulgare and show that all identified members are expressed in developing barley endosperm. We used fluorescently tagged core ESCRT-III members HvSNF7a/CHMP4 and HvVPS24/CHMP3 and the associated ESCRT-III component HvVPS60a/CHMP5 for transient localization studies in barley endosperm. In vivo confocal microscopic analyses show that the localization of recombinantly expressed HvSNF7a, HvVPS24 and HvVPS60a differs within barley endosperm. Whereas HvSNF7a induces large agglomerations, HvVPS24 shows mainly cytosolic localization in aleurone and subaleurone. In contrast, HvVPS60a localizes strongly at the plasma membrane in aleurone. In subaleurone, HvVPS60a was found to a lesser extent at the plasma membrane and at vacuolar membranes. These results indicate that the steady-state association of ESCRT-III may be influenced by cell layer-specific protein deposition or trafficking and remodelling of the endomembrane system in endosperm. We show that sorting of an artificially mono-ubiquitinated Arabidopsis plasma membrane protein is inhibited by HvVPS60a in aleurone. The involvement of HvVPS60a in different cell layer-specific trafficking pathways, reflected by localization of HvVPS60a at the plasma membrane in aleurone and at the PSV membrane in subaleurone, is discussed. PMID:25796522

  2. Influenza virus-like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice.

    PubMed

    D'Aoust, Marc-André; Lavoie, Pierre-Olivier; Couture, Manon M-J; Trépanier, Sonia; Guay, Jean-Martin; Dargis, Michèle; Mongrand, Sébastien; Landry, Nathalie; Ward, Brian J; Vézina, Louis-P

    2008-12-01

    A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains. PMID:19076615

  3. Regulation of transient receptor potential vanilloid 1 expression in trigeminal ganglion neurons via methyl-CpG binding protein 2 signaling contributes tongue heat sensitivity and inflammatory hyperalgesia in mice

    PubMed Central

    Suzuki, Azumi; Honda, Kuniya; Shirakawa, Tetsuro; Iwata, Koichi

    2016-01-01

    Background Pain hypoalgesia has been reported in Rett syndrome patients, a severe neurodevelopmental disorder which can be attributed to mutations in the methyl-CpG binding protein 2 (MeCP2). Here, we examined the role of MeCP2 signaling in tongue heat sensitivity in the normal and inflamed state using Mecp2 heterozygous (Mecp2+/−) mice. Results Heat hypoalgesia of the tongue occurred in Mecp2+/− mice and submucosal injection of complete Freund’s adjuvant into the tongue produced a long-lasting heat hyperalgesia at the inflamed site in wild-type mice but not in Mecp2+/− mice. Transient receptor potential vanilloid 1 was expressed in a large number of MeCP2-immunoreactive trigeminal ganglion neurons innervating the tongue in both wild-type and Mecp2+/− mice (70.9% in wild type; 72.1% in Mecp2+/−). The number of transient receptor potential vanilloid 1-immunoreactive trigeminal ganglion neurons innervating the tongue was smaller in Mecp2+/− mice relative to wild-type mice (30.5% in wild type; 20.2% in Mecp2+/−). Following complete Freund’s adjuvant injection, the number of transient receptor potential vanilloid 1- and MeCP2-immunoreactive trigeminal ganglion neurons innervating the tongue, as well as MeCP2 protein expression in trigeminal ganglion, was significantly increased in wild-type mice but not in Mecp2+/− mice. Additionally, tongue heat hyperalgesia following complete Freund’s adjuvant injection was completely suppressed by the administration of SB366791, a transient receptor potential vanilloid 1 antagonist, in the tongue. Conclusions These findings indicate that tongue heat sensitivity and hypersensitivity are dependent on the expression of transient receptor potential vanilloid 1 which is regulated via MeCP2 signaling in trigeminal ganglion neurons innervating the tongue. PMID:27030715

  4. Proliferative events experimentally induced by a transient cold shock in the brain of adult terrestrial heterothermic vertebrates: preliminary analysis of PCNA expression in Podarcis sicula.

    PubMed

    Margotta, Vito

    2014-01-01

    In past studies on the encephalic regenerative phenomena some authors adopted a pre-surgi- cal stratagem (drastic, sudden, transient thermal stimulus) to adult brain-injured newts to limit death rate upon surgery and with this method, unexpected tissue reparation was obtained. This procedure became a routine technicality also in frog and lizard to stimulate an increase in the neurogenesis, attributable to putative stem cells which appear either in clusters ("matrix areas" or "matrix zones"), mostly at or near the telencephalic ventricular surfaces, or scattered ("matrix cells") within other cerebral districts. On the basis of this literature background, planning an immunocytochemical re-evaluation of the survival of latent proliferative properties in these adult cold shocked organisms, as already studied in Triturus carnifex, the actual investigation was carried out on the brain of Podarcis sicula not subjected to cerebral injury. The immunohistochem- ical expression of the proliferating cell nuclear antigen (PCNA) seemed moderate only in those encephalic portions better provided with cells in stand-by: the olfactory peduncles and the telen- cephalic zonae germinativae ventrales. This scenario appeared rather disappointed and inadequate with respect to the unexpected, widespread restoration of the removed portions observed by previous authors. These findings could be due both to a small effectiveness of a relatively mild thermal stress, depending on the difference between the applied temperature and seasonal one, to the lack of other experimental conditions (surgical trauma, encephalic injury) adopted in the past, and to the interspecific differences in sensitivity, since lacertilian Reptiles are less endowed with proliferative/regenerative power than Amphibians, mainly Urodela, and Teleosts. PMID:25665278

  5. Expression of store-operated Ca2+ entry and transient receptor potential canonical and vanilloid-related proteins in rat distal pulmonary venous smooth muscle.

    PubMed

    Peng, Gongyong; Lu, Wenju; Li, Xiaoyan; Chen, Yuqin; Zhong, Nanshan; Ran, Pixin; Wang, Jian

    2010-11-01

    Chronic hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins. Although pulmonary arteries have been studied extensively in these disorders, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension remain poorly understood. In pulmonary arterial smooth muscle, we have previously demonstrated that influx of Ca(2+) through store-operated calcium channels (SOCC) thought to be composed of transient receptor potential (TRP) proteins is likely to play an important role in development of chronic hypoxic pulmonary hypertension. To determine whether this mechanism could also be operative in pulmonary venous smooth muscle, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) by fura-2 fluorescence microscopy in primary cultures of pulmonary venous smooth muscle cells (PVSMC) isolated from rat distal pulmonary veins. In cells perfused with Ca(2+)-free media containing cyclopiazonic acid (10 μM) and nifedipine (5 μM) to deplete sarcoplasmic reticulum Ca(2+) stores and block voltage-dependent Ca(2+) channels, restoration of extracellular Ca(2+) (2.5 mM) caused marked increases in [Ca(2+)](i), whereas MnCl(2) (200 μM) quenched fura-2 fluorescence, indicating store-operated Ca(2+) entry (SOCE). SKF-96365 and NiCl(2), antagonists of SOCC, blocked SOCE at concentrations that did not alter Ca(2+) responses to 60 mM KCl. Of the seven known canonical TRP (TRPC1-7) and six vanilloid-related TRP channels (TRPV1-6), real-time PCR revealed mRNA expression of TRPC1 > TRPC6 > TRPC4 > TRPC2 ≈ TRPC5 > TRPC3, TRPV2 > TRPV4 > TRPV1 in distal PVSMC, and TRPC1 > TRPC6 > TRPC3 > TRPC4 ≈ TRPC5, TRPV2 ≈ TRPV4 > TRPV1 in rat distal pulmonary vein (PV) smooth muscle. Western blotting confirmed protein expression of TRPC1, TRPC6, TRPV2, and TRPV4 in both PVSMC and PV. Our results suggest that SOCE through Ca(2+) channels composed of TRP proteins may

  6. Transient nucleation in condensed systems

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.; Greer, A. L.; Thompson, C. V.

    1983-01-01

    Using classical nucleation theory we consider transient nucleation occurring in a one-component, condensed system under isothermal conditions. We obtain an exact closed-form expression for the time dependent cluster populations. In addition, a more versatile approach is developed: a numerical simulation technique which models directly the reactions by which clusters are produced. This simulation demonstrates the evolution of cluster populations and nucleation rate in the transient regime. Results from the simulation are verified by comparison with exact analytical solutions for the steady state. Experimental methods for measuring transient nucleation are assessed, and it is demonstrated that the observed behavior depends on the method used. The effect of preexisting cluster distributions is studied. Previous analytical and numerical treatments of transient nucleation are compared to the solutions obtained from the simulation. The simple expressions of Kashchiev are shown to give good descriptions of the nucleation behavior.

  7. Repression of global protein synthesis by Eif1a-like genes that are expressed specifically in the two-cell embryos and the transient Zscan4-positive state of embryonic stem cells.

    PubMed

    Hung, Sandy S C; Wong, Raymond C B; Sharov, Alexei A; Nakatake, Yuhki; Yu, Hong; Ko, Minoru S H

    2013-08-01

    Mouse embryonic stem (ES) cells are prototypical stem cells that remain undifferentiated in culture for long periods, yet maintain the ability to differentiate into essentially all cell types. Previously, we have reported that ES cells oscillate between two distinct states, which can be distinguished by the transient expression of Zscan4 genes originally identified for its specific expression in mouse two-cell stage embryos. Here, we report that the nascent protein synthesis is globally repressed in the Zscan4-positive state of ES cells, which is mediated by the transient expression of newly identified eukaryotic translation initiation factor 1A (Eif1a)-like genes. Eif1a-like genes, clustered on Chromosome 12, show the high sequence similarity to the Eifa1 and consist of 10 genes (Eif1al1-Eif1al10) and 9 pseudogenes (Eif1al-ps1-Eif1al-ps9). The analysis of the expressed sequence tag database showed that Eif1a-like genes are expressed mostly in the two-cell stage mouse embryos. Microarray analyses and quantitative real-time polymerase chain reaction analyses show that Eif1a-like genes are expressed specifically in the Zscan4-positive state of ES cells. These results indicate a novel mechanism to repress protein synthesis by Eif1a-like genes and a unique mode of protein synthesis regulation in ES cells, which undergo a transient and reversible repression of global protein synthesis in the Zscan4-positive state. PMID:23649898

  8. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene.

    PubMed

    Wang, Lin; Samac, Deborah A; Shapir, Nir; Wackett, Lawrence P; Vance, Carroll P; Olszewski, Neil E; Sadowsky, Michael J

    2005-09-01

    Atrazine is one of the most widely used herbicides in the USA. Atrazine chlorohydrolase (AtzA), the first enzyme in a six-step pathway leading to the mineralization of atrazine in Gram-negative soil bacteria, catalyses the hydrolytic dechlorination and detoxification of atrazine to hydroxyatrazine. In this study, we investigated the potential use of transgenic plants expressing atzA to take up, dechlorinate and detoxify atrazine. Alfalfa, Arabidopsis thaliana and tobacco were transformed with a modified bacterial atzA gene, p-atzA, under the control of the cassava vein mosaic virus promoter. All transgenic plant species actively expressed p-atzA and grew over a wide range of atrazine concentrations. Thin layer chromatography analyses indicated that in planta expression of p-atzA resulted in the production of hydroxyatrazine. Hydroponically grown transgenic tobacco and alfalfa dechlorinated atrazine to hydroxyatrazine in leaves, stems and roots. Moreover, p-atzA was found to be useful as a conditional-positive selection system to isolate alfalfa and Arabidopsis transformants following Agrobacterium-mediated transformation. Our work suggests that the in planta expression of p-atzA may be useful for the development of plants for the phytoremediation of atrazine-contaminated soils and soil water, and as a marker gene to select for the integration of exogenous DNA into the plant genome. PMID:17173634

  9. The expression of analgesic-antitumor peptide (AGAP) from Chinese Buthus martensii Karsch in transgenic tobacco and tomato.

    PubMed

    Lai, Linlin; Huang, Tingting; Wang, Yi; Liu, Yanfeng; Zhang, Jinghai; Song, Yongbo

    2009-05-01

    The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze-thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants. PMID:18546060

  10. Cloning and expression analysis of a stress-induced GmIMT1 gene in soybean (Glycine max).

    PubMed

    Wang, H T; Guo, N; Zhao, J M; Karthikeyan, A; Xue, D; Xue, C C; Xu, J Y; Xu, Z H; Gai, J Y; Xing, H

    2014-01-01

    Here, we aimed to clone and identify the GmIMT1 gene related to the salt stress response in soybean. The full-length cDNA sequence of the GmIMT1 gene was amplified in soybean using degenerate primers of Mesembrythmum crystallium. To understand the stress response, the GmIMT1 gene was cloned and sequenced. Then, the expression vectors of the gene were constructed, and introduced into the model plant Arabidopsis thaliana through Agrobacterium mediated transformation, and the salt tolerance was analyzed in the transgenic plants. In addition, the expression patterns of GmIMT1 gene in soybean were analyzed. The expression was examined in different organs (roots, leaves, flower seeds, and stem) and under different stress conditions (drought, high salt, low temperature, salicylic acid, ethane, abscisic acid, and methyl jasmonate) by real-time fluorescent quantitative polymerase chain reaction analysis. The results showed that the root, leaves, and stems exhibited high level of GmIMT1 gene expression, whereas there was no expression in the seeds. In addition, the GmIMT1 gene expression was upregulated under all stress conditions. Overall, the results clearly indicate that GmIMT1 might be involved in multiple plant response pathways to the different environmental conditions. Furthermore transgenic plants exhibited higher salt-tolerance compared to wild type plants. PMID:24615045

  11. Modification of oil and glucosinolate content in canola seeds with altered expression of Brassica napus LEAFY COTYLEDON1.

    PubMed

    Elahi, Nosheen; Duncan, Robert W; Stasolla, Claudio

    2016-03-01

    Over the last few decades, research focusing on canola (Brassica napus L.) seed oil content and composition has expanded. Oil production and accumulation are influenced by genes participating in embryo and seed development. The Arabidopsis LEAFY COTYLEDON1 (LEC1) is a well characterized regulator of embryo development that also enhances the expression of genes involved in fatty acid (FA) synthesis. B. napus lines over-expressing or down-regulating BnLEC1 were successfully generated by Agrobacterium-mediated transformation. The constitutive expression of BnLEC1 in B. napus var. Polo, increased seed oil content by 7-16%, while the down-regulation of BnLEC1 in B. napus var. Topas reduced oil content by 9-12%. Experimental manipulation of BnLEC1 caused transcriptional changes in enzymes participating in sucrose metabolism, glycolysis, and FA biosynthesis, suggesting an enhanced carbon flux towards FA biosynthesis in tissues over-expressing BnLEC1. The increase in oil content induced by BnLEC1 was not accompanied by alterations in FA composition, oil nutritional value or glucosinolate (GLS) levels. Suppression of BnLEC1 reduced seed oil accumulation and elevated the level of GLS possibly through the transcriptional regulation of BnST5a (Sulphotransferase5a), the last GLS biosynthetic enzyme. Collectively, these findings demonstrate that experimental alterations of BnLEC1 expression can be used to influence oil production and quality in B. napus. PMID:26773545

  12. Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen.

    PubMed

    Lee, H J; Lee, S B; Chung, J S; Han, S U; Han, O; Guh, J O; Jeon, J S; An, G; Back, K

    2000-06-01

    Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed. PMID:10945344

  13. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

    PubMed Central

    Bertran, J; Miller, J L; Yang, Y; Fenimore-Justman, A; Rueda, F; Vanin, E F; Nienhuis, A W

    1996-01-01

    Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. PMID:8794313

  14. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    SciTech Connect

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  15. Expression of ZmGA20ox cDNA alters plant morphology and increases biomass production of switchgrass (Panicum virgatum L.).

    PubMed

    Do, Phat T; De Tar, Joann R; Lee, Hyeyoung; Folta, Michelle K; Zhang, Zhanyuan J

    2016-07-01

    Switchgrass (Panicum virgatum L.) is considered a model herbaceous energy crop for the USA, for its adaptation to marginal land, low rainfall and nutrient-deficient soils; however, its low biomass yield is one of several constraints, and this might be rectified by modulating plant growth regulator levels. In this study, we have determined whether the expression of the Zea mays gibberellin 20-oxidase (ZmGA20ox) cDNA in switchgrass will improve biomass production. The ZmGA20ox gene was placed under the control of constitutive CaMV35S promoter with a strong TMV omega enhancer, and introduced into switchgrass via Agrobacterium-mediated transformation. The transgene integration and expression levels of ZmGA20ox in T0 plants were analysed using Southern blot and qRT-PCR. Under glasshouse conditions, selected transgenic plants exhibited longer leaves, internodes and tillers, which resulted in twofold increased biomass. These phenotypic alterations correlated with the levels of transgene expression and the particular gibberellin content. Expression of ZmGA20ox also affected the expression of genes coding for key enzymes in lignin biosynthesis. Our results suggest that the employment of ectopic ZmGA20ox and selection for natural variants with high level expression of endogenous GA20ox are appropriate approaches to increase biomass production of switchgrass and other monocot biofuel crops. PMID:26801525

  16. Stimulatory and inhibitory mechanisms of slow muscle-specific myosin heavy chain gene expression in fish: Transient and transgenic analysis of torafugu MYH{sub M86-2} promoter in zebrafish embryos

    SciTech Connect

    Asaduzzaman, Md.; Kinoshita, Shigeharu; Bhuiyan, Sharmin Siddique; Asakawa, Shuichi; Watabe, Shugo

    2013-04-01

    The myosin heavy chain gene, MYH{sub M86-2}, exhibited restricted expression in slow muscle fibers of torafugu embryos and larvae, suggesting its functional roles for embryonic and larval muscle development. However, the transcriptional mechanisms involved in its expression are still ambiguous. The present study is the first extensive analysis of slow muscle-specific MYH{sub M86-2} promoter in fish for identifying the cis-elements that are crucial for its expression. Combining both transient transfection and transgenic approaches, we demonstrated that the 2614 bp 5′-flanking sequences of MYH{sub M86-2} contain a sufficient promoter activity to drive gene expression specific to superficial slow muscle fibers. By cyclopamine treatment, we also demonstrated that the differentiation of such superficial slow muscle fibers depends on hedgehog signaling activity. The deletion analyses defined an upstream fragment necessary for repressing ectopic MYH{sub M86-2} expression in the fast muscle fibers. The transcriptional mechanism that prevents MYH{sub M86-2} expression in the fast muscle fibers is mediated through Sox6 binding elements. We also demonstrated that Sox6 may function as a transcriptional repressor of MYH{sub M86-2} expression. We further discovered that nuclear factor of activated T cells (NFAT) binding elements plays a key role and myocyte enhancer factor-2 (MEF2) binding elements participate in the transcriptional regulation of MYH{sub M86-2} expression. - Highlights: ► MYH{sub M86-2} is highly expressed in slow muscle fibers of torafugu embryos and larvae. ► MYH{sub M86-2} promoter activity depends on the hedgehog signaling. ► Sox6 binding elements inhibits MYH{sub M86-2} expression in fast muscle fibers. ► Sox6 elements function as transcriptional repressor of MYH{sub M86-2} promoter activity. ► NFAT and MEF2 binding elements play a key role for directing MYH{sub M86-2} expression.

  17. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene.

    PubMed

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-06-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  18. 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

  19. Development and bioassay of transgenic Chinese cabbage expressing potato proteinase inhibitor II gene

    PubMed Central

    Zhang, Junjie; Liu, Fan; Yao, Lei; Luo, Chen; Yin, Yue; Wang, Guixiang; Huang, Yubi

    2012-01-01

    Lepidopteran larvae are the most injurious pests of Chinese cabbage production. We attempted the development of transgenic Chinese cabbage expressing the potato proteinase inhibitor II gene (pinII) and bioassayed the pest-repelling ability of these transgenic plants. Cotyledons with petioles from aseptic seedlings were used as explants for Agrobacterium-mediated in vitro transformation. Agrobacterium tumefaciens C58 contained the binary vector pBBBasta-pinII-bar comprising pinII and bar genes. Plants showing vigorous PPT resistance were obtained by a series concentration selection for PPT resistance and subsequent regeneration of leaf explants dissected from the putative chimera. Transgenic plants were confirmed by PCR and genomic Southern blotting, which showed that the bar and pinII genes were integrated into the plant genome. Double haploid homozygous transgenic plants were obtained by microspore culture. The pinII expression was detected using quantitative real time polymerase chain reaction (qRT-PCR) and detection of PINII protein content in the transgenic homozygous lines. Insect-feeding trials using the larvae of cabbage worm (Pieris rapae) and the larvae of the diamondback moth (Plutella xylostella) showed higher larval mortality, stunted larval development, and lower pupal weights, pupation rates, and eclosion rates in most of the transgenic lines in comparison with the corresponding values in the non-transformed wild-type line. PMID:23136521

  20. [Expression of cecropin P1 gene increases resistance of Camelina sativa (L.) plants to microbial phytopathogenes].

    PubMed

    Zakharchenko, N S; Kaliaeva, M A; Bur'ianov, Ia I

    2013-05-01

    Transgenic plants of camelina (Camelina sativa (L.) Crantz) with the synthetic gene of antimicrobial peptide cecropin P1 (cecP1) were obtained. Agrobacterium-mediated transformation is performed using the binary vector pGA482::cecP1 by vacuum infiltration of flower buds. The presence of the cecP1 gene in the genome of plants was confirmed by PCR. cecP1 gene expression in transgenic plants was shown by Western blot analysis and by antimicrobial activity of plant extracts against the bacterial phytopathogene Erwinia carotovora. The plants of F0 and F1 generations had the normal phenotype and retained the ability to form viable seeds in self-pollination. cecP1 plants exhibit enhanced resistance to bacterial and fungal phytopathogens: Erwinia carotovora and Fusarium sporotrichioides. The increased sustainability of cecropin P1-expressing plants against salt stress is shown. The possibility of the integration of the cecP1 gene into the overall protective system of plants against biotic and abiotic stresses is discussed. PMID:24159802

  1. Preservation and Faithful Expression of Transgene via Artificial Seeds in Alfalfa

    PubMed Central

    Liu, Wenting; Liang, Zongsuo; Wang, Xinhua; Sibbald, Susan; Hunter, David; Tian, Lining

    2013-01-01

    Proper preservation of transgenes and transgenic materials is important for wider use of transgenic technology in plants. Here, we report stable preservation and faithful expression of a transgene via artificial seed technology in alfalfa. DNA constructs containing the uid reporter gene coding for β-glucuronidase (GUS) driven by a 35S promoter or a tCUP promoter were introduced into alfalfa via Agrobacterium-mediated genetic transformation. Somatic embryos were subsequently induced from transgenic alfalfa plants via in vitro technology. These embryos were treated with abscisic acid to induce desiccation tolerance and were subjected to a water loss process. After the desiccation procedure, the water content in dried embryos, or called artificial seeds, was about 12–15% which was equivalent to that in true seeds. Upon water rehydration, the dried somatic embryos showed high degrees of viability and exhibited normal germination. Full plants were subsequently developed and recovered in a greenhouse. The progeny plants developed from artificial seeds showed GUS enzyme activity and the GUS expression level was comparable to that of plants developed from somatic embryos without the desiccation process. Polymerase chain reaction analysis indicated that the transgene was well retained in the plants and Southern blot analysis showed that the transgene was stably integrated in plant genome. The research showed that the transgene and the new trait can be well preserved in artificial seeds and the progeny developed. The research provides a new method for transgenic germplasm preservation in different plant species. PMID:23690914

  2. Changes in Gene Expression Patterns of Circadian-Clock, Transient Receptor Potential Vanilloid-1 and Nerve Growth Factor in Inflamed Human Esophagus

    PubMed Central

    Yang, Shu-Chuan; Chen, Chien-Lin; Yi, Chih-Hsun; Liu, Tso-Tsai; Shieh, Kun-Ruey

    2015-01-01

    Circadian rhythm is driven by the molecular circadian-clock system and regulates many physiological functions. Diurnal rhythms in the gastrointestinal tract are known to be related to feeding pattern, but whether these rhythms are also related to the gastrointestinal damage or injuries; for example, gastroesophageal reflux disease (GERD), is unclear. This study was conducted to determine whether expression of circadian-clock genes or factors involved in vagal stimulation or sensitization were altered in the esophagus of GERD patients. Diurnal patterns of PER1, PER2, BMAL1, CRY2, TRPV1, and NGF mRNA expression were found in patient controls, and these patterns were altered and significantly correlated to the GERD severity in GERD patients. Although levels of CRY1, TIM, CB1, NHE3, GDNF, and TAC1 mRNA expression did not show diurnal patterns, they were elevated and also correlated with GERD severity in GERD patients. Finally, strong correlations among PER1, TRPV1, NGF and CRY2 mRNA expression, and among PER2, TRPV1 and CRY2 expression were found. Expression levels of CRY1 mRNA highly correlated with levels of TIM, CB1, NHE3, GDNF and TAC1. This study suggests that the circadian rhythm in the esophagus may be important for the mediation of and/or the response to erosive damage in GERD patients. PMID:26337663

  3. An Upregulation in the Expression of Vanilloid Transient Potential Channels 2 Enhances Hypotonicity-Induced Cytosolic Ca2+ Rise in Human Induced Pluripotent Stem Cell Model of Hutchinson Gillford Progeria

    PubMed Central

    Ho, Jenny Chung-Yee; Siu, Chung-Wah; Cheung, Sin-Ying; Tang, Nelson L.; Yu, Shan; Tse, Hung-Fat; Yao, Xiaoqiang

    2014-01-01

    Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca2+ ([Ca2+]i) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca2+]i rise in iPSC-ECs from normal individuals but a sustained [Ca2+]i elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca2+]i rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca2+]i rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca2+]i elevation in HGPS-iPSC-ECs under hypotonicity, consequently

  4. Schisandrin B inhibits the proliferation of airway smooth muscle cells via microRNA-135a suppressing the expression of transient receptor potential channel 1.

    PubMed

    Zhang, Xiao-Yu; Zhang, Luo-Xian; Guo, Ya-Li; Zhao, Li-Min; Tang, Xue-Yi; Tian, Cui-Jie; Cheng, Dong-Jun; Chen, Xian-Liang; Ma, Li-Jun; Chen, Zhuo-Chang

    2016-07-01

    Airway smooth muscle cell (ASMC) was known to involve in the pathophysiology of asthma. Schisandrin B was reported to have anti-asthmatic effects in a murine asthma model. However, the molecular mechanism involving in the effect of Schisandrin B on ASMCs remains poorly understood. Sprague-Dawley rats were divided into three groups: rats as the control (Group 1), sensitized rats (Group 2), sensitized rats and intragastric-administrated Schisandrin B (Group 3). The expression of miR-135a and TRPC1 was detected in the rats from three groups. Platelet-derived growth factor (PDGF)-BB was used to induce the proliferation of isolated ASMCs, and the expression of miR-135a and TRPC1 was detected in PDGF-BB-treated ASMCs. Cell viability was examined in ASMCs transfected with miR-135a inhibitor or si-TRPC1. The expression of TRPC1 was examined in A10 cells pretreated with miR-135a inhibitor or miR-135a mimic. In this study, we found that Schisandrin B attenuated the inspiratory and expiratory resistances in sensitized rats. Schisandrin B upregulated the mRNA level of miR-135a and decreased the expression of TRPC1 in sensitized rats. In addition, Schisandrin B reversed the expression of miR-135a and TRPC1 in PDGF-BB-induced ASMCs. Si-TRPC1 abrogated the increasing proliferation of ASMCs induced by miR-135a inhibitor. We also found that miR-135a regulated the expression of TRPC1 in the A10 cells. These results demonstrate that Schisandrin B inhibits the proliferation of ASMCs via miR-135a suppressing the expression of TRPC1. PMID:26916957

  5. Expression and Functional Characterization of the Agrobacterium VirB2 Amino Acid Substitution Variants in T-pilus Biogenesis, Virulence, and Transient Transformation Efficiency

    PubMed Central

    Wu, Hung-Yi; Chen, Chao-Ying; Lai, Erh-Min

    2014-01-01

    Agrobacterium tumefaciens is a phytopathogenic bacterium that causes crown gall disease by transferring transferred DNA (T-DNA) into the plant genome. The translocation process is mediated by the type IV secretion system (T4SS) consisting of the VirD4 coupling protein and 11 VirB proteins (VirB1 to VirB11). All VirB proteins are required for the production of T-pilus, which consists of processed VirB2 (T-pilin) and VirB5 as major and minor subunits, respectively. VirB2 is an essential component of T4SS, but the roles of VirB2 and the assembled T-pilus in Agrobacterium virulence and the T-DNA transfer process remain unknown. Here, we generated 34 VirB2 amino acid substitution variants to study the functions of VirB2 involved in VirB2 stability, extracellular VirB2/T-pilus production and virulence of A. tumefaciens. From the capacity for extracellular VirB2 production (ExB2+ or ExB2−) and tumorigenesis on tomato stems (Vir+ or Vir−), the mutants could be classified into three groups: ExB2−/Vir−, ExB2−/Vir+, and ExB2+/Vir+. We also confirmed by electron microscopy that five ExB2−/Vir+ mutants exhibited a wild-type level of virulence with their deficiency in T-pilus formation. Interestingly, although the five T-pilus−/Vir+ uncoupling mutants retained a wild-type level of tumorigenesis efficiency on tomato stems and/or potato tuber discs, their transient transformation efficiency in Arabidopsis seedlings was highly attenuated. In conclusion, we have provided evidence for a role of T-pilus in Agrobacterium transformation process and have identified the domains and amino acid residues critical for VirB2 stability, T-pilus biogenesis, tumorigenesis, and transient transformation efficiency. PMID:24971727

  6. GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is Regulated by a Novel NOBOX Transcript

    PubMed Central

    Bayne, Rosemary A. L.; Kinnell, Hazel L.; Coutts, Shiona M.; He, Jing; Childs, Andrew J.; Anderson, Richard A.

    2015-01-01

    During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations. PMID:25790371

  7. Over-expression of fHbp in Arabdopsis for development of meningococcal serogroup B subunit vaccine.

    PubMed

    Xu, Nuo; Wang, Yunpeng; Ma, Jisheng; Jin, Libo; Xing, Shaochen; Jiang, Chao; Li, Xiaokun

    2016-07-01

    Due to lack of commercial vaccine against the serogroup B (MenB) of Neisseria meningitides, the incidence of meningococcal disease remains high. To solve the issue, transgenic plants are used as bioreactors to produce a plant-derived fHbp subunit vaccine. In this study, the fHbp gene was optimized according to the codon usage bias of Arabidopsis thaliana, synthesized artificially, cloned into an expression vector, driven by a seed-specific promoter, and introduced into A. thaliana by Agrobacterium-mediated floral-dip transformation. Transgenic plants were identified by glufosinate selection, quickstix strips for PAT/bar tests and PCR analysis. The five plants showing higher expression of recombinant fHbp were screened through indirect ELISA. Southern blot analysis showed that the transgenic line rHF-22 had a single-copy integration and the highest expression of fHbp. Recombinant fHbp was purified from seeds of rHF-22 by nitrilotriacetic acid-mediated affinity chromatography, and the purity was 82.5%. BALB/c mice were tested for fHbp vaccine protection from lethal MenB infection, and the relative percent survival was found to be 80%. This study indicates that the recombinant fHbp produced from seeds of rHF-22 is a potential candidate for commercial MenB vaccine. It also provides a reference for safe, cheap and large-scale production of other plant-made vaccines. PMID:27119621

  8. Transient tachypnea - newborn

    MedlinePlus

    TTN; Wet lungs - newborns; Retained fetal lung fluid; Transient RDS; Prolonged transition; Neonatal - transient tachypnea ... As the baby grows in the womb, the lungs make a special fluid. This fluid fills the ...

  9. Discrepancy in Expression of β-Secretase and Amyloid-β Protein Precursor in Alzheimer-Related Genes in the Rat Medial Temporal Lobe Cortex Following Transient Global Brain Ischemia.

    PubMed

    Pluta, Ryszard; Kocki, Janusz; Ułamek-Kozioł, Marzena; Petniak, Alicja; Gil-Kulik, Paulina; Januszewski, Sławomir; Bogucki, Jacek; Jabłoński, Mirosław; Brzozowska, Judyta; Furmaga-Jabłońska, Wanda; Bogucka-Kocka, Anna; Czuczwar, Stanisław J

    2016-01-01

    Brain ischemia may be causally related with Alzheimer's disease. Presumably, β-secretase and amyloid-β protein precursor gene expression changes may be associated with Alzheimer's disease neuropathology. Consequently, we have examined quantitative changes in both β-secretase and amyloid-β protein precursor genes in the medial temporal lobe cortex with the use of quantitative rtPCR analysis following 10-min global brain ischemia in rats with survival of 2, 7, and 30 days. The greatest significant overexpression of β-secretase gene was noted on the 2nd day, while on days 7-30 the expression of this gene was only modestly downregulated. Amyloid-β protein precursor gene was downregulated on the 2nd day, but on days 7-30 postischemia, there was a significant reverse tendency. Thus, the demonstrated alterations indicate that the considerable changes of expression of β-secretase and amyloid-β protein precursor genes may be connected with a response of neurons in medial temporal lobe cortex to transient global brain ischemia. Finally, the ischemia-induced gene changes may play a key role in a late and slow onset of Alzheimer-type pathology. PMID:26890784

  10. Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation.

    PubMed

    Jaari, S; van Dijk, K W; Olkkonen, V M; van der Zee, A; Metso, J; Havekes, L; Jauhiainen, M; Ehnholm, C

    2001-04-01

    The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process. PMID:11290460

  11. Effect of Transient Inactivation of Ventral Tegmental Area on the Expression and Acquisition of Nicotine-Induced Conditioned Place Preference in Rats

    PubMed Central

    Chalabi-Yani, Durna; Sahraei, Hedayat; Meftahi, Gholam Hossein; Hosseini, Seydeh Bentolhuda; Sadeghi-Gharajehdaghi, Sara; Ali Beig, Hengameh; Bourbour, Zahra; Ranjabaran, Mina

    2015-01-01

    Background: Nicotine can activate dopaminergic neurons within the ventral tegmental area (VTA). However, there is no evidence about complete inhibition of VTA on nicotine reinforcement. Methods: in the present study, we used conditioned-place preference (CPP) method to study the effect of transient inhibition of left and/or right side of the VTA by lidocaine on nicotine reward properties. Male Wistar rats seven days after recovery from surgery and cannulation were conditioned to nicotine (1.5 mg/kg) in an unbiased designed CPP apparatus. Five min before each nicotine injection in conditioning phase, lidocaine (2%) was administered either uni- or bi-laterally into the VTA (0.5µl/rat). Results: results revealed that lidocaine administration into the left but not right side of the VTA reduced nicotine CPP significantly. The reduction was potentiated when lidocaine injected in to both sides of the VTA. In addition, the number of compartment crossing was reduced when lidocaine injected in both side of VTA as well as left side. On the other hand, rearing was reduced when lidocaine injected to the right but not left side of VTA. At last, sniffing was reduced only in the group in which received lidocaine in both side of VTA. Sniffing and rearing increased in the group in which received lidocaine in right side. Conclusion: It is concluded that the right and left side of VTA play different role in nicotine-induced activity and reward. PMID:26210948

  12. Transient drainage summary report

    SciTech Connect

    1996-09-01

    This report summarizes the history of transient drainage issues on the Uranium Mill Tailings Remedial Action (UMTRA) Project. It defines and describes the UMTRA Project disposal cell transient drainage process and chronicles UMTRA Project treatment of the transient drainage phenomenon. Section 4.0 includes a conceptual cross section of each UMTRA Project disposal site and summarizes design and construction information, the ground water protection strategy, and the potential for transient drainage.

  13. OPTIMIZING EFFICIENCY AND FLEXIBILITY OF AGROBACTERIUM-MEDIATED TRANSFORMATION OF RED CLOVER (TRIFOLIUM PRATENSE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic modification of plants by the insertion of transgenes can be a powerful experimental approach to answer basic questions about gene product function. This technology can also be used to make improved crop varieties for use in the field. To apply this powerful tool to red clover (Trifolium pra...

  14. Agrobacterium-mediated inoculation of chrysanthemum (Chrysanthemum morifolium) plants with chrysanthemum stunt viroid.

    PubMed

    Nabeshima, Tomoyuki; Doi, Motoaki; Hosokawa, Munetaka

    2016-08-01

    Agroinfiltration was tested as a method of inoculation of chrysanthemum plants with chrysanthemum stunt viroid (CSVd). Binary vectors harboring dimeric CSVd sequences in sense and antisense orientations were constructed, and Agrobacterium transfected with these binary vectors was infiltrated into chrysanthemum leaves. Northern blotting and reverse transcription polymerase chain reaction analysis showed that local infection was established within 7 days and systemic infection within 20 days. CSVd polarities showed no difference in infectivity. This study showed that agroinfiltration of chrysanthemum plants is an easy, rapid, and cost-effective method for CSVd inoculation. PMID:27155239

  15. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat

    PubMed Central

    Jones, Huw D; Doherty, Angela; Wu, Huixia

    2005-01-01

    Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties. PMID:16270934

  16. Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat.

    PubMed

    Jones, Huw D; Doherty, Angela; Wu, Huixia

    2005-09-01

    Since the first report of wheat transformation by Agrobacterium tumefaciens in 1997, various factors that influence T-DNA delivery and regeneration in tissue culture have been further investigated and modified. This paper reviews the current methodology literature describing Agrobacterium transformation of wheat and provides a complete protocol that we have developed and used to produce over one hundred transgenic lines in both spring and winter wheat varieties. PMID:16270934

  17. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd 21-3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brachypodium distachyon (Brachypodium) is a small grass with biological attributes (rapid generation time, small genome, diploid accessions, small stature and simple growth requirements) that make it suitable for use as a model system. In addition, a growing list of genomic resources have been devel...

  18. Genetic transformation of Metroxylon sagu (Rottb.) cultures via Agrobacterium-mediated and particle bombardment.

    PubMed

    Ibrahim, Evra Raunie; Hossain, Md Anowar; Roslan, Hairul Azman

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  19. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera.

    PubMed

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar - Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  20. Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

    PubMed Central

    Ibrahim, Evra Raunie

    2014-01-01

    Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

  1. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell.

    PubMed

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  2. Agrobacterium-Mediated Stable Genetic Transformation of Populus angustifolia and Populus balsamifera

    PubMed Central

    Maheshwari, Priti; Kovalchuk, Igor

    2016-01-01

    The present study demonstrates Agrobacterium tumefaciens-mediated stable genetic transformation of two species of poplar – Populus angustifolia and Populus balsamifera. The binary vector pCAMBIA-Npro-long-Luc containing the luciferase reporter gene was used to transform stem internode and axillary bud explants. Putative transformants were regenerated on selection-free medium using our previously established in vitro regeneration method. Explant type, genotype, effect of pre-culture, Agrobacterium concentration, a time period of infection and varying periods of co-culture with bacteria were tested for the transformation frequency. The highest frequency of transformation was obtained with stem internode explants pre-cultured for 2 days, infected with Agrobacterium culture at the concentration of OD600 = 0.5 for 10 min and co-cultivated with Agrobacterium for 48 h. Out of the two genotypes tested, P. balsamifera exhibited a higher transformation rate in comparison to P. angustifolia. The primary transformants that exhibited luciferase activity in a bioluminescence assay under the CCD camera when subjected to polymerase chain reaction and Southern blot analysis revealed a stable single-copy integration of luc in their genomes. The reported protocol is highly reproducible and can be applied to other species of poplar; it will also be useful for future genetic engineering of one of the most important families of woody plants for sustainable development. PMID:27014319

  3. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    PubMed Central

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  4. Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated β-adrenoceptor activation- induced cardiac hypertrophic growth in experimental rats.

    PubMed

    Venkatesan, Balaji; Tumala, Anusha; Subramanian, Vimala; Vellaichamy, Elangovan

    2016-07-01

    Natriuretic peptide receptor-C (NPR-C) is considered as a clearance receptor that maintains the circulatory levels of natriuretic peptides. It has been suggested that augmented expression of NPR-C as a cause for the diminished anti-hypertrophic action of natriuretic peptides in the failing heart. Hence, we sought to determine the level of Npr3 gene (coding for NPR-C) expression in the Isoproterenol (ISO) treated Wistar rats. In addition, we studied the effect of Npr3 gene silencing on the hypertrophic growth. A significant increase in heart weight-to-body weight ratio (HW/BW-24%,P<0.01), an indicator of cardiac hypertrophic growth was observed in the ISO (10mg/kg BW/day,i.p for 7 days) treated rats. As expected, the cardiac NPR-C protein expression was significantly increased by 4 fold as compared to control rats. In parallel, the circulatory atrial natriuretic peptide (ANP) level was significantly decreased (2 fold) in ISO treated rats. Upon treatment with siRNA-Npr3, a significant decrease in the cardiac NPR-C protein expression (70%,P<0.01), HW/BW ratio (70%,P<0.01) and hypertrophic marker genes (α-Sk, β-MHC, c-fos, P<0.01, respectively) mRNA expression were observed. Interestingly, the circulatory ANP level was increased by 1.5 fold in the siRNA-Npr3 treated rats as compared to ISO treated rats. Moreover, the cardiac collagen content, matrixmetalloprotinases-2 (MMP-2) and enzymatic antioxidant status (P<0.01, respectively) were found to be restored back to near normal upon siRNA-Npr3 treatment. Taken together, the results of this study indicates that specific down-regulation of Npr3 gene improves the circulatory levels of ANP and antioxidant system and there by attenuates the β-adrenoceptor over-activation mediated cardiac hypertrophic growth in experimental rats. PMID:27108789

  5. Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana.

    PubMed

    Chan, Hui-Ting; Chia, Min-Yuan; Pang, Victor Fei; Jeng, Chian-Ren; Do, Yi-Yin; Huang, Pung-Ling

    2013-04-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV. PMID:23116484

  6. Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens.

    PubMed

    Ma, Liang; Zhang, Jun; Zou, Gen; Wang, Chengshu; Zhou, Zhihua

    2011-09-10

    Trichoderma reesei is a well-known cellulase producer and widely applied in enzyme industry. To increase its ability to efficiently decompose cellulose, the beta-glucosidase activity of its enzyme cocktail needs to be enhanced. In this study, a beta-glucosidase I coding sequence from Penicillium decumbens was ligated with the cellobiohydrolase I (cbh1) promoter of T. reesei and introduced into the genome of T. reesei strain Rut-C30 by Agrobacterium-mediated transformation. In comparison to that from the parent strain, the beta-glucosidase activity of the enzyme complexes from two selected transformants increased 6- to 8-fold and their filter paper activity (FPAs) was enhanced by 30% on average. The transformant's saccharifying ability towards pretreated cornstalk was also significantly enhanced. To further confirm the effect of heterologous beta-glucosidase on the cellulase activity of T. reesei, the heterologously expressed pBGL1 was purified and added to the enzyme complex produced by T. reesei Rut-C30. Supplementation of the Rut-C30 enzyme complex with pBGL1 brought about 80% increase of glucose yield during the saccharification of pretreated cornstalk. Our results indicated that the heterologous expression of a beta-glucosidase gene in T. reesei might produce balanced cellulase preparation. PMID:22112562

  7. Tobamoviral movement protein transiently expressed in a single epidermal cell functions beyond multiple plasmodesmata and spreads multicellularly in an infection-coupled manner.

    PubMed

    Tamai, A; Meshi, T

    2001-02-01

    Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome. PMID:11204775

  8. Ablation of Chop Transiently Enhances Photoreceptor Survival but Does Not Prevent Retinal Degeneration in Transgenic Mice Expressing Human P23H Rhodopsin

    PubMed Central

    Chiang, Wei-Chieh; Joseph, Victory; Matthes, Michael T.; Lewin, Alfred S.; Gorbatyuk, Marina S.; Ahern, Kelly; LaVail, Matthew M.

    2016-01-01

    RHO (Rod opsin) encodes a G-protein coupled receptor that is expressed exclusively by rod photoreceptors of the retina and forms the essential photopigment, rhodopsin, when coupled with 11-cis-retinal. Many rod opsin disease mutations cause rod opsin protein misfolding and trigger endoplasmic reticulum (ER) stress, leading to activation of the Unfolded Protein Response (UPR) signal transduction network. Chop is a transcriptional activator that is induced by ER stress and promotes cell death in response to chronic ER stress. Here, we examined the role of Chop in transgenic mice expressing human P23H rhodopsin (hP23H Rho Tg) that undergo retinal degeneration. With the exception of one time point, we found no significant induction of Chop in these animals and no significant change in retinal degeneration by histology and electrophysiology when hP23H Rho Tg animals were bred into a Chop−/− background. Our results indicate that Chop does not play a significant causal role during retinal degeneration in these animals. We suggest that other modules of the ER stress-induced UPR signaling network may be involved photoreceptor disease induced by P23H rhodopsin. PMID:26427410

  9. Mapping and identification of cassava mosaic geminivirus DNA-A and DNA-B genome sequences for efficient siRNA expression and RNAi based virus resistance by transient agro-infiltration studies.

    PubMed

    Patil, Basavaprabhu L; Bagewadi, Basavaraj; Yadav, Jitender S; Fauquet, Claude M

    2016-02-01

    Geminiviruses are among the most serious pathogens of many economically important crop plants and RNA interference (RNAi) is an important strategy for their control. Although any fragment of a viral genome can be used to generate a double stranded (ds) RNA trigger, the precursor for generation of siRNAs, the exact sequence and size requirements for efficient gene silencing and virus resistance have so far not been investigated. Previous efforts to control geminiviruses by gene silencing mostly targeted AC1, the gene encoding replication-associated protein. In this study we made RNAi constructs for all the genes of both the genomic components (DNA-A and DNA-B) of African cassava mosaic virus (ACMV-CM), one of the most devastating geminiviruses causing cassava mosaic disease (CMD) in Africa. Using transient agro-infiltration studies, RNAi constructs were evaluated for their ability to trigger gene silencing against the invading virus and protection against it. The results show that the selection of the DNA target sequence is an important determinant for the amount of siRNA produced and the extent of resistance. The ACMV genes AC1, AC2, AC4 from DNA-A and BC1 from DNA-B were effective targets for RNAi-mediated resistance and their siRNA expression was higher compared to other RNAi constructs. The RNAi construct targeting AC2, the suppressor of gene silencing of ACMV-CM gave highest level of resistance in the transient studies. This is the first report of targeting DNA-B to confer resistance to a bipartite geminivirus infection. PMID:26581664

  10. Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.

    PubMed

    Gómez, Evangelina; Lucero, María Soledad; Chimeno Zoth, Silvina; Carballeda, Juan Manuel; Gravisaco, María José; Berinstein, Analía

    2013-05-28

    Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV. PMID:23583894

  11. Express

    Integrated Risk Information System (IRIS)

    Express ; CASRN 101200 - 48 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effect

  12. Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea.

    PubMed

    Coca, María; Bortolotti, Cristina; Rufat, Mar; Peñas, Gisela; Eritja, Ramón; Tharreau, Didier; del Pozo, Alvaro Martinez; Messeguer, Joaquima; San Segundo, Blanca

    2004-01-01

    The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea. PMID:15159626

  13. Ectopic expression of two MADS box genes from orchid (Oncidium Gower Ramsey) and lily (Lilium longiflorum) alters flower transition and formation in Eustoma grandiflorum.

    PubMed

    Thiruvengadam, Muthu; Yang, Chang-Hsien

    2009-10-01

    Lisianthus [Eustoma grandiflorum (Raf.) Shinn] is a popular cut flower crop throughout the world, and the demand for this plant for cut flowers and potted plants has been increasing worldwide. Recent advances in genetic engineering have enabled the transformation and regeneration of plants to become a powerful tool for improvement of lisianthus. We have established a highly efficient plant regeneration system and Agrobacterium-mediated genetic transformation of E. grandiflorum. The greatest shoot regeneration frequency and number of shoot buds per explant are observed on media supplemented with 6-Benzylaminopurine (BAP) and alpha-Naphthalene acetic acid (NAA). We report an efficient plant regeneration system using leaf explants via organogenesis with high efficiency of transgenic plants (15%) in culture of 11 weeks' duration. Further ectopic expression of two MADS box genes, LMADS1-M from lily (Lilium longiflorum) and OMADS1 from orchid (Oncidium Gower Ramsey), was performed in E. grandiflorum. Conversion of second whorl petals into sepal-like structures and alteration of third whorl stamen formation were observed in the transgenic E. grandiflorum plants ectopically expressing 35S::LMADS1-M. 35S::OMADS1 transgenic E. grandiflorum plants flowered significantly earlier than non-transgenic plants. This is the first report on the ectopic expression of two MADS box genes in E. grandiflorum using a simple and highly efficient gene transfer protocol. Our results reveal the potential for floral modification in E. grandiflorum through genetic transformation. PMID:19639326

  14. Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

    PubMed Central

    2011-01-01

    Background Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses. Results All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct. Conclusions Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case. PMID

  15. Optical & NIR Transient Surveys

    NASA Astrophysics Data System (ADS)

    Cross, Nicholas J. G.; Djorgovski, S. G.

    2012-04-01

    A workshop on Optical & Near Infrared Transients took place during the first afternoon of the Symposium. It ran for two sessions. The first was given over to talks about various current optical and near-infrared transient surveys, focussing on the Vista surveys, the Catalina Real-Time Transient Survey, Pan-STARRS, Gaia, TAOS and TAOS2. The second session was a panel-led discussion about coordinating multi-wavelength surveys and associated follow-ups.

  16. Cross-linked low molecular weight glycopeptide-mediated gene delivery: relationship between DNA metabolic stability and the level of transient gene expression in vivo.

    PubMed

    Yang, Y; Park, Y; Man, S; Liu, Y; Rice, K G

    2001-12-01

    DNA co-condensates were formed by reacting [125I]DNA with an admixture of a high-mannose glycopeptide (Man9-CWK(18)) and either of two poly(ethylene glycol) peptides (PEG-VS-CWK(18) or PEG-SS-CWK(18)) followed by cross-linking with 6-50 mol equiv of glutaraldehyde. [125I]DNA co-condensates were administered intravenously in mice to determine the influence of peptide DNA formulation parameters on specific targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9-CWK(18) and PEG-CWK(18) to mediate specific recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross-linked Man9-CWK(18)/PEG-CWK(18) DNA co-condensates was receptor mediated since Kupffer cell targeting was inhibited by pre-administration of Man-bovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver-targeted DNA localized to Kupffer cells. Cross-linking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half-live- of 4, 6, and 39 h, respectively. Tail vein dosing of 50 microg of DNA co-condensates cross-linked with 6 mol equiv of glutaraldehyde produced detectable levels of human alpha1-antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human alpha1-antitrypsin was elevated two-fold each day when dosing with DNA co-condensates cross-linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co-condensates cross-linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half-life, providing insight into a means to target DNA condensates to peripheral tissues. PMID:11745760

  17. Ectopic expression of Arabidopsis thaliana Na+(K+)/H+ antiporter gene, AtNHX5, enhances soybean salt tolerance.

    PubMed

    Wu, X X; Li, J; Wu, X D; Liu, Q; Wang, Z K; Liu, S S; Li, S N; Ma, Y L; Sun, J; Zhao, L; Li, H Y; Li, D M; Li, W B; Su, A Y

    2016-01-01

    Drought and salt stresses are the two major factors influencing the yield and quality of crops worldwide. Na(+)(K(+))/H(+) antiporters (NHXs) are ubiquitous membrane proteins that play important roles in maintaining the cellular pH and Na(+)(K(+)) homeostasis. The model plant Arabidopsis potentially encodes six NHX genes, namely AtNHX1 to 6. In the present study, AtNHX5, a comparatively less well-studied NHX, was cloned and transferred into a soybean variety, Dongnong-50, via Agrobacterium-mediated cotyledonary node transformation to assess its role in improving salt tolerance of the transgenic plants. The transgenic soybean plants were tolerant to the presence of 300 mM NaCl whereas the non-transgenic plants were not. Furthermore, after NaCl treatment, the transgenic plants had a higher content of free proline but lower content of malondialdehyde compared to the non-transgenic plants. Our results revealed that that AtNHX5 possibly functioned by efficiently transporting Na(+) and K(+) ions from the roots to the leaves. Overall, the results obtained in this study suggest that soybean salt tolerance could be improved through the over expression of Arabidopsis AtNHX5. PMID:27323012

  18. Spectral relaxation in pump-probe transients

    NASA Astrophysics Data System (ADS)

    Farrow, Darcie A.; Yu, Anchi; Jonas, David M.

    2003-05-01

    The relationship between pump-probe transients and the transition frequency correlation function, M(t), is examined. Calculations of pump-probe transients are carried out with a full-quantum expression for a displaced harmonic oscillator coupled to a heat bath. Pump-probe transients for a slowly decaying, overdamped, Brownian oscillator are shown to resemble a power series in M(t), where the slowest time scale is always equal to the slowest decay in M(t). This equality is consistent with a semiclassical model of pump-probe and valid over the full range of temperature, pulse duration, and detuning explored. The contribution of time scales faster than M(t) to the pump-probe transient increases with increasing temperature, pulse duration, and detuning of the pulse center frequency below resonance. Pump-probe transients for a critically damped oscillator that decays on a femtosecond time scale also have faster early time decay at higher temperatures. Based on these calculations a bootstrap method is suggested for extracting M(t) from pump-probe data starting with the slowest decay. Comparisons are made between simulations of pump-probe and three pulse echo peak shift (3PEPS) transients for a single oscillator and for multiple oscillator systems. Additional fast relaxations similar to those in pump-probe are also present in the 3PEPS transients. For the models investigated, pump-probe is comparable to 3PEPS for the extraction of M(t).

  19. Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3.

    PubMed Central

    Huston, E; Lumb, S; Russell, A; Catterall, C; Ross, A H; Steele, M R; Bolger, G B; Perry, M J; Owens, R J; Houslay, M D

    1997-01-01

    5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram ¿4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone¿, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed

  20. Perception of acoustic transients

    NASA Astrophysics Data System (ADS)

    Howard, J. H., Jr.

    1984-01-01

    The research investigates the role of knowledge based or top-down processing in the perception of nonlinguistic, transient signals. The experiments address issues in transient pattern classification, target observation, attentional focusing, auditory induction, and computer based performance aids. The theoretical significance and naval relevance of the research is considered.

  1. Transgenic indica rice lines, expressing Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1), exhibit enhanced resistance to major pathogens.

    PubMed

    Sadumpati, Vijayakumar; Kalambur, Muralidharan; Vudem, Dashavantha Reddy; Kirti, Pulugurtha Bharadwaja; Khareedu, Venkateswara Rao

    2013-07-10

    Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into commercial indica rice varieties by Agrobacterium-mediated genetic transformation. Transgenic rice plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in various transgenic rice lines. Transgenes NPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Homozygous transgenic rice lines expressing BjNPR1 protein displayed enhanced resistance to rice blast, sheath blight and bacterial leaf blight diseases. Rice transformants with higher levels of NPR1 revealed notable increases in plant height, panicle length, flag-leaf length, number of seeds/panicle and seed yield/plant as compared to the untransformed plants. The overall results amply demonstrate the profound impact of BjNPR1 in imparting resistance against major pathogens of rice. The multipotent BjNPR1, as such, seems promising as a prime candidate gene to fortify crop plants with durable resistance against various pathogens. PMID:23664883

  2. Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton

    PubMed Central

    Bajwa, Kamran S.; Shahid, Ahmad A.; Rao, Abdul Q.; Bashir, Aftab; Aftab, Asia; Husnain, Tayyab

    2015-01-01

    Cotton fiber is multigenic trait controlled by number of genes. Previous studies suggest that one of these genes may be responsible for switching cotton fiber growth on and off to influence the fiber quality produced from a cotton seed. In the present study, the Gossypium hirsutum GhEXPA8 fiber expansin gene was introduced into local cotton variety NIAB 846 by using an Agrobacterium-mediated gene transformation. The neomycin phosphotransferase (NPTII) gene was used as a selection marker for screening of putative transgenic cotton plants. Integration and expression of the fiber expansin gene in cotton plants was confirmed with molecular techniques including Southern blot analyses, real-time PCR. Cellulose assay was used for measurement of cellulose contents of transgenic cotton fiber. The data collected from 3 years of field performance of the transgenic cotton plants expressing GhEXPA8 showed that significant improvement has been made in fiber lengths and micronaire values as compared to control G. hirsutum variety NIAB 846 cotton plants. Statistical techniques were also used for analysis of fiber and agronomic characteristics. The results of this study support improvement of cotton fiber through genetic modification. PMID:26583018

  3. Enhanced tolerance and remediation to mixed contaminates of PCBs and 2,4-DCP by transgenic alfalfa plants expressing the 2,3-dihydroxybiphenyl-1,2-dioxygenase.

    PubMed

    Wang, Yan; Ren, Hejun; Pan, Hongyu; Liu, Jinliang; Zhang, Lanying

    2015-04-01

    Polychlorinated biphenyls (PCBs) and 2,4-dichlorophenol (2,4-DCP) generally led to mixed contamination of soils as a result of commercial and agricultural activities. Their accumulation in the environment poses great risks to human and animal health. Therefore, the effective strategies for disposal of these pollutants are urgently needed. In this study, genetic engineering to enhance PCBs/2,4-DCP phytoremediation is a focus. We cloned the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC.B) from a soil metagenomic library, which is the key enzyme of aerobic catabolism of a variety of aromatic compounds, and then it was expressed in alfalfa driven by CaMV 35S promoter using Agrobacterium-mediated transformation. Transgenic line BB11 was selected out through PCR, Western blot analysis and enzyme activity assays. Its disposal and tolerance to both PCBs and 2,4-DCP were examined. The tolerance capability of transgenic line BB11 towards complex contaminants of PCBs/2,4-DCP significantly increased compared with non-transgenic plants. Strong dissipation of PCBs and high removal efficiency of 2,4-DCP were exhibited in a short time. It was confirmed expressing BphC.B would be a feasible strategy to help achieving phytoremediation in mixed contaminated soils with PCBs and 2,4-DCP. PMID:25590820

  4. The elicitor-inducible alfalfa isoflavone reductase promoter confers different patterns of developmental expression in homologous and heterologous transgenic plants.

    PubMed Central

    Oommen, A; Dixon, R A; Paiva, N L

    1994-01-01

    In legumes, the synthesis of infection- and elicitor-inducible antimicrobial phytoalexins occurs via the isoflavonoid branch of the phenylpropanoid pathway. To study transcriptional regulation of isoflavonoid pathway-specific genes, we have isolated the gene encoding isoflavone reductase (IFR), which is the enzyme that catalyzes the penultimate step in the synthesis of the phytoalexin medicarpin in alfalfa. Chimeric gene fusions were constructed between 765- and 436-bp promoter fragments of the IFR gene and the beta-glucuronidase reporter gene and transferred to alfalfa and tobacco by Agrobacterium-mediated transformation. Both promoter fragments conferred elicitor-mediated expression in cell suspension cultures derived from transgenic plants of both species and fungal infection-mediated expression in leaves of transgenic alfalfa. Developmental expression directed by both promoter fragments in transgenic alfalfa was observed only in the root meristem, cortex, and nodules, which is consistent with the accumulation of endogenous IFR transcripts. However, in transgenic tobacco, expression from the 765-bp promoter was observed in vegetative tissues (root meristem and cortex, inner vascular tissue of stems and petioles, leaf tips, and stem peripheries adjacent to petioles) and in reproductive tissues (stigma, placenta, base of the ovary, receptacle, seed, tapetal layer, and pollen grains), whereas the 436-bp promoter was expressed only in fruits, seed, and pollen. These data indicate that infection/elicitor inducibility of the IFR promoter in both species and developmental expression in alfalfa are determined by sequences downstream of position -436, whereas sequences between -436 and -765 confer a complex pattern of strong ectopic developmental expression in the heterologous species that lacks the isoflavonoid pathway. PMID:7866024

  5. High-energy transients.

    PubMed

    Gehrels, Neil; Cannizzo, John K

    2013-06-13

    We present an overview of high-energy transients in astrophysics, highlighting important advances over the past 50 years. We begin with early discoveries of γ-ray transients, and then delve into physical details associated with a variety of phenomena. We discuss some of the unexpected transients found by Fermi and Swift, many of which are not easily classifiable or in some way challenge conventional wisdom. These objects are important insofar as they underscore the necessity of future, more detailed studies. PMID:23630376

  6. Transient nucleation in glasses

    NASA Technical Reports Server (NTRS)

    Kelton, K. F.

    1991-01-01

    Nucleation rates in condensed systems are frequently not at their steady state values. Such time dependent (or transient) nucleation is most clearly observed in devitrification studies of metallic and silicate glasses. The origin of transient nucleation and its role in the formation and stability of desired phases and microstructures are discussed. Numerical models of nucleation in isothermal and nonisothermal situations, based on the coupled differential equations describing cluster evolution within the classical theory, are presented. The importance of transient nucleation in glass formation and crystallization is discussed.

  7. BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses

    PubMed Central

    Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; Foster, Cliff E.; Vogel, John P.; Karlen, Steven D.; Ralph, John; Sedbrook, John C.

    2016-01-01

    Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is

  8. Production of marker-free transgenic Jatropha curcas expressing hybrid Bacillus thuringiensis δ-endotoxin Cry1Ab/1Ac for resistance to larvae of tortrix moth (Archips micaceanus)

    PubMed Central

    2014-01-01

    Background The potential biofuel plant Jatropha curcas L. is affected by larvae of Archips micaceanus (Walker), a moth of the family Tortricidae. The hybrid Bacillus thuringiensis (Bt) δ-endotoxin protein Cry1Ab/1Ac confers resistance to lepidopteran insects in transgenic rice. Results Here, we report the production of a marker-free transgenic line of J. curcas (L10) expressing Cry1Ab/1Ac using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L10 carries a single copy of marker-free T-DNA that contains the Cry1Ab/1Ac gene under the control of a maize phosphoenolpyruvate carboxylase gene promoter (P Pepc :Cry1Ab/1Ac:T Nos ). The P Pepc :Cry1Ab/1Ac:T Nos gene was highly expressed in leaves of L10 plants. Insecticidal bioassays using leaf explants of L10 resulted in 80-100% mortality of larvae of A. micaceanus at 4 days after infestation. Conclusion The results demonstrate that the hybrid Bt δ-endotoxin protein Cry1Ab/1Ac expressed in Jatropha curcas displays strong insecticidal activity to A. micaceanus. The marker-free transgenic J. curcas line L10 can be used for breeding of insect resistance to A. micaceanus. PMID:24808924

  9. Altered expression of CmNRRa changes flowering time of Chrysanthemum morifolium.

    PubMed

    Zhang, Yuman; Lian, Lijuan; Liu, Qing; Xiao, Na; Fang, Rongxiang; Liu, Qinglin; Chen, Xiaoying

    2013-04-01

    Flowering time is an important ornamental trait for chrysanthemum (Chrysanthemum morifolium, Dendranthema x grandiflorum) floricultural production. In this study, CmNRRa, an orthologous gene of OsNRRa that regulates root growth in response to nutrient stress in rice, was identified from Chrysanthemum and its role in flowering time was studied. The entire CmNRRa cDNA sequence was determined using a combinatorial PCR approach along with 5' and 3' RACE methods. CmNRRa expression levels in various tissues were monitored by real-time RT-PCR. CmNRRa was strongly expressed in flower buds and peduncles, suggesting that CmNRRa plays a regulatory role in floral development. To investigate the biological function of CmNRRa in chrysanthemums, overexpression and knockdown of CmNRRa were carried out using transgenic Chrysanthemum plants generated through Agrobacterium-mediated transformation. CmNRRa expression levels in the transgenic plants were assayed by real-time RT-PCR and Northern blot analysis. The transgenic plants showed altered flowering times compared with nontransgenic plants. CmNRRa-RNAi transgenic plants flowered 40-64 days earlier, while CmNRRa-overexpressing plants exhibited a delayed flowering phenotype. These results revealed a negative effect of CmNRRa on flowering time modulation. Alteration of CmNRRa expression levels might be an effective means of controlling flowering time in Chrysanthemum. These results possess potential application in molecular breeding of chrysanthemums that production year-round, and may improve commercial chrysanthemum production in the flower industry. PMID:23190188

  10. High energy transients

    NASA Technical Reports Server (NTRS)

    Woosley, S. E.

    1984-01-01

    A meeting was convened on the campus of the University of California at Santa Cruz during the two-week interval July 11 through July 22, 1983. Roughly 100 participants were chosen so as to give broad representation to all aspects of high energy transients. Ten morning review sessions were held in which invited speakers discussed the current status of observations and theory of the above subjects. Afternoon workshops were also held, usually more than one per day, to informally review various technical aspects of transients, confront shortcomings in theoretical models, and to propose productive courses for future research. Special attention was also given to the instrumentation used to study high energy transient and the characteristics and goals of a dedicated space mission to study transients in the next decade were determined. A listing of articles written by various members of the workshop is included.

  11. Gamma ray transients

    NASA Technical Reports Server (NTRS)

    Cline, Thomas L.

    1987-01-01

    The discovery of cosmic gamma ray bursts was made with systems designed at Los Alamos Laboratory for the detection of nuclear explosions beyond the atmosphere. HELIOS-2 was the first gamma ray burst instrument launched; its initial results in 1976, seemed to deepen the mystery around gamma ray transients. Interplanetary spacecraft data were reviewed in terms of explaining the behavior and source of the transients.

  12. Four RNA families with functional transient structures.

    PubMed

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5' flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5' UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM. All

  13. Four RNA families with functional transient structures

    PubMed Central

    Zhu, Jing Yun A; Meyer, Irmtraud M

    2015-01-01

    Protein-coding and non-coding RNA transcripts perform a wide variety of cellular functions in diverse organisms. Several of their functional roles are expressed and modulated via RNA structure. A given transcript, however, can have more than a single functional RNA structure throughout its life, a fact which has been previously overlooked. Transient RNA structures, for example, are only present during specific time intervals and cellular conditions. We here introduce four RNA families with transient RNA structures that play distinct and diverse functional roles. Moreover, we show that these transient RNA structures are structurally well-defined and evolutionarily conserved. Since Rfam annotates one structure for each family, there is either no annotation for these transient structures or no such family. Thus, our alignments either significantly update and extend the existing Rfam families or introduce a new RNA family to Rfam. For each of the four RNA families, we compile a multiple-sequence alignment based on experimentally verified transient and dominant (dominant in terms of either the thermodynamic stability and/or attention received so far) RNA secondary structures using a combination of automated search via covariance model and manual curation. The first alignment is the Trp operon leader which regulates the operon transcription in response to tryptophan abundance through alternative structures. The second alignment is the HDV ribozyme which we extend to the 5′ flanking sequence. This flanking sequence is involved in the regulation of the transcript's self-cleavage activity. The third alignment is the 5′ UTR of the maturation protein from Levivirus which contains a transient structure that temporarily postpones the formation of the final inhibitory structure to allow translation of maturation protein. The fourth and last alignment is the SAM riboswitch which regulates the downstream gene expression by assuming alternative structures upon binding of SAM

  14. Co-expression of RCH10 and AGLU1 confers rice resistance to fungal sheath blight Rhizoctonia solani and blast Magnorpathe oryzae and reveals impact on seed germination.

    PubMed

    Mao, Bizeng; Liu, Xuehui; Hu, Dongwei; Li, Debao

    2014-04-01

    Rice sheath blight and blast caused by Rhizoctonia solani Kühn and Magnorpathe oryzae respectively, are the two most destructive fungal diseases in rice. With no genetic natural traits conferring resistance to sheath blight, transgenic manipulation provides an obvious approach. In this study, the rice basic chitinase gene (RCH10) and the alfalfa β-1,3-glucanase gene (AGLU1) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate a double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation. More than 160 independent transformants were obtained and confirmed by PCR. Northern analysis of inheritable progenies revealed similar levels of both RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was challenged in open field inoculation. Immunogold detection revealed that RCH10 and AGLU1 proteins were initially located mainly in the chloroplasts and were delivered to the vacuole and cell wall upon infection, suggesting that these subcellular compartments act as the gathering and execution site for these anti-fungal proteins. We also observed that transgenic seeds display lower germination rate and seedling vigor, indicating that defense enhancement might be achieved at the expense of development. PMID:24197785

  15. Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression1[w

    PubMed Central

    Townsend, Belinda J.; Poole, Andrew; Blake, Christopher J.; Llewellyn, Danny J.

    2005-01-01

    In cotton (Gossypium hirsutum) the enzyme (+)-δ-cadinene synthase (CDNS) catalyzes the first committed step in the biosynthesis of cadinane-type sesquiterpenes, such as gossypol, that provide constitutive and inducible protection against pests and diseases. A cotton cDNA clone encoding CDNS (cdn1-C4) was isolated from developing embryos and functionally characterized. Southern analysis showed that CDNS genes belong to a large multigene family, of which five genomic clones were studied, including three pseudogenes and one gene that may represent another subfamily of CDNS. CDNS expression was shown to be induced in cotton infected with either the bacterial blight or verticillium wilt pathogens. Constructs for the constitutive or seed-specific antisense suppression of cdn1-C4 were introduced into cotton by Agrobacterium-mediated transformation. Gossypol levels were not reduced in the seeds of transformants with either construct, nor was the induction of CDNS expression affected in stems of the constitutive antisense plants infected with Verticillium dahliae Kleb. However, the induction of CDNS mRNA and protein in response to bacterial blight infection of cotyledons was completely blocked in the constitutive antisense plants. These results suggest that cdn1-C4 may be involved specifically in the bacterial blight response and that the CDNS multigene family comprises a complex set of genes differing in their temporal and spatial regulation and responsible for different branches of the cotton sesquiterpene pathway. PMID:15849309

  16. The LOFAR Transients Pipeline

    NASA Astrophysics Data System (ADS)

    Swinbank, John D.; Staley, Tim D.; Molenaar, Gijs J.; Rol, Evert; Rowlinson, Antonia; Scheers, Bart; Spreeuw, Hanno; Bell, Martin E.; Broderick, Jess W.; Carbone, Dario; Garsden, Hugh; van der Horst, Alexander J.; Law, Casey J.; Wise, Michael; Breton, Rene P.; Cendes, Yvette; Corbel, Stéphane; Eislöffel, Jochen; Falcke, Heino; Fender, Rob; Grießmeier, Jean-Mathias; Hessels, Jason W. T.; Stappers, Benjamin W.; Stewart, Adam J.; Wijers, Ralph A. M. J.; Wijnands, Rudy; Zarka, Philippe

    2015-06-01

    Current and future astronomical survey facilities provide a remarkably rich opportunity for transient astronomy, combining unprecedented fields of view with high sensitivity and the ability to access previously unexplored wavelength regimes. This is particularly true of LOFAR, a recently-commissioned, low-frequency radio interferometer, based in the Netherlands and with stations across Europe. The identification of and response to transients is one of LOFAR's key science goals. However, the large data volumes which LOFAR produces, combined with the scientific requirement for rapid response, make automation essential. To support this, we have developed the LOFAR Transients Pipeline, or TraP. The TraP ingests multi-frequency image data from LOFAR or other instruments and searches it for transients and variables, providing automatic alerts of significant detections and populating a lightcurve database for further analysis by astronomers. Here, we discuss the scientific goals of the TraP and how it has been designed to meet them. We describe its implementation, including both the algorithms adopted to maximize performance as well as the development methodology used to ensure it is robust and reliable, particularly in the presence of artefacts typical of radio astronomy imaging. Finally, we report on a series of tests of the pipeline carried out using simulated LOFAR observations with a known population of transients.

  17. The Transient Universe

    NASA Astrophysics Data System (ADS)

    Shappee, Benjamin John

    When one looks at the night sky, one usually gets the impression of a static and constant universe. Quite apart from appearances, the sky is teeming with violent, variable, and transient events that shape our universe. These capricious objects are not only penetrating probes into physical conditions too extreme for earthbound laboratories, but they are also useful tools to measure the universe. In this dissertation, I investigate the observational and theoretical properties of three classes of transient/variable objects: thermonuclear supernovae, Cepheid variable stars, and active galactic nuclei.

  18. Transient Uncoupling Induces Synchronization

    NASA Astrophysics Data System (ADS)

    Schröder, Malte; Mannattil, Manu; Dutta, Debabrata; Chakraborty, Sagar; Timme, Marc

    2015-07-01

    Finding conditions that support synchronization is a fertile and active area of research with applications across multiple disciplines. Here we present and analyze a scheme for synchronizing chaotic dynamical systems by transiently uncoupling them. Specifically, systems coupled only in a fraction of their state space may synchronize even if fully coupled they do not. While for many standard systems coupling strengths need to be bounded to ensure synchrony, transient uncoupling removes this bound and thus enables synchronization in an infinite range of effective coupling strengths. The presented coupling scheme therefore opens up the possibility to induce synchrony in (biological or technical) systems whose parameters are fixed and cannot be modified continuously.

  19. Transient Uncoupling Induces Synchronization.

    PubMed

    Schröder, Malte; Mannattil, Manu; Dutta, Debabrata; Chakraborty, Sagar; Timme, Marc

    2015-07-31

    Finding conditions that support synchronization is a fertile and active area of research with applications across multiple disciplines. Here we present and analyze a scheme for synchronizing chaotic dynamical systems by transiently uncoupling them. Specifically, systems coupled only in a fraction of their state space may synchronize even if fully coupled they do not. While for many standard systems coupling strengths need to be bounded to ensure synchrony, transient uncoupling removes this bound and thus enables synchronization in an infinite range of effective coupling strengths. The presented coupling scheme therefore opens up the possibility to induce synchrony in (biological or technical) systems whose parameters are fixed and cannot be modified continuously. PMID:26274420

  20. Enhanced salt stress tolerance in transgenic potato plants expressing IbMYB1, a sweet potato transcription factor.

    PubMed

    Cheng, Yu-Jie; Kim, Myoung-Duck; Deng, Xi-Ping; Kwak, Sang-Soo; Chen, Wei

    2013-12-01

    IbMYB1, a transcription factor (TF) for R2R3-type MYB TFs, is a key regulator of anthocyanin biosynthesis during storage of sweet potatoes. Anthocyanins provide important antioxidants of nutritional value to humans, and also protect plants from oxidative stress. This study aimed to increase transgenic potatoes' (Solanum tuberosum cv. LongShu No.3) tolerance to environmental stress and enhance their nutritional value. Transgenic potato plants expressing IbMYB1 genes under the control of an oxidative stress-inducible peroxidase (SWPA2) promoter (referred to as SM plants) were successfully generated through Agrobacterium-mediated transformation. Two representative transgenic SM5 and SM12 lines were evaluated for enhanced tolerance to salinity, UV-B rays, and drought conditions. Following treatment of 100 mM NaCl, seedlings of SM5 and SM12 lines showed less root damage and more shoot growth than control lines expressing only an empty vector. Transgenic potato plants in pots treated with 400 mM NaCl showed high amounts of secondary metabolites, including phenols, anthocyanins, and flavonoids, compared with control plants. After treatment of 400 mM NaCl, transgenic potato plants also showed high DDPH radical scavenging activity and high PS II photochemical efficiency compared with the control line. Furthermore, following treatment of NaCl, UV-B, and drought stress, the expression levels of IbMYB1 and several structural genes in the flavonoid biosynthesis such as CHS, DFR, and ANS in transgenic plants were found to be correlated with plant phenotype. The results suggest that enhanced IbMYB1 expression affects secondary metabolism, which leads to improved tolerance ability in transgenic potatoes. PMID:24378636

  1. Expression of β-glucosidase increases trichome density and artemisinin content in transgenic Artemisia annua plants.

    PubMed

    Singh, Nameirakpam Dolendro; Kumar, Shashi; Daniell, Henry

    2016-03-01

    Artemisinin is highly effective against multidrug-resistant strains of Plasmodium falciparum, the aetiological agent of the most severe form of malaria. However, a low level of accumulation of artemisinin in Artemisia annua is a major limitation for its production and delivery to malaria endemic areas of the world. While several strategies to enhance artemisinin have been extensively explored, enhancing storage capacity in trichome has not yet been considered. Therefore, trichome density was increased with the expression of β-glucosidase (bgl1) gene in A. annua through Agrobacterium-mediated transformation. Transgene (bgl1) integration and transcript were confirmed by molecular analysis. Trichome density increased up to 20% in leaves and 66% in flowers of BGL1 transgenic plants than Artemisia control plants. High-performance liquid chromatography, time of flight mass spectrometer data showed that artemisinin content increased up to 1.4% in leaf and 2.56% in flowers (per g DW), similar to the highest yields achieved so far through metabolic engineering. Artemisinin was enhanced up to five-fold in BGL1 transgenic flowers. This study opens the possibility of increasing artemisinin content by manipulating trichomes' density, which is a major reservoir of artemisinin. Combining biosynthetic pathway engineering with enhancing trichome density may further increase artemisinin yield in A. annua. Because oral feeding of Artemisia plant cells reduced parasitemia more efficiently than the purified drug, reduced drug resistance and cost of prohibitively expensive purification process, enhanced expression should play a key role in making this valuable drug affordable to treat malaria in a large global population that disproportionally impacts low-socioeconomic areas and underprivileged children. PMID:26360801

  2. Stable Expression of mtlD Gene Imparts Multiple Stress Tolerance in Finger Millet

    PubMed Central

    Hema, Ramanna; Vemanna, Ramu S.; Sreeramulu, Shivakumar; Reddy, Chandrasekhara P.; Senthil-Kumar, Muthappa; Udayakumar, Makarla

    2014-01-01

    Finger millet is susceptible to abiotic stresses, especially drought and salinity stress, in the field during seed germination and early stages of seedling development. Therefore developing stress tolerant finger millet plants combating drought, salinity and associated oxidative stress in these two growth stages is important. Cellular protection through osmotic adjustment and efficient free radical scavenging ability during abiotic stress are important components of stress tolerance mechanisms in plants. Mannitol, an osmolyte, is known to scavenge hydroxyl radicals generated during various abiotic stresses and thereby minimize stress damage in several plant species. In this study transgenic finger millet plants expressing the mannitol biosynthetic pathway gene from bacteria, mannitol-1-phosphate dehydrogenase (mtlD), were developed through Agrobacterium tumefaciens-mediated genetic transformation. mtlD gene integration in the putative transgenic plants was confirmed by Southern blot. Further, performance of transgenic finger millet under drought, salinity and oxidative stress was studied at plant level in T1 generation and in T1 and T2 generation seedlings. Results from these experiments showed that transgenic finger millet had better growth under drought and salinity stress compared to wild-type. At plant level, transgenic plants showed better osmotic adjustment and chlorophyll retention under drought stress compared to the wild-type. However, the overall increase in stress tolerance of transgenics for the three stresses, especially for oxidative stress, was only marginal compared to other mtlD gene expressing plant species reported in the literature. Moreover, the Agrobacterium-mediated genetic transformation protocol developed for finger millet in this study can be used to introduce diverse traits of agronomic importance in finger millet. PMID:24922513

  3. Measuring Transient Memory Load

    ERIC Educational Resources Information Center

    Wanner, Eric; Shiner, Sandra

    1976-01-01

    Two experiments are reported in which subjects performed simple mental arithmetic problems which were presented visually in a sequential fashion. At some point in the presentation of each problem, the sequential display was interrupted and a memory task introduced. The purpose was to validate a measure of transient memory load. (Author/RM)

  4. Rotor transient analysis

    NASA Technical Reports Server (NTRS)

    Allaire, P. E.; Choy, K. C.; Gunter, E. J.

    1980-01-01

    Undamped modes approximate dynamic behavior of rotors and bearings. Application of modal analysis to uncouple equations of motion simplifies stability, steady-state unbalance response, and transient response analysis of system; nonlinear stability is predicted from calculated frequency spectra. Analysis provides designers with complete information without involving large-scale computational costs. Programs are written in FORTRAN IV for use on CDC 6600 computer.

  5. Long-term stability of attacin E expression in transformed apple after 12 years in the field and effect of the expression of this gene on the fruit characteristics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. The future use of transformed trees on a commercial basis depends upon thorough evaluation of the potential environmental and public health impact of the modified plants, transgene stability over ...

  6. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.].

    PubMed

    Ellul, P; Ríos, G; Atarés, A; Roig, L A; Serrano, R; Moreno, V

    2003-08-01

    An optimised Agrobacterium-mediated gene transfer protocol was developed in order to obtain watermelon transgenic plants [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Transformation efficiencies ranged from 2.8% to 5.3%, depending on the cultivar. The method was applied to obtain genetically engineered watermelon plants expressing the Saccharomyces cerevisiae HAL1 gene related to salt tolerance. In order to enhance its constitutive expression in plants, the HAL1 gene was cloned in a pBiN19 plasmid under control of the 35S promoter with a double enhancer sequence from the cauliflower mosaic virus and the RNA4 leader sequence of the alfalfa mosaic virus. This vector was introduced into Agrobacterium tumefaciens strain LBA4404 for further inoculation of watermelon half-cotyledon explants. The introduction of both the neomycin phosphotransferase II and HAL1 genes was assessed in primary transformants (TG1) by polymerase chain reaction analysis and Southern hybridisation. The expression of the HAL1 gene was determined by Northern analysis, and the diploid level of transgenic plants was confirmed by flow cytometry. The presence of the selectable marker gene in the expected Mendelian ratios was demonstrated in TG2 progenies. The TG2 kanamycin-resistant plantlets elongated better and produced new roots and leaves in culture media supplemented with NaCl compared with the control. Salt tolerance was confirmed in a semi-hydroponic system (EC=6 dS m(-1)) on the basis of the higher growth performance of homozygous TG3 lines with respect to their respective azygous control lines without the transgene. The halotolerance observed confirmed the inheritance of the trait and supports the potential usefulness of the HAL1 gene of S. cerevisiae as a molecular tool for genetic engineering of salt-stress protection in other crop species. PMID:12783167

  7. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    SciTech Connect

    Chou, Hong L.; Dai, Ziyu; Hsieh, Chia W.; Ku, Maurice S.

    2011-12-10

    Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. In this study, the cellulose hydrolytic enzyme {beta}-1, 4-endoglucanase (E1) from the thermophilic bacterium Acidothermus cellulolyticus was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer with the signal peptide of tobacco pathogenesis-related protein for targeting the protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial enzyme were obtained, which expressed the gene at high levels with a normal phenotype. The specific activities of E1 in the leaves of the highest expressing transgenic rice lines were about 20 fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. Zymogram and temperature-dependent activity analyses demonstrated the thermostability of the enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid yielded almost twice more reducing sugars than wild type straw. Taken together, these data suggest that transgenic rice can effectively serve as a bioreactor for large-scale production of active, thermostable cellulose hydrolytic enzymes. As a feedstock, direct expression of large amount of cellulases in

  8. Transient neonatal zinc deficiency.

    PubMed

    Krieger, I; Alpern, B E; Cunnane, S C

    1986-06-01

    We report an infant who developed clinical manifestations of zinc deficiency during the first month of life although the diet was adequate for zinc and no other causes could be ascertained. The diagnosis was confirmed by low plasma-zinc concentrations and a positive response to zinc treatment. The fatty acid profile of plasma phospholipids was typical of zinc deficiency (ie, arachidonic acid was markedly decreased). The transient nature of this disorder was evident when no relapse occurred after cessation of zinc therapy and plasma-zinc and arachidonic acid concentrations remained normal. Several explanations for the development of transient neonatal zinc deficiency are offered. The observation demonstrates that occasional infants may have requirements for zinc that are beyond the intakes of the conventional RDA. PMID:3717070

  9. Transient infrared emission spectroscopy

    SciTech Connect

    Jones, R.W.; McClelland, J.F.

    1989-04-01

    Transient infrared emission spectroscopy (TIRES) is a new method that produces analytically useful emission spectra from optically thick, solid samples by greatly reducing self-absorption of emitted radiation. The method reduces self-absorption by creating a thin, short-lived, heated layer at the sample surface and collecting the transient emission from this layer. The technique requires no sample preparation and may be applied to both moving and stationary samples. The single-ended, noncontact TIRES measurement geometry is ideal for on-line and other remote-sensing applications. TIRES spectra acquired via a Fourier transform infrared spectrometer on moving samples of coal, plastic, and paint are presented and compared to photoacoustic absorption spectra of these materials. The TIRES and photoacoustic results are in close agreement as predicted by Kirchhoff's law.

  10. The VAO Transient Facility

    NASA Astrophysics Data System (ADS)

    Graham, Matthew J.; Djorgovski, S. G.; Drake, Andrew; Mahabal, Ashish; Williams, Roy; Seaman, Rob

    2012-04-01

    The time-domain community wants robust and reliable tools to enable the production of, and subscription to, community-endorsed event notification packets (VOEvent). The Virtual Astronomical Observatory (VAO) Transient Facility (VTF) is being designed to be the premier brokering service for the community, both collecting and disseminating observations about time-critical astronomical transients but also supporting annotations and the application of intelligent machine-learning to those observations. Two types of activity associated with the facility can therefore be distinguished: core infrastructure, and user services. We review the prior art in both areas, and describe the planned capabilities of the VTF. In particular, we focus on scalability and quality-of-service issues required by the next generation of sky surveys such as LSST and SKA.

  11. Reactor System Transient Code.

    1999-07-14

    RELAP3B describes the behavior of water-cooled nuclear reactors during postulated accidents or power transients, such as large reactivity excursions, coolant losses or pump failures. The program calculates flows, mass and energy inventories, pressures, temperatures, and steam qualities along with variables associated with reactor power, reactor heat transfer, or control systems. Its versatility allows one to describe simple hydraulic systems as well as complex reactor systems.

  12. Two Transients discovered by PSST

    NASA Astrophysics Data System (ADS)

    Smith, K. W.; Wright, D.; Smartt, S. J.; Young, D. R.; Huber, M.; Chambers, K. C.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.

    2016-08-01

    Two transients have been discovered as part of the Pan-STARRS Survey for Transients (PSST). Information on all objects discovered by the Pan-STARRS Survey for Transients is available at http://star.pst.qub.ac.uk/ps1threepi/ (see Huber et al. ATel #7153).

  13. Bright Transients discovered by PSST

    NASA Astrophysics Data System (ADS)

    Smith, K. W.; Wright, D.; Smartt, S. J.; Young, D. R.; Huber, M.; Chambers, K. C.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.

    2016-08-01

    Six bright transients have been discovered as part of the Pan-STARRS Survey for Transients (PSST). Information on all objects discovered by the Pan-STARRS Survey for Transients is available at http://star.pst.qub.ac.uk/ps1threepi/ (see Huber et al. ATel #7153).

  14. Bright Transients discovered by PSST

    NASA Astrophysics Data System (ADS)

    Smith, K. W.; Wright, D.; Smartt, S. J.; Huber, M.; Chambers, K. C.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.

    2016-04-01

    Seven bright transients have been discovered as part of the Pan-STARRS Survey for Transients (PSST). Information on all objects discovered by the Pan-STARRS Survey for Transients is available at http://star.pst.qub.ac.uk/ps1threepi/ (see Huber et al. ATel #7153).

  15. ON DETECTING TRANSIENT PHENOMENA

    SciTech Connect

    Belanger, G.

    2013-08-10

    Transient phenomena are interesting and potentially highly revealing of details about the processes under observation and study that could otherwise go unnoticed. It is therefore important to maximize the sensitivity of the method used to identify such events. In this article, we present a general procedure based on the use of the likelihood function for identifying transients which is particularly suited for real-time applications because it requires no grouping or pre-processing of the data. The method makes use of all the information that is available in the data throughout the statistical decision-making process, and is suitable for a wide range of applications. Here we consider those most common in astrophysics, which involve searching for transient sources, events or features in images, time series, energy spectra, and power spectra, and demonstrate the use of the method in the case of a weak X-ray flare in a time series and a short-lived quasi-periodic oscillation in a power spectrum. We derive a fit statistic that is ideal for fitting arbitrarily shaped models to a power density distribution, which is of general interest in all applications involving periodogram analysis.

  16. Advanced PFBC transient analysis

    SciTech Connect

    White, J.S.; Bonk, D.L.

    1997-05-01

    Transient modeling and analysis of advanced Pressurized Fluidized Bed Combustion (PFBC) systems is a research area that is currently under investigation by the US Department of Energy`s Federal Energy Technology Center (FETC). The object of the effort is to identify key operating parameters that affect plant performance and then quantify the basic response of major sub-systems to changes in operating conditions. PC-TRAX{trademark}, a commercially available dynamic software program, was chosen and applied in this modeling and analysis effort. This paper describes the development of a series of TRAX-based transient models of advanced PFBC power plants. These power plants burn coal or other suitable fuel in a PFBC, and the high temperature flue gas supports low-Btu fuel gas or natural gas combustion in a gas turbine topping combustor. When it is utilized, the low-Btu fuel gas is produced in a bubbling bed carbonizer. High temperature, high pressure combustion products exiting the topping combustor are expanded in a modified gas turbine to generate electrical power. Waste heat from the system is used to raise and superheat steam for a reheat steam turbine bottoming cycle that generates additional electrical power. Basic control/instrumentation models were developed and modeled in PC-TRAX and used to investigate off-design plant performance. System performance for various transient conditions and control philosophies was studied.

  17. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress.

    PubMed

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P; Dobaria, Jentilal R

    2016-01-01

    Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

  18. Improvement in the stability and functionality of Nicotiana tabacum produced recombinant TRAIL through employment of endoplasmic reticulum expression and ascorbate buffer mediated extraction strategies

    PubMed Central

    Heidari, Hamid Reza; Bandehpour, Mojgan; Vahidi, Hossein; Barar, Jaleh; Kazemi, Bahram; Naderi-Manesh, Hossein

    2014-01-01

    Introduction: In order to employ Nicotiana tabacum cells as a profitable natural bioreactor for production of bio-functional "Soluble human TRAIL" (ShTRAIL), endoplasmic reticulum (ER) targeted expression and innovative extraction procedures were exploited. Methods: At first, the ShTRAIL encoding gene was sub-cloned into designed H2 helper vector to equip it with potent TMV omega leader sequences, ER sorting signal peptide, poly-histidine tag and ER retention signal peptide (KDEL). Then, the ER targeted ShTRAIL cassette was sequentially sub-cloned into "CaMV-35S" helper and "pGreen-0179" final expression vectors. Afterward, Agrobacterium mediated transformation method was adopted to express the ShTRAIL in the ER of N. tabacum . Next, the ShTRAIL protein was extracted through both phosphate and innovative ascorbate extraction buffers. Subsequently, oligomerization state of the ShTRAIL was evaluated through cross-linking assay and western blot analysis. Then, semi-quantitative western blot analysis was performed to estimate the ShTRAIL production. Finally, biological activity of the ShTRAIL was evaluated through MTT assay. Results: The phosphate buffer extracted ShTRAIL was produced in dimmer form, whereas the ShTRAIL extracted with ascorbate buffer generated trimer form. The ER targeted ShTRAIL strategy increased the ShTRAIL’s production level up to about 20 μg/g of fresh weight of N. tabacum . MTT assay indicated that ascorbate buffer extracted ShTRAIL could prohibit proliferation of A549 cell line. Conclusion: Endoplasmic reticulum expression and reductive ascorbate buffer extraction procedure can be employed to enhance the stability and overall production level of bio-functional recombinant ShTRAIL from transgenic N. tabacum cells. PMID:25337465

  19. Stress Inducible Expression of AtDREB1A Transcription Factor in Transgenic Peanut (Arachis hypogaea L.) Conferred Tolerance to Soil-Moisture Deficit Stress

    PubMed Central

    Sarkar, Tanmoy; Thankappan, Radhakrishnan; Kumar, Abhay; Mishra, Gyan P.; Dobaria, Jentilal R.

    2016-01-01

    Peanut, an important oilseed crop, is gaining priority for the development of drought tolerant genotypes in recent times, since the area under drought is constantly on the rise. To achieve this, one of the important strategies is to genetically engineer the ruling peanut varieties using transcription factor regulating the expression of several downstream, abiotic-stress responsive gene(s). In this study, eight independent transgenic peanut (cv. GG20) lines were developed using AtDREB1A gene, encoding for a transcription factor, through Agrobacterium-mediated genetic transformation. The transgene insertion was confirmed in (T0) using PCR and Dot-blot analysis, while copy-number(s) was ascertained using Southern-blot analysis. The inheritance of AtDREB1A gene in individual transgenic plants (T1 and T2) was confirmed using PCR. In homozygous transgenic plants (T2), under soil-moisture deficit stress, elevated level of AtDREB1A transgene expression was observed by RT-PCR assay. The transgenic plants at 45-d or reproductive growth stage showed tolerance to severe soil-moisture deficit stress. Physio-biochemical parameters such as proline content, osmotic potential, relative water content, electrolytic leakage, and total-chlorophyll content were found positively correlated with growth-related traits without any morphological abnormality, when compared to wild-type. qPCR analysis revealed consistent increase in expression of AtDREB1A gene under progressive soil-moisture deficit stress in two homozygous transgenic plants. The transgene expression showed significant correlation with improved physio-biochemical traits. The improvement of drought-stress tolerance in combination with improved growth-related traits is very essential criterion for a premium peanut cultivar like GG20, so that marginal farmers of India can incur the economic benefits during seasonal drought and water scarcity. PMID:27446163

  20. Workshop on Radio Transients

    NASA Astrophysics Data System (ADS)

    Croft, Steve; Gaensler, Bryan

    2012-04-01

    abstract-type="normal">SummaryWe are entering a new era in the study of variable and transient radio sources. This workshop discussed the instruments and the strategies employed to study those sources, how they are identified and classified, how results from different surveys can be compared, and how radio observations tie in with those at other wavelengths. The emphasis was on learning what common ground there is between the plethora of on-going projects, how methods and code can be shared, and how best practices regarding survey strategy could be adopted. The workshop featured the four topics below. Each topic commenced with a fairly brief introductory talk, which then developed into discussion. By way of preparation, participants had been invited to upload and discuss one slide per topic to a wiki ahead of the workshop. 1. Telescopes, instrumentation and survey strategy. New radio facilities and on-going projects (including upgrades) are both studying the variability of the radio sky, and searching for transients. The discussion first centred on the status of those facilities, and on projects with a time-domain focus, both ongoing and planned, before turning to factors driving choices of instrumentation, such as phased array versus single pixel feeds, the field of view, spatial and time resolution, frequency and bandwidth, depth, area, and cadence of the surveys. 2. Detection, pipelines, and classification. The workshop debated (a) the factors that influence decisions to study variability in the (u,v) plane, in images, or in catalogues, (b) whether, and how much, pipeline code could potentially be shared between one project and another, and which software packages are best for different approaches, (c) how data are stored and later accessed, and (d) how transients and variables are defined and classified. 3. Statistics, interpretation, and synthesis. It then discussed how (i) the choice of facility and strategy and (ii) detection and classification schemes

  1. Thermal transient anemometer

    DOEpatents

    Bailey, J.L.; Vresk, J.

    1989-07-18

    A thermal transient anemometer is disclosed having a thermocouple probe which is utilized to measure the change in temperature over a period of time to provide a measure of fluid flow velocity. The thermocouple probe is located in the fluid flow path and pulsed to heat or cool the probe. The cooling of the heated probe or the heating of the cooled probe from the fluid flow over a period of time is measured to determine the fluid flow velocity. The probe is desired to be locally heated near the tip to increase the efficiency of devices incorporating the probe. 12 figs.

  2. Thermal transient anemometer

    DOEpatents

    Bailey, James L.; Vresk, Josip

    1989-01-01

    A thermal transient anemometer having a thermocouple probe which is utilized to measure the change in temperature over a period of time to provide a measure of fluid flow velocity. The thermocouple probe is located in the fluid flow path and pulsed to heat or cool the probe. The cooling of the heated probe or the heating of the cooled probe from the fluid flow over a period of time is measured to determine the fluid flow velocity. The probe is desired to be locally heated near the tip to increase the efficiency of devices incorporating the probe.

  3. The Rapid Transient Surveyor

    NASA Astrophysics Data System (ADS)

    Baranec, Christoph; Tonry, John; Wright, Shelley; Tully, R. Brent; Lu, Jessica R.; Takamiya, Marianne Y.; Hunter, Lisa

    2016-01-01

    The next decade of astronomy will be dominated by large area surveys (see the detailed discussion in the Astro-2010 Decadal survey and NRC's recent OIR System Report). Ground-based optical transient surveys, e.g., LSST, ZTF and ATLAS and space-based exoplanet, supernova, and lensing surveys such as TESS and WFIRST will join the Gaia all-sky astrometric survey in producing a flood of data that will enable leaps in our understanding of the universe. There is a critical need for further characterization of these discoveries through high angular resolution images, deeper images, spectra, or observations at different cadences or periods than the main surveys. Such follow-up characterization must be well matched to the particular surveys, and requires sufficient additional observing resources and time to cover the extensive number of targets.We describe plans for the Rapid Transient Surveyor (RTS), a permanently mounted, rapid-response, high-cadence facility for follow-up characterization of transient objects on the U. of Hawai'i 2.2-m telescope on Maunakea. RTS will comprise an improved robotic laser adaptive optics system, based on the prototype Robo-AO system (formerly at the Palomar 1.5-m and now at the Kitt Peak 2.2-m telescope), with simultaneous visible and near-infrared imagers as well as a near-infrared integral field spectrograph (R~100, λ = 850 - 1830 nm, 0.15″ spaxels, 8.7″×6.0″ FoV). RTS will achieve an acuity of ~0.07″ in visible wavelengths and < 0.16″ in the near infrared leading to an increase of the infrared point-source sensitivity against the sky background by a factor of ~9, crucial for efficient near-infrared spectroscopy.RTS will allow us to map the dark matter distribution in the z < 0.1 local universe with ten times better accuracy and precision than previous experiments. ATLAS will discover several thousand SNIae per year, measuring SNIa peak brightness, and decline rates, while RTS will measure reddening by dust, confirm SN type and

  4. Measurand transient signal suppressor

    NASA Technical Reports Server (NTRS)

    Bozeman, Richard J., Jr. (Inventor)

    1994-01-01

    A transient signal suppressor for use in a controls system which is adapted to respond to a change in a physical parameter whenever it crosses a predetermined threshold value in a selected direction of increasing or decreasing values with respect to the threshold value and is sustained for a selected discrete time interval is presented. The suppressor includes a sensor transducer for sensing the physical parameter and generating an electrical input signal whenever the sensed physical parameter crosses the threshold level in the selected direction. A manually operated switch is provided for adapting the suppressor to produce an output drive signal whenever the physical parameter crosses the threshold value in the selected direction of increasing or decreasing values. A time delay circuit is selectively adjustable for suppressing the transducer input signal for a preselected one of a plurality of available discrete suppression time and producing an output signal only if the input signal is sustained for a time greater than the selected suppression time. An electronic gate is coupled to receive the transducer input signal and the timer output signal and produce an output drive signal for energizing a control relay whenever the transducer input is a non-transient signal which is sustained beyond the selected time interval.

  5. Characterizing Nanoscale Transient Communication.

    PubMed

    Chen, Yifan; Anwar, Putri Santi; Huang, Limin; Asvial, Muhamad

    2016-04-01

    We consider the novel paradigm of nanoscale transient communication (NTC), where certain components of the small-scale communication link are physically transient. As such, the transmitter and the receiver may change their properties over a prescribed lifespan due to their time-varying structures. The NTC systems may find important applications in the biomedical, environmental, and military fields, where system degradability allows for benign integration into life and environment. In this paper, we analyze the NTC systems from the channel-modeling and capacity-analysis perspectives and focus on the stochastically meaningful slow transience scenario, where the coherence time of degeneration Td is much longer than the coding delay Tc. We first develop novel and parsimonious models to characterize the NTC channels, where three types of physical layers are considered: electromagnetism-based terahertz (THz) communication, diffusion-based molecular communication (DMC), and nanobots-assisted touchable communication (TouchCom). We then revisit the classical performance measure of ϵ-outage channel capacity and take a fresh look at its formulations in the NTC context. Next, we present the notion of capacity degeneration profile (CDP), which describes the reduction of channel capacity with respect to the degeneration time. Finally, we provide numerical examples to demonstrate the features of CDP. To the best of our knowledge, the current work represents a first attempt to systematically evaluate the quality of nanoscale communication systems deteriorating with time. PMID:26955048

  6. Transient Observations with LSST

    NASA Astrophysics Data System (ADS)

    Walkowicz, Lucianne

    2010-12-01

    In the coming decade, LSST';s combination of all-sky coverage, consistent long-term monitoring, and flexible criteria for event identification will revolutionize studies of a wide variety of astrophysical phenomena. The umbrella of time domain science with LSST encompasses objects both familiar and exotic, from classical variables within our Galaxy to explosive cosmological events. LSST will make localization for gravity wave events possible, identify counterparts to GRBs and X-ray flashes, and discover new supernovae. Increased sample sizes of known-but-rare observational phenomena will quantify their distributions for the first time, thus challenging existing theory. Perhaps most excitingly, LSST will provide the opportunity to sample previously untouched regions of parameter space, where transient events are expected on theoretical grounds, but have not yet been observed. LSST will generate "alerts" within 60 seconds of detecting a new transient, permitting the community t o follow up unusual events in greater detail. Here, I highlight some of the scientific opportunities LSST will provide, as well as the challenges we face and opportunities for community involvement.

  7. Engineered Promoters for Potent Transient Overexpression

    PubMed Central

    Ideses, Diana; Tikotzki, Ravid; Shir-Shapira, Hila; Shefi, Orit; Juven-Gershon, Tamar

    2016-01-01

    The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3 (SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as gene-therapy-related applications that require potent expression for extended time periods. PMID:26872062

  8. Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes1[w

    PubMed Central

    Wu, Shuiqin; Schoenbeck, Mark A.; Greenhagen, Bryan T.; Takahashi, Shunji; Lee, Sungbeom; Coates, Robert M.; Chappell, Joseph

    2005-01-01

    A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing β-glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for α-barbatene, thujopsene, and β-chamigrene biosynthesis. PMID:15965019

  9. Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice.

    PubMed

    Ravikumar, G; Manimaran, P; Voleti, S R; Subrahmanyam, D; Sundaram, R M; Bansal, K C; Viraktamath, B C; Balachandran, S M

    2014-06-01

    The cultivation of rice (Oryza sativa L.), a major food crop, requires ample water (30 % of the fresh water available worldwide), and its productivity is greatly affected by drought, the most significant environmental factor. Much research has focussed on identifying quantitative trait loci, stress-regulated genes and transcription factors that will contribute towards the development of climate-resilient/tolerant crop plants in general and rice in particular. The transcription factor DREB1A, identified from the model plant Arabidopsis thaliana, has been reported to enhance stress tolerance against drought stress. We developed transgenic rice plants with AtDREB1A in the background of indica rice cultivar Samba Mahsuri through Agrobacterium-mediated transformation. The AtDREB1A gene was stably inherited and expressed in T1 and T2 plants and in subsequent generations, as indicated by the results of PCR, Southern blot and RT-PCR analyses. Expression of AtDREB1A was induced by drought stress in transgenic rice lines, which were highly tolerant to severe water deficit stress in both the vegetative and reproductive stages without affecting their morphological or agronomic traits. The physiological studies revealed that the expression of AtDREB1A was associated with an increased accumulation of the osmotic substance proline, maintenance of chlorophyll, increased relative water content and decreased ion leakage under drought stress. Most of the homozygous lines were highly tolerant to drought stress and showed significantly a higher grain yield and spikelet fertility relative to the nontransgenic control plants under both stressed and unstressed conditions. The improvement in drought stress tolerance in combination with agronomic traits is very essential in high premium indica rice cultivars, such as Samba Mahsuri, so that farmers can benefit in times of seasonal droughts and water scarcity. PMID:24398893

  10. The joy of transient chaos

    SciTech Connect

    Tél, Tamás

    2015-09-15

    We intend to show that transient chaos is a very appealing, but still not widely appreciated, subfield of nonlinear dynamics. Besides flashing its basic properties and giving a brief overview of the many applications, a few recent transient-chaos-related subjects are introduced in some detail. These include the dynamics of decision making, dispersion, and sedimentation of volcanic ash, doubly transient chaos of undriven autonomous mechanical systems, and a dynamical systems approach to energy absorption or explosion.

  11. The Zwicky Transient Facility

    NASA Astrophysics Data System (ADS)

    Kulkarni, Shrinivas R.

    2016-01-01

    The Zwicky Transient Facility (ZTF) has been designed with a singular focus: a systematic exploration of the night sky at a magnitude level well suited for spectral classification and follow up with the existing class of 4-m to 10-m class telescopes. ZTF is the successor to the Palomar Transient Factory (PTF). The discovery engine for ZTF is a 47 square degree camera (realized through 16 e2V monolithic CCDs) that fills the entire focal plane of the 48-inch Oschin telescope of the Palomar Observatory. Single 30-s epoch sensitivity is about 20.5 in g and R bands. The Infarared Processing & Analysis Center (IPAC) is the data center for ZTF. ZTF is a public-private partnership with equal contributions from a consortium of world-wide partners and an NSF MSIP grant. Forty percent of ZTF time is set aside for two major community surveys: a 3-day cadence survey of high latitudes (to mimic LSST) and a time domain survey of the entire Northern Galactic plane. We expect first light in February 2017 and begin a 3-year survey starting summer of 2017. The first year will be spent on building up deep reference images of the sky (a must for transient surveys). During the second year IPAC will deliver near archival quality photometric products within 12 hours of observations. By comparison to reference images photometric alerts will be sent out. Year 3 will see the near real-time release of image differencing products. A Community Science Advisory Committee (CSAC), chaired by S. Ridgway (NOAO), has been set up to both advise the PI and to ensure that the US community's interests are well served. Astronomers interested in getting a head start on ZTF may wish to peruse the data releases from PTF. Young people (or young at heart) may wish to attend the annual summer school on PTF/ZTF (August, Caltech campus). The Principal Investigator (PI) for the project is S. Kulkarni and the Project Scientist is Eric Bellm.For further details please consult http://www.ptf.caltech.edu/ztf

  12. Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

    PubMed

    Zhao, Yanhong; Liao, Xiaofang; Huang, Zhipeng; Chen, Peng; Zhou, Bujin; Liu, Dongmei; Kong, Xiangjun; Zhou, Ruiyang

    2015-08-01

    Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In

  13. Transient complex peroxisomal interactions

    PubMed Central

    Bonekamp, Nina A.; Schrader, Michael

    2012-01-01

    Mitochondria and peroxisomes are ubiquitous subcellular organelles that fulfill essential metabolic functions, rendering them indispensable for human development and health. Both are highly dynamic organelles that can undergo remarkable changes in morphology and number to accomplish cellular needs. While mitochondrial dynamics are also regulated by frequent fusion events, the fusion of mature peroxisomes in mammalian cells remained a matter of debate. In our recent study, we clarified systematically that there is no complete fusion of mature peroxisomes analogous to mitochondria. Moreover, in contrast to key division components such as DLP1, Fis1 or Mff, mitochondrial fusion proteins were not localized to peroxisomes. However, we discovered and characterized novel transient, complex interactions between individual peroxisomes which may contribute to the homogenization of the often heterogeneous peroxisomal compartment, e.g., by distribution of metabolites, signals or other “molecular information” via interperoxisomal contact sites. PMID:23336019

  14. Optical transient monitor

    NASA Astrophysics Data System (ADS)

    Bernas, Martin; Páta, Petr; Hudec, René; Soldán, Jan; Rezek, Tomáš; Castro-Tirado, Alberto J.

    1998-05-01

    Although there are several optical GRB follow-up systems in operation and/or in development, some of them with a very short response time, they will never be able to provide true simultaneous (no delay) and pre-burst optical data for GRBs. We report on the development and tests of a monitoring experiment expected to be put into test operation in 1998. The system should detect Optical Transients down to mag 6-7 (few seconds duration assumed) over a wide field of view. The system is based on the double CCD wide-field cameras ST8. For the real time evaluation of the signal from both cameras, two TMS 320C40 processors are used. Using two channels differing in spectral sensitivity and processing of temporal sequence of images allows us to eliminate man-made objects and defects of the CCD electronics. The system is controlled by a standard PC computer.

  15. Transient Dentritic Solidification Experiment

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The Transient Dentritic Solidification Experiment (TDSE) is being developed as a candidate for flight aboard the International Space Station. TDSE will study the growth of dentrites (treelike crystalline structures) in a transparent material (succinonitrile or SCN) that mimics the behavior of widely used iron-based metals. Basic work by three Space Shuttle flights (STS-62, STS-75, and STS-87) of the Isothermal Dendritic Growth Experiment (IDGE) is yielding new insights into virtually all industrially relevant metal and alloy forming operations. The TDSE is similar to IDGE, but will maintain a constant temperature while varying pressure on the dentrites. Shown here is a cutaway of the isothermal bath containing its growth cell at the heart of the TDSE. The principal investigator is Matthew Koss of College of the Holy Cross in Worcester, MA. Note: an Acrobat PDF version is available from http://microgravity.nasa.gov/gallery

  16. Agrobacterium-mediated transformation of Mexican lime (Citrus aurantifolia Swingle) using optimized systems for epicotyls and cotelydons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epicotyl and internodal stem segments provide the predominantly used explants for regeneration of transgenic citrus plants following co-cultivation with Agrobacterium. Previous reports using epicotyls segments from Mexican lime have shown low affinity for Agrobacterium tumefaciens infection which re...

  17. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.

    PubMed

    Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha

    2015-08-01

    The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 10(7) conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi. PMID:26051491

  18. Agrobacterium mediated transfer of a mutant Arabidopsis acetolactate synthase gene confers resistance to chlorsulfuron in chicory (Cichorium intybus L.).

    PubMed

    Vermeulen, A; Vaucheret, H; Pautot, V; Chupeau, Y

    1992-06-01

    Leaf discs of C. intybus were inoculated with an Agrobacterium tumefaciens strain harboring a neomycin phosphotransferase (neo) gene for kanamycin resistance and a mutant acetolactate synthase gene (csr1-1) from Arabidopsis thaliana conferring resistance to sulfonylurea herbicides. A regeneration medium was optimized which permitted an efficient shoot regeneration from leaf discs. Transgenic shoots were selected on rooting medium containing 100 mg/l kanamycin sulfate. Integration of the csr1-1 gene into genomic DNA of kanamycin resistant chicory plants was confirmed by Southern blot hybridizations. Analysis of the selfed progenies (S1 and S2) of two independent transformed clones showed that kanamycin and chlorsulfuron resistances were inherited as dominant Mendelian traits. The method described here for producing transformed plants will allow new opportunities for chicory breeding. PMID:24203132

  19. Bacterial Transposons Are Co-Transferred with T-DNA to Rice Chromosomes during Agrobacterium-Mediated Transformation

    PubMed Central

    Kim, Sung-Ryul; An, Gynheung

    2012-01-01

    Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant’s genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying T-DNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences. PMID:22570148

  20. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation.

    PubMed

    Paz, Margie M; Martinez, Juan Carlos; Kalvig, Andrea B; Fonger, Tina M; Wang, Kan

    2006-03-01

    The utility of transformation for soybean improvement requires an efficient system for production of stable transgenic lines. We describe here an improved cotyledonary node method using an alternative explant for Agrobacterium tumefaciens-mediated soybean transformation. We use the term "half-seed" to refer to this alternative cotyledonary explant that is derived from mature seed of soybean following an overnight imbibition and to distinguish it from cotyledonary node derived from 5-7-day-old seedlings. Transformation efficiencies using half-seed explants ranged between 1.4 and 8.7% with an overall efficiency of 3.8% based on the number of transformed events that have been confirmed in the T1 generation by phenotypic assay using the herbicide Liberty (active ingredient glufosinate) and by Southern analysis. This efficiency is 1.5-fold higher than the cotyledonary node method used in our laboratory. Significantly, the half-seed system is simple and does not require deliberate wounding of explants, which is a critical and technically demanding step in the cotyledonary node method. PMID:16249869

  1. Transient heliosheath modulation

    NASA Astrophysics Data System (ADS)

    Quenby, J. J.; Webber, W. R.

    2015-10-01

    Voyager 1 has explored the solar wind-interstellar medium interaction region between the terminal shock and heliopause, following the intensity distribution of Galactic cosmic ray protons above 200 MeV energy. Before this component reached the expected galactic flux level at 121.7 au from the Sun, four episodes of rapid intensity change occurred with a behaviour similar to that found in Forbush Decreases in the inner Solar system, rather than that expected from a mechanism related to models for the long-term modulation found closer to the Sun. Because the mean solar wind flow is both expected and observed to be perpendicular to the radial direction close to the heliopause, an explanation is suggested in terms of transient radial flows related to possible heliopause boundary flapping. It is necessary that the radial flows are of the order either of the sound speed found for conditions downstream of the terminal shock or of the fluctuations found near the boundary by the Voyager 1 Low Energy Charged Particle detector and that the relevant cosmic ray diffusion perpendicular to the mean field is controlled by `slab' fluctuations accounting for about 20 per cent of the total power in the field variance. However, additional radial drift motion related to possible north to south gradients in the magnetic field may allow the inclusion of some diffusion according to the predictions of a theory based upon the presence of 2D turbulence. The required field gradients may arise due to field variation in the field carried by solar plasma flow deflected away from the solar equatorial plane. Modulation amounting to a total 30 per cent drop in galactic intensity requires explanation by a combination of transient effects.

  2. Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

    PubMed Central

    Kim, Hye Jeong; Pak, Jung Hun; Lee, Jai-Heon; Kim, Doh-Hoon; Choi, Hong Kyu; Jung, Ho Won; Lee, Jeong-Dong; Chung, Young-Soo; Ha, Sun-Hwa

    2012-01-01

    The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T2 generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock. PMID:23118971

  3. Genetic transformation and expression of transgenic lines of Populus x euramericana with insect-resistance and salt-tolerance genes.

    PubMed

    Yang, R L; Wang, A X; Zhang, J; Dong, Y; Yang, M S; Wang, J M

    2016-01-01

    We characterized new transgenic varieties of poplar with multiple insect-resistant and salt stress tolerant genes. Two insect-resistant Bacillus thuringiensis (Bt) genes, Cry1Ac and Cry3A, and a salt-tolerant gene, Betaine aldehyde dehydrogenase (BADH) were inserted into a vector, p209-Cry1Ac-Cry3A-BADH. The clone of Populus x euramericana was transformed by the vector using the Agrobacterium-mediated method. Three transgenic lines were assessed using genetic detection and resistance expression analysis. PCR revealed that exogenous genes Cry1Ac, Cry3A, BADH and selective marker gene NPTII were present in three transgenic lines. Quantitative real-time PCR (qPCR) showed significant differences in the transcriptional abundance of three exogenous genes in different lines. Results of assays for Bt toxic proteins showed that the Cry1Ac and Cry3A toxic protein content of each line was 12.83-26.32 and 2108.91-2724.79 ng/g, respectively. The Cry1Ac toxic protein content of different lines was significantly different; the Cry3A toxic protein content was about 100 times higher than that of the Cry1Ac toxic protein. The insect-resistance test revealed the mortality rate of transgenic lines to Hyphantria cunea L1 larvae varied by 42.2-66.7%, which was significantly higher than non-transgenic lines. The mortality rate of L1 and L2 Plagiodera versicolora larvae was 100%. The insecticidal effect of transgenic lines to P. versicolora larvae was higher than that to H. cunea larvae. NaCl stress tolerance of three transgenic lines under 3-6% NaCl concentration was significantly higher than that of non-transgenic lines. PMID:27173305

  4. Development of Transgenic Cotton Lines Expressing Allium sativum Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

    PubMed Central

    Nunna, Hariprasad Rao; Puligundla, Sateesh Kumar; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

    2013-01-01

    Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects. PMID:24023750

  5. Transients by substructuring with DMAP

    NASA Technical Reports Server (NTRS)

    Butler, T. G.

    1978-01-01

    Automated substructuring in level 16 of NASTRAN was employed as a preface to the solution of a direct transient analysis. The DMAP ALTER statements written to adapt the substructuring for transient purposes are explained. Data recovery was accomplished with transfer functions. Proof of the success of the method is presented with an application to a missile structure.

  6. Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa.

    PubMed

    Jiwan, Derick; Roalson, Eric H; Main, Dorrie; Dhingra, Amit

    2013-12-01

    Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae. PMID:23728780

  7. Epstein-Barr Virus Induced Epigenetic Alterations Following Transient Infection

    PubMed Central

    Queen, Krista J.; Shi, Mingxia; Zhang, Fangfang; Cvek, Urska; Scott, Rona S.

    2012-01-01

    Epstein-Barr virus (EBV) is a known tumor virus associated with an increasing array of malignancies; however, the association of the virus with certain malignancies is often erratic. To determine EBV’s contributions to tumorigenesis in a setting of incomplete association, a transient model of infection was established where a clonal CCL185 carcinoma cell line infected with recombinant EBV was allowed to lose viral genomes by withdrawal of selection pressure. Global gene expression comparing EBV-negative, transiently infected clones to uninfected controls identified expression changes in over 1000 genes. Among downregulated genes, several genes known to be DNA methylated in cancer were identified including E-cadherin and PYCARD. A cadherin switch, increased motility and enhanced cellular invasiveness present in EBV-positive cells were retained following viral loss indicating an epigenetic effect. Repression of PYCARD expression was due to increased promoter CpG methylation, whereas loss of E-cadherin expression after transient EBV infection did not correlate with increased DNA methylation of the E-cadherin promoter. Rather, repression of E-cadherin was consistent with formation of a repressive chromatin state. Decreased histone 3 or 4 acetylation at the promoter and 5’ end of the E-cadherin gene was observed in an EBV-negative, transiently infected clone relative to the uninfected controls. These results suggest that EBV can stably alter gene expression in a heritable fashion in formerly infected cells, while its own contribution to the oncogenic process is masked. PMID:23047626

  8. Bounds on Transient Instability for Complex Ecosystems.

    PubMed

    Caravelli, Francesco; Staniczenko, Phillip P A

    2016-01-01

    Stability is a desirable property of complex ecosystems. If a community of interacting species is at a stable equilibrium point then it is able to withstand small perturbations to component species' abundances without suffering adverse effects. In ecology, the Jacobian matrix evaluated at an equilibrium point is known as the community matrix, which describes the population dynamics of interacting species. A system's asymptotic short- and long-term behaviour can be determined from eigenvalues derived from the community matrix. Here we use results from the theory of pseudospectra to describe intermediate, transient dynamics. We first recover the established result that the transition from stable to unstable dynamics includes a region of 'transient instability', where the effect of a small perturbation to species' abundances-to the population vector-is amplified before ultimately decaying. Then we show that the shift from stability to transient instability can be affected by uncertainty in, or small changes to, entries in the community matrix, and determine lower and upper bounds to the maximum amplitude of perturbations to the population vector. Of five different types of community matrix, we find that amplification is least severe when predator-prey interactions dominate. This analysis is relevant to other systems whose dynamics can be expressed in terms of the Jacobian matrix. PMID:27327511

  9. Bounds on Transient Instability for Complex Ecosystems

    PubMed Central

    2016-01-01

    Stability is a desirable property of complex ecosystems. If a community of interacting species is at a stable equilibrium point then it is able to withstand small perturbations to component species’ abundances without suffering adverse effects. In ecology, the Jacobian matrix evaluated at an equilibrium point is known as the community matrix, which describes the population dynamics of interacting species. A system’s asymptotic short- and long-term behaviour can be determined from eigenvalues derived from the community matrix. Here we use results from the theory of pseudospectra to describe intermediate, transient dynamics. We first recover the established result that the transition from stable to unstable dynamics includes a region of ‘transient instability’, where the effect of a small perturbation to species’ abundances—to the population vector—is amplified before ultimately decaying. Then we show that the shift from stability to transient instability can be affected by uncertainty in, or small changes to, entries in the community matrix, and determine lower and upper bounds to the maximum amplitude of perturbations to the population vector. Of five different types of community matrix, we find that amplification is least severe when predator-prey interactions dominate. This analysis is relevant to other systems whose dynamics can be expressed in terms of the Jacobian matrix. PMID:27327511

  10. Transient catalytic combustor model

    NASA Technical Reports Server (NTRS)

    Tien, J. S.

    1981-01-01

    A quasi-steady gas phase and thermally thin substrate model is used to analyze the transient behavior of catalytic monolith combustors in fuel lean operation. The combustor response delay is due to the substrate thermal inertia. Fast response is favored by thin substrate, short catalytic bed length, high combustor inlet and final temperatures, and small gas channel diameters. The calculated gas and substrate temperature time history at different axial positions provides an understanding of how the catalytic combustor responds to an upstream condition change. The computed results also suggest that the gas residence times in the catalytic bed in the after bed space are correlatable with the nondimensional combustor response time. The model also performs steady state combustion calculations; and the computed steady state emission characteristics show agreement with available experimental data in the range of parameters covered. A catalytic combustor design for automotive gas turbine engine which has reasonably fast response ( 1 second) and can satisfy the emission goals in an acceptable total combustor length is possible.

  11. Agrobacterium tumefaciens-mediated transient transformation of Arabidopsis thaliana leaves.

    PubMed

    Mangano, Silvina; Gonzalez, Cintia Daniela; Petruccelli, Silvana

    2014-01-01

    Transient assays provide a convenient alternative to stable transformation. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Nevertheless, at difference of tobacco and other plant species, Arabidopsis thaliana remains recalcitrant to routine transient assays. In this chapter, we describe a transient expression assay using simple infiltration of intact Arabidopsis leaves with Agrobacterium tumefaciens carrying a plasmid expressing a reporter fluorescent protein. In this protocol, Agrobacterium aggressiveness was increased by a prolonged treatment in an induction medium deficient in nutrients and containing acetosyringone. Besides, Arabidopsis plants were cultivated in intermediate photoperiod (12 h light-12 h dark) to promote leaf growth. PMID:24057365

  12. Initiation of a coronal transient

    SciTech Connect

    Low, B.C.; Munro, R.H.; Fisher, R.R.

    1982-03-01

    This paper analyzes the coronal transient/eruptive prominence event of 1980 August 5 observed by the Mauna Loa experiment system. This event yielded data on the early development of the transient in the low corona between 1.2 R/sub sun/ and 2.2 R/sub sun/, information which was not available when earlier attempts were made to explain transient phenomena. The transient's initial appearance in the form of a rising density-depleted structure, prior to the eruption of the associated prominence, can be explained as an effect of magnetic buoyancy. The data indicate that this transient has a density depletion of 17% to 33% relative to an undisturbed corona which is approximately isothermal with a temperature of 1.5 x 10/sup 6/ K and a coronal density of 1.0 x 10/sup 9/ cm/sup -3/ at the base of the corona. The height versus base length relationship of the evolving transient resembles, remarkably well, the theoretical predictions obtained from a quasi-static model of a margnetically buoyant loop system. By matching this relationship with the theoretical model, we estimate the magnetic field at the base of the transient to be between 2 and 3 gauss. It is also shown that the initial, nearly constant speed of the top of the transient, 80 +- 20 km s/sup -1/, is consistent with a theoretical estimate calculated from the quasi-static model. These results suggest that some transients are not initiated impulsively, the initial stage of the development being driven by a quasi-static response to a slow change in magnetic field conditions at the base of the corona.

  13. Canonical transient receptor potential 5.

    PubMed

    Beech, D J

    2007-01-01

    Canonical transient receptor potential 5 TRPC5 (also TrpC5, trp-5 or trp5) is one of the seven mammalian TRPC proteins. Its known functional property is that of a mixed cationic plasma membrane channel with calcium permeability. It is active alone or as a heteromultimeric assembly with TRPC1; TRPC4 and TRPC3 may also be involved. Multiple activators of TRPC5 are emerging, including various G protein-coupled receptor agonists, lysophospholipids, lanthanide ions and, in some contexts, calcium store depletion. Intracellular calcium has complex impact on TRPC5, including a permissive role for other activators, as well as inhibition at high concentrations. Protein kinase C is inhibitory and mediates desensitisation following receptor activation. Tonic TRPC5 activity is detected and may reflect the presence of constitutive activation signals. The channel has voltage dependence but the biological significance of this is unknown; it is partially due to intracellular magnesium blockade at aspartic acid residue 633. Protein partners include calmodulin, CaBP1, enkurin, Na(+)-H+ exchange regulatory factor (NHERF) and stathmin. TRPC5 is included in local vesicular trafficking regulated by growth factors through phosphatidylinositol (PI)-3-kinase, Rac1 and PIP-5-kinase. Inhibition of myosin light chain kinase suppresses TRPC5, possibly via an effect on trafficking. Biological roles of TRPC5 are emerging but more reports on this aspect are needed. One proposed role is as a mediator of calcium entry and excitation in smooth muscle, another as an inhibitor of neuronal growth cone extension. The latter is intriguing in view of the original cloning of the human TRPC5 gene from a region of the X chromosome linked to mental retardation. TRPC5 is a broadly expressed calcium channel with capability to act as an integrator of extracellular and intracellular signals at the level of calcium entry. PMID:17217053

  14. Transient upsets in microprocessor controllers

    NASA Technical Reports Server (NTRS)

    Glaser, R. E.; Masson, G. M.

    1981-01-01

    The modeling and analysis of transient faults in microprocessor based controllers are discussed. Such controllers typically consist of a microprocessor, read only memory storing and application program, random access memory for data storage, and input/output devices for external communications. The effects of transient faults on the performance of the controller are reviewed. An instruction level perspective of performance is taken which is the basis of a useful high level program state description of the microprocessor controller. A transition matrix is defined which determines the controller's response to transient fault arrivals.

  15. Transient Photoconduction in Phthalocyanines

    NASA Astrophysics Data System (ADS)

    Petty, David Matthew

    1991-02-01

    Available from UMI in association with The British Library. Requires signed TDF. Photoconduction occurs when illumination interacts with a sample to cause a change in the flow of current through that sample. The work described in this thesis is centred on the investigation of the processes which effect photoconduction. These processes include the way in which charge carriers move the electrodes, the way in which charge carriers transit the bulk and the way in which charge carriers are excited and relax. The thesis is divided into three parts. Part I is essentially a literature survey of the theories which describe photoconduction. An overview of photoconduction at a microscopic level in all types of sample is given. By categorising the sample as neutral or supporting a single carrier space charge, the mathematical description of photoconduction behaviour can be simplified. In Part II existing theories which describe photoconduction in charged samples are reviewed. A new theory yields two important equations: (i) the Plasma equation which describes the conditions for a neutral sample and those for a single carrier space charge, (ii) the BUIC (Bulk Uniform Injected Charge) equation which describes the time and field dependence of a photoconductor which supports a single carrier space charge. The BUIC equation is compared with existing theories and its ability to describe photoconduction in sandwich cells of phthalocyanine compounds is discussed. Part III contains an investigation of photoconduction in phthalocyanines. Steady state and chopped light transient photoconduction experiments are carried out on nine different Phthalocyanine compounds under a variety of conditions. The results obtained are interpreted using the BUIC, Plasma equations and neutral photoconductor theories.

  16. Catalina/Palomar Optical Transient

    NASA Astrophysics Data System (ADS)

    Ganeshalingam, M.; Griffith, C. V.; Filippenko, A. V.; Foley, R. J.

    2008-07-01

    Inspection of a CCD spectrum (range 330-1000 nm), obtained on July 7 UT with the 3-m Shane reflector (+ Kast spectrograph) at Lick Observatory, shows that the transient reported in ATel 1604 has a very blue, featureless continuum. This could be a very young Type II supernova (though at this time no clear hydrogen Balmer features are present), or some kind of Galactic transient. Further observations are encouraged.

  17. Transient Tsunamis in Lakes

    NASA Astrophysics Data System (ADS)

    Couston, L.; Mei, C.; Alam, M.

    2013-12-01

    A large number of lakes are surrounded by steep and unstable mountains with slopes prone to failure. As a result, landslides are likely to occur and impact water sitting in closed reservoirs. These rare geological phenomena pose serious threats to dam reservoirs and nearshore facilities because they can generate unexpectedly large tsunami waves. In fact, the tallest wave experienced by contemporary humans occurred because of a landslide in the narrow bay of Lituya in 1958, and five years later, a deadly landslide tsunami overtopped Lake Vajont's dam, flooding and damaging villages along the lakefront and in the Piave valley. If unstable slopes and potential slides are detected ahead of time, inundation maps can be drawn to help people know the risks, and mitigate the destructive power of the ensuing waves. These maps give the maximum wave runup height along the lake's vertical and sloping boundaries, and can be obtained by numerical simulations. Keeping track of the moving shorelines along beaches is challenging in classical Eulerian formulations because the horizontal extent of the fluid domain can change over time. As a result, assuming a solid slide and nonbreaking waves, here we develop a nonlinear shallow-water model equation in the Lagrangian framework to address the problem of transient landslide-tsunamis. In this manner, the shorelines' three-dimensional motion is part of the solution. The model equation is hyperbolic and can be solved numerically by finite differences. Here, a 4th order Runge-Kutta method and a compact finite-difference scheme are implemented to integrate in time and spatially discretize the forced shallow-water equation in Lagrangian coordinates. The formulation is applied to different lake and slide geometries to better understand the effects of the lake's finite lengths and slide's forcing mechanism on the generated wavefield. Specifically, for a slide moving down a plane beach, we show that edge-waves trapped by the shoreline and free

  18. Transient Alerts in LSST

    NASA Astrophysics Data System (ADS)

    Kantor, J.

    During LSST observing, transient events will be detected and alerts generated at the LSST Archive Center at NCSA in Champaign-Illinois. As a very high rate of alerts is expected, approaching ˜ 10 million per night, we plan for VOEvent-compliant Distributor/Brokers (http://voevent.org) to be the primary end-points of the full LSST alert streams. End users will then use these Distributor/Brokers to classify and filter events on the stream for those fitting their science goals. These Distributor/Brokers are envisioned to be operated as a community service by third parties who will have signed MOUs with LSST. The exact identification of Distributor/Brokers to receive alerts will be determined as LSST approaches full operations and may change over time, but it is in our interest to identify and coordinate with them as early as possible. LSST will also operate a limited Distributor/Broker with a filtering capability at the Archive Center, to allow alerts to be sent directly to a limited number of entities that for some reason need to have a more direct connection to LSST. This might include, for example, observatories with significant follow-up capabilities whose observing may temporarily be more directly tied to LSST observing. It will let astronomers create simple filters that limit what alerts are ultimately forwarded to them. These user defined filters will be possible to specify using an SQL-like declarative language, or short snippets of (likely Python) code. We emphasize that this LSST-provided capability will be limited, and is not intended to satisfy the wide variety of use cases that a full-fledged public Event Distributor/Broker could. End users will not be able to subscribe to full, unfiltered, alert streams coming directly from LSST. In this session, we will discuss anticipated LSST data rates, and capabilities for alert processing and distribution/brokering. We will clarify what the LSST Observatory will provide versus what we anticipate will be a

  19. Isolating parameter sensitivity in reach scale transient storage modeling

    NASA Astrophysics Data System (ADS)

    Schmadel, Noah M.; Neilson, Bethany T.; Heavilin, Justin E.; Wörman, Anders

    2016-03-01

    Parameter sensitivity analyses, although necessary to assess identifiability, may not lead to an increased understanding or accurate representation of transient storage processes when associated parameter sensitivities are muted. Reducing the number of uncertain calibration parameters through field-based measurements may allow for more realistic representations and improved predictive capabilities of reach scale stream solute transport. Using a two-zone transient storage model, we examined the spatial detail necessary to set parameters describing hydraulic characteristics and isolate the sensitivity of the parameters associated with transient storage processes. We represented uncertain parameter distributions as triangular fuzzy numbers and used closed form statistical moment solutions to express parameter sensitivity thus avoiding copious model simulations. These solutions also allowed for the direct incorporation of different levels of spatial information regarding hydraulic characteristics. To establish a baseline for comparison, we performed a sensitivity analysis considering all model parameters as uncertain. Next, we set hydraulic parameters as the reach averages, leaving the transient storage parameters as uncertain, and repeated the analysis. Lastly, we incorporated high resolution hydraulic information assessed from aerial imagery to examine whether more spatial detail was necessary to isolate the sensitivity of transient storage parameters. We found that a reach-average hydraulic representation, as opposed to using detailed spatial information, was sufficient to highlight transient storage parameter sensitivity and provide more information regarding the potential identifiability of these parameters.

  20. Improving phosphorus acquisition of white clover (Trifolium repens L.) by transgenic expression of plant-derived phytase and acid phosphatase genes.

    PubMed

    Ma, Xue-Feng; Wright, Elane; Ge, Yaxin; Bell, Jeremey; Xi, Yajun; Bouton, Joseph H; Wang, Zeng-Yu

    2009-04-01

    Phosphate is one of the least available macronutrients restricting crop production in many ecosystems. A phytase gene (MtPHY1) and a purple acid phosphatase gene (MtPAP1), both isolated from the model legume Medicago truncatula, were introduced into white clover (Trifolium repens L.) by Agrobacterium-mediated transformation. The transgenes were driven by the constitutive CaMV35S promoter or the root-specific MtPT1 promoter. Transcripts were detected in roots of the transgenic plants. Phytase or acid phosphatase (APase) activities in root apoplasts of the transgenic plants were increased up to three-fold compared to the wild type control. After the plants were grown 80 days in sand pots supplied with organic phosphorus (Po) as the sole P source, dry weights of shoot tissues of the best performing transgenic plants almost doubled that of the control and were comparable to the counterparts supplied with inorganic phosphorus (Pi). Relative biomass production of the transgenics under Po treatment was over 90% and 80% of that from the Pi treatment when the plants were grown in hydroponics (40 days) and sand pots (80 days), respectively. In contrast, biomass of the wild type controls under Po treatment was only about 50% of the Pi treatment in either hydroponic cultures or sand pots. In addition, shoot P concentrations of the transgenic plants were significantly increased compared to the control. Transgenic plants accumulated much higher amounts of total P (up to 2.6-fold after 80 days of growth) than the control in Po supplied sand pots. The results showed that transgenic expression of MtPHY1 or MtPAP1 in white clover plants increased their abilities of utilizing organic phosphorus in response to P deficiency. PMID:26493137

  1. BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

    DOE PAGESBeta

    Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; Foster, Cliff E.; Vogel, John P.; Karlen, Steven D.; Ralph, John; Sedbrook, John C.

    2016-02-04

    Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the

  2. Rapid in vivo analysis of synthetic promoters for plant pathogen phytosensing

    PubMed Central

    2011-01-01

    Background We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing. Results For rapid analysis, an Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly. Conclusions These observations demonstrate that the Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants. PMID:22093754

  3. Gaia transient detection efficiency: hunting for nuclear transients

    NASA Astrophysics Data System (ADS)

    Blagorodnova, N.; Van Velzen, S.; Harrison, D. L.; Koposov, S.; Mattila, S.; Campbell, H.; Walton, N. A.; Wyrzykowski, Ł.

    2016-01-01

    We present a study of the detectability of transient events associated with galaxies for the Gaia European Space Agency astrometric mission. We simulated the on-board detections, and on-ground processing for a mock galaxy catalogue to establish the properties required for the discovery of transient events by Gaia, specifically tidal disruption events (TDEs) and supernovae (SNe). Transients may either be discovered by the on-board detection of a new source or by the brightening of a previously known source. We show that Gaia transients can be identified as new detections on-board for offsets from the host galaxy nucleus of 0.1-0.5 arcsec, depending on magnitude and scanning angle. The Gaia detection system shows no significant loss of SNe at close radial distances to the nucleus. We used the detection efficiencies to predict the number of transients events discovered by Gaia. For a limiting magnitude of 19, we expect around 1300 SNe per year: 65 per cent SN Ia, 28 per cent SN II and 7 per cent SN Ibc, and ˜20 TDEs per year.

  4. Spike-dip transformation of Setaria viridis.

    PubMed

    Saha, Prasenjit; Blumwald, Eduardo

    2016-04-01

    Traditional method of Agrobacterium-mediated transformation through the generation of tissue culture had limited success for Setaria viridis, an emerging C4 monocot model. Here we present an efficient in planta method for Agrobacterium-mediated genetic transformation of S. viridis using spike dip. Pre-anthesis developing spikes were dipped into a solution of Agrobacterium tumefaciens strain AGL1 harboring the β-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to standardize and optimize conditions for transient as well as stable transformations. A transformation efficiency of 0.8 ± 0.1% was obtained after dipping of 5-day-old S3 spikes for 20 min in Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025% Silwet L-77 and 200 μm acetosyringone. Reproducibility of this method was demonstrated by generating stable transgenic lines expressing β-glucuronidase plus (GUSplus), green fluorescent protein (GFP) and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or intron-interrupted maize ubiquitin (Ubi) promoters from three S. viridis genotypes. Expression of these reporter genes in transient assays as well as in T1 stable transformed plants was monitored using histochemical, fluorometric GUS activity and fluorescence microscopy. Molecular analysis of transgenic lines revealed stable integration of transgenes into the genome, and inherited transgenes expressed in the subsequent generations. This approach provides opportunities for the high-throughput transformation and potentially facilitates translational research in a monocot model plant. PMID:26932666

  5. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses.

    PubMed

    Raikwar, Shailendra; Srivastava, Vineet K; Gill, Sarvajeet S; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression. PMID:26734018

  6. Emerging Importance of Helicases in Plant Stress Tolerance: Characterization of Oryza sativa Repair Helicase XPB2 Promoter and Its Functional Validation in Tobacco under Multiple Stresses

    PubMed Central

    Raikwar, Shailendra; Srivastava, Vineet K.; Gill, Sarvajeet S.; Tuteja, Renu; Tuteja, Narendra

    2015-01-01

    Genetic material always remains at the risk of spontaneous or induced damage which challenges the normal functioning of DNA molecule, thus, DNA repair is vital to protect the organisms against genetic damage. Helicases, the unique molecular motors, are emerged as prospective molecules to engineer stress tolerance in plants and are involved in nucleic acid metabolism including DNA repair. The repair helicase, XPB is an evolutionary conserved protein present in different organisms, including plants. Availability of few efficient promoters for gene expression in plants provoked us to study the promoter of XPB for better understanding of gene regulation under stress conditions. Here, we report the in silico analysis of novel stress inducible promoter of Oryza sativa XPB2 (OsXPB2). The in vivo validation of functionality/activity of OsXPB2 promoter under abiotic and hormonal stress conditions was performed by Agrobacterium-mediated transient assay in tobacco leaves using OsXPB2::GUS chimeric construct. The present research revealed that OsXPB2 promoter contains cis-elements accounting for various abiotic stresses (salt, dehydration, or cold) and hormone (Auxin, ABA, or MeJA) induced GUS expression/activity in the promoter-reporter assay. The promoter region of OsXPB2 contains CACG, GTAACG, CACGTG, CGTCA CCGCCGCGCT cis acting-elements which are reported to be salt, dehydration, cold, MeJA, or ABA responsive, respectively. Functional analysis was done by Agrobacterium-mediated transient assay using agroinfiltration in tobacco leaves, followed by GUS staining and fluorescence quantitative analyses. The results revealed high induction of GUS activity under multiple abiotic stresses as compared to mock treated control. The present findings suggest that OsXPB2 promoter is a multi-stress inducible promoter and has potential applications in sustainable crop production under abiotic stresses by regulating desirable pattern of gene expression. PMID:26734018

  7. Heater drain system transient monitoring

    SciTech Connect

    Voll, B.J.; Farsaci, C.D.

    1995-12-01

    Feedwater heater drain systems are susceptible to unstable, two phase flow conditions. These instabilities are difficult to predict and are dependent on plant-specific system designs and operating conditions. Therefore, significant vibrations and transient events can occur that the systems are not specifically designed for. This paper describes how heater drain system responses due to unanticipated transient events at a nuclear plant were captured and quantified using a digital data acquisition system. The setup of the data acquisition system, including the determination of what parameters to monitor and how to effectively capture potential transient events, is discussed. This paper also discusses the monitoring results and their relevance to system modification evaluations and root cause evaluations.

  8. Transient Faults in Computer Systems

    NASA Technical Reports Server (NTRS)

    Masson, Gerald M.

    1993-01-01

    A powerful technique particularly appropriate for the detection of errors caused by transient faults in computer systems was developed. The technique can be implemented in either software or hardware; the research conducted thus far primarily considered software implementations. The error detection technique developed has the distinct advantage of having provably complete coverage of all errors caused by transient faults that affect the output produced by the execution of a program. In other words, the technique does not have to be tuned to a particular error model to enhance error coverage. Also, the correctness of the technique can be formally verified. The technique uses time and software redundancy. The foundation for an effective, low-overhead, software-based certification trail approach to real-time error detection resulting from transient fault phenomena was developed.

  9. Transient voltage oscillations in coils

    SciTech Connect

    Chowdhuri, P.

    1985-01-01

    Magnet coils may be excited into internal voltage oscillations by transient voltages. Such oscillations may electrically stress the magnet's dielectric components to many times its normal stress. This may precipitate a dielectric failure, and the attendant prolonged loss of service and costly repair work. Therefore, it is important to know the natural frequencies of oscillations of a magnet during the design stage, and to determine whether the expected switching transient voltages can excite the magnet into high-voltage internal oscillations. The series capacitance of a winding significantly affects its natural frequencies. However, the series capacitance is difficult to calculate, because it may comprise complex capacitance network, consisting of intra- and inter-coil turn-to-turn capacitances of the coil sections. A method of calculating the series capacitance of a winding is proposed. This method is rigorous but simple to execute. The time-varying transient voltages along the winding are also calculated.

  10. Studies of Transient Meteor Activity

    NASA Technical Reports Server (NTRS)

    Jenniskens, Peter M. M.

    2002-01-01

    Meteoroids bombard Earth's atmosphere daily, but occasionally meteor rates increase to unusual high levels when Earth crosses the relatively fresh ejecta of comets. These transient events in meteor activity provide clues about the whereabouts of Earth-threatening long-period comets, the mechanisms of large-grain dust ejection from comets, and the particle composition and size distribution of the cometary ejecta. Observations of these transient events provide important insight in natural processes that determine the large grain dust environment of comets, in natural phenomena that were prevalent during the time of the origin of life, and in processes that determine the hazard of civilizations to large impacts and of man-made satellites to the periodic blizzard of small meteoroids. In this proposal, three tasks form a coherent program aimed at elucidating various aspects of meteor outbursts, with special reference to planetary astronomy and astrobiology. Task 1 was a ground-based effort to observe periods of transient meteor activity. This includes: (1) stereoscopic imaging of meteors during transient meteor events for measurements of particle size distribution, meteoroid orbital dispersions and fluxes; and (2) technical support for Global-MS-Net, a network of amateur-operated automatic counting stations for meteor reflections from commercial VHF radio and TV broadcasting stations, keeping a 24h vigil on the level of meteor activity for the detection of new meteor streams. Task 2 consisted of ground-based and satellite born spectroscopic observations of meteors and meteor trains during transient meteor events for measurements of elemental composition, the presence of organic matter in the meteoroids, and products generated by the interaction of the meteoroid with the atmosphere. Task 3 was an airborne effort to explore the 2000 Leonid meteor outbursts, which are anticipated to be the most significant of transient meteor activity events in the remainder of the

  11. Transient diabetes insipidus in pregnancy

    PubMed Central

    Gunawardana, Kavinga; Grossman, Ashley

    2015-01-01

    Summary Gestational diabetes insipidus (DI) is a rare complication of pregnancy, usually developing in the third trimester and remitting spontaneously 4–6 weeks post-partum. It is mainly caused by excessive vasopressinase activity, an enzyme expressed by placental trophoblasts which metabolises arginine vasopressin (AVP). Its diagnosis is challenging, and the treatment requires desmopressin. A 38-year-old Chinese woman was referred in the 37th week of her first single-gestation due to polyuria, nocturia and polydipsia. She was known to have gestational diabetes mellitus diagnosed in the second trimester, well-controlled with diet. Her medical history was unremarkable. Physical examination demonstrated decreased skin turgor; her blood pressure was 102/63 mmHg, heart rate 78 beats/min and weight 53 kg (BMI 22.6 kg/m2). Laboratory data revealed low urine osmolality 89 mOsmol/kg (350–1000), serum osmolality 293 mOsmol/kg (278–295), serum sodium 144 mmol/l (135–145), potassium 4.1 mmol/l (3.5–5.0), urea 2.2 mmol/l (2.5–6.7), glucose 3.5 mmol/l and HbA1c 5.3%. Bilirubin, alanine transaminase, alkaline phosphatase and full blood count were normal. The patient was started on desmopressin with improvement in her symptoms, and normalisation of serum and urine osmolality (280 and 310 mOsmol/kg respectively). A fetus was delivered at the 39th week without major problems. After delivery, desmopressin was stopped and she had no further evidence of polyuria, polydipsia or nocturia. Her sodium, serum/urine osmolality at 12-weeks post-partum were normal. A pituitary magnetic resonance imaging (MRI) revealed the neurohypophyseal T1-bright spot situated ectopically, with a normal adenohypophysis and infundibulum. She remains clinically well, currently breastfeeding, and off all medication. This case illustrates some challenges in the diagnosis and management of transient gestational DI. Learning points Gestational DI is a rare complication of

  12. Transient classification and novae ejecta

    NASA Astrophysics Data System (ADS)

    Williams, Robert

    2016-07-01

    A third parameter, in addition to luminosity and rate of brightness decline, derived from the spectra of transients is suggested as a means of more accurately classifying objects in outburst. Principal component analysis of the spectra of transients is suggested as the best way to determine the third parameter. A model is suggested for novae ejecta that is based on the ballistic ejection of an ensemble of clouds having a wide range of sizes. Short term brightness fluctuations of novae, the formation of dust, and the production of X-ray emission follow naturally from such a picture of the evolving clouds.

  13. Modeling a Transient Catalytic Combustor

    NASA Technical Reports Server (NTRS)

    Tien, J. S.

    1985-01-01

    Transient model of monolith catalytic combustor presented in report done under NASA/DOE contract. Model assumes quasi-steady gas phase and thermally "thin" solid. In gas-phase treatment, several quasi-global chemical reactions assumed capable of describing CO and unburnt hydrocarbon emissions in fuel-lean operations. In steady-state computation presented, influence of selected operating and design parameters on minimum combustor length studied. When fast transient responses required, both steady and unsteady studies made to achieve meaningful compromise in design.

  14. Transient-mediated fate determination in a transcriptional circuit of HIV

    SciTech Connect

    Weinberger, Leor S.; Dar, Roy D.; Simpson, Michael L

    2008-01-01

    Steady-state behavior and bistability have been proposed as mechanisms for decision-making in gene circuits1-3. However, transient gene expression has also been proposed to control cell fate4, 5 with the decision arbitrated by the lifetime of the expression transient. Here, we report that transcriptional positive-feedback plays a critical role in determining HIV infected cell-fate by extending the duration of Tat expression transients6, 7 far beyond what protein half-life modulation can achieve. To directly quantify feedback strength and its effects on the duration of Tat transcriptional pulses, we exploit the noise inherent to gene-expression and measure shifts in the autocorrelation of expression noise. The results indicate that transcriptional positive-feedback extends the single-cell Tat expression lifetime by 2-to 6-fold for both minimal Tat circuits and full-length, actively-replicating HIV-1. Importantly, artificial weakening of Tat positive-feedback shortened the duration of Tat expression transients and biased the probability in favor of latency. Thus, transcriptional positive-feedback appears to modulate transient expression lifetime and thereby control cell-fate in HIV.

  15. Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests

    PubMed Central

    Yarasi, Bharathi; Sadumpati, Vijayakumar; Immanni, China Pasalu; Vudem, Dasavantha Reddy; Khareedu, Venkateswara Rao

    2008-01-01

    Background Rice (Oryza sativa) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of Allium sativum leaf agglutinin (asal) gene, and its expression in elite indica rice cultivars using Agrobacterium-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests. Results Allium sativum leaf lectin gene (asal), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with Agrobacterium harbouring pSB111 super-binary vector comprising garlic lectin gene asal along with the herbicide resistance gene bar, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. In planta insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects. Conclusion In planta insect bioassays were carried out on asal transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high

  16. Radio transients: an antediluvian review

    NASA Astrophysics Data System (ADS)

    Fender, R. P.; Bell, M. E.

    2011-09-01

    We are at the dawn of a new golden age for radio astronomy, with a new generation of facilities under construction and the global community focused on the Square Kilometre Array as its goal for the next decade. These new facilities offer orders of magnitude improvements in survey speed compared to existing radio telescopes and arrays. Furthermore, the study of transient and variable radio sources, and what they can tell us about the extremes of astrophysics as well as the state of the diffuse intervening media, have been embraced as key science projects for these new facilities. In this paper we review the studies of the populations of radio transients made to date, largely based upon archival surveys. Many of these radio transients and variables have been found in the image plane, and their astrophysical origin remains unclear. We take this population and combine it with sensitivity estimates for the next generation arrays to demonstrate that in the coming decade we may find ourselves detecting 10^5 image plane radio transients per year, providing a vast and rich field of research and an almost limitless set of targets for multi-wavelength follow up.

  17. Detection of transient fluorine atoms

    NASA Astrophysics Data System (ADS)

    Loge, Gary W.; Nereson, Norris; Fry, Herbert A.

    1987-09-01

    A KrF laser with a fluence of 50 mJ/cm2 was used to photolyze either uranium hexafluoride or molecular fluorine, yielding a transient number density of fluorine atoms. The rise and decay of the atomic fluorine density was observed by transient absorption of a 25-μm Pb-salt diode laser. To prevent the diode laser wavelength from drifting out of resonance with the atomic fluorine line, part of the beam was split off and sent through a microwave discharge fluorine atom cell. This allowed a wavelength modulation-feedback technique to be used to lock the diode laser wavelength onto the atomic line. The remaining diode laser beam was made collinear with the excimer laser beam using a LiF window with a 45° angle of incidence to reflect the infrared beam while transmitting most of the uv beam. Using this setup along with a transient digitizer to average between 100 and 200 transient absorption profiles, fluorine atom number densities on the order of 1014 cm-3 in a 1.7 m pathlength were detected. The signal observed were about a factor of two less than expected from known photolysis and atomic fluorine absorption cross-sections.

  18. ATLAS discoveries of optical transients

    NASA Astrophysics Data System (ADS)

    Tonry, J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Rest, A.; Smith, K. W.; Smartt, S. J.

    2016-03-01

    We report the following transients found by the ATLAS survey (Tonry et al. ATel #8680). ATLAS is a twin 0.5m telescope system on Haleakala and Mauna Loa. The first unit is robotically operational on Haleakala (see http://www.fallingstar.com).

  19. Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria.

    PubMed

    Wu, Tingquan; Tang, Dingzhong; Chen, Weida; Huang, Hexun; Wang, Rui; Chen, Yongfang

    2013-09-15

    Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed

  20. Expression of the Rice Arginase Gene OsARG in Cotton Influences the Morphology and Nitrogen Transition of Seedlings

    PubMed Central

    Zhang, Rui; Liang, Chengzhen; Wan, Jianmin; Wang, Yanling; Zhai, Honghong; Guo, Sandui

    2015-01-01

    Arginase is the only enzyme capable of producing urea in plants. This enzyme also contributes to many important biological functions during plant growth and development, such as seed development, root development and plant nitrogen using. The unique rice arginase gene OsARG is known to affect nitrogen use efficiency and is also associated with higher yields in rice. In this study, we transformed OsARG into upland cotton R18 by Agrobacterium-mediated genetic transformation and analyzed the function of OsARG in transgenic cotton. Two independent OsARG expression transgenic cotton lines, ARG-26 and ARG-38, were obtained via transformation. Southern blot analysis indicated that two copies and one copy of the OsARG gene were integrated into the ARG-26 and ARG-38 genomes, respectively. Enzyme activity and RNA transcription analysis revealed that the OsARG gene is highly expressed in cotton. The nitric oxide content and the morphology of ARG-26 and ARG-38 seedlings were both affected by expression of the OsARG gene. Field experiments indicated that the polyamine and nitrogen content increased by more than two-fold in the T3 generation plants of the transgenic cotton lines ARG-26-2, ARG-26-7, ARG-38-8, and ARG-38-11, as compared with the control plants. After harvesting cotton fibers grown in field conditions, we analyzed the quality of fiber and found that the fiber length was increased in the transgenic lines. The average cotton fiber length for all of the transgenic cotton lines was two millimeters longer than the fibers of the control plants; the average cotton fiber lengths were 31.94 mm, 32.00 mm, 32.68 mm and 32.84 mm in the ARG-26ARG-26-2, ARG-26-7, ARG-38-8 and ARG-38-11 lines, respectively, but the average fiber length of the control plants was 29.36mm. Our results indicate that the OsARG gene could potentially be used to improve cotton fiber length traits. PMID:26528551

  1. Spectroscopic Classifications of Optical Transients with SOAR

    NASA Astrophysics Data System (ADS)

    Hounsell, R. A.; Miller, J. A.; Pan, Y.-C.; Foley, R. J.; Jha, S. W.; Rest, A.; Scolnic, D.; Smith, K. W.; Wright, D.; Smartt, S. J.; Huber, M.; Chambers, K. C.; Flewelling, H.; Willman, M.; Primak, N.; Schultz, A.; Gibson, B.; Magnier, E.; Waters, C.; Tonry, J.; Wainscoat, R. J.

    2016-06-01

    We report the following classifications of optical transients from spectroscopic observations with the Goodman spectrograph on the SOAR 4-m telescope. Targets were supplied by the Pan-STARRS Survey for Transients (PSST).

  2. Design of chimeric antigen receptors with integrated controllable transient functions.

    PubMed

    Juillerat, Alexandre; Marechal, Alan; Filhol, Jean-Marie; Valton, Julien; Duclert, Aymeric; Poirot, Laurent; Duchateau, Philippe

    2016-01-01

    The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug. PMID:26750734

  3. Superresolution microscopy with transient binding.

    PubMed

    Molle, Julia; Raab, Mario; Holzmeister, Susanne; Schmitt-Monreal, Daniel; Grohmann, Dina; He, Zhike; Tinnefeld, Philip

    2016-06-01

    For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution. PMID:26773299

  4. Transient simulation of absorption machines

    NASA Astrophysics Data System (ADS)

    Anand, D. K.; Allen, R. W.; Kumar, B.

    A model for a water-cooled Lithium-Bromide/water absorption chiller is presented. Its transient response both during the start-up phase and during the shut-off period is predicted. The simulation model incorporates such influencing factors as the thermodynamic properties of the working fluid, the absorbent, the heat-transfer configuration of different components of the chiller and related physical data. The time constants of different components are controlled by a set of key parameters that have been identified. The results show a variable but at times significant amount of time delay before the chiller capacity gets close to its steady-state value. The model is intended to provide an insight into the mechanism of build-up to steady-state performance. By recognizing the significant factors contributing to transient degradation, steps can be taken to reduce such degradation.

  5. Transient Ablation of Teflon Hemispheres

    NASA Technical Reports Server (NTRS)

    Arai, Norio; Karashima, Kei-ichi; Sato, Kiyoshi

    1997-01-01

    For high-speed entry of space vehicles into atmospheric environments, ablation is a practical method for alleviating severe aerodynamic heating. Several studies have been undertaken on steady or quasi-steady ablation. However, ablation is a very complicated phenomenon in which a nonequilibrium chemical process is associated with an aerodynamic process that involves changes in body shape with time. Therefore, it seems realistic to consider that ablation is an unsteady phenomenon. In the design of an ablative heat-shield system, since the ultimate purpose of the heat shield is to keep the internal temperature of the space vehicle at a safe level during entry, the transient heat conduction characteristics of the ablator may be critical in the selection of the material and its thickness. This note presents an experimental study of transient ablation of Teflon, with particular emphasis on the change in body shape, the instantaneous internal temperature distribution, and the effect of thermal expansion on ablation rate.

  6. Geometric analysis of transient bursts

    NASA Astrophysics Data System (ADS)

    Osinga, Hinke M.; Tsaneva-Atanasova, Krasimira T.

    2013-12-01

    We consider the effect of a brief stimulation from the rest state of a minimal neuronal model with multiple time scales. Such transient dynamics brings out the intrinsic bursting capabilities of the system. Our main goal is to show that a minimum of three dimensions is enough to generate spike-adding phenomena in transient responses, and that the onset of a new spike can be tracked using existing continuation packages. We take a geometric approach to illustrate how the underlying fast subsystem organises the spike adding in much the same way as for spike adding in periodic bursts, but the bifurcation analysis for spike onset is entirely different. By using a generic model, we further strengthen claims made in our earlier work that our numerical method for spike onset can be used for a broad class of systems.

  7. Model refinement using transient response

    SciTech Connect

    Dohrmann, C.R.; Carne, T.G.

    1997-12-01

    A method is presented for estimating uncertain or unknown parameters in a mathematical model using measurements of transient response. The method is based on a least squares formulation in which the differences between the model and test-based responses are minimized. An application of the method is presented for a nonlinear structural dynamic system. The method is also applied to a model of the Department of Energy armored tractor trailer. For the subject problem, the transient response was generated by driving the vehicle over a bump of prescribed shape and size. Results from the analysis and inspection of the test data revealed that a linear model of the vehicle`s suspension is not adequate to accurately predict the response caused by the bump.

  8. ATLAS discoveries of optical transients

    NASA Astrophysics Data System (ADS)

    Tonry, J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Rest, A.; Smith, K. W.; Smartt, S. J.

    2016-09-01

    We report the following transients found by the ATLAS survey (see Tonry et al. ATel #8680). ATLAS is a twin 0.5m telescope system on Haleakala and Mauna Loa. The first unit is operational on Haleakala is robotically surveying the sky. Two filters are used, cyan and orange (denoted c and o, all mags in AB system), more information is on http://www.fallingstar.com.

  9. Radio Counterparts to SXR Transients

    NASA Astrophysics Data System (ADS)

    Gary, D. E.

    1999-12-01

    By now several studies have been done on small-scale brightenings seen at radio, UV, EUV, and soft X-ray wavelengths. These are to be reviewed by Kundu in these proceedings. In this talk we concentrate on the radio counterpart of a particular type of brightening---the soft X-ray transient brightenings of Shimizu. These brightenings are associated with active regions, and a study of radio counterparts by White et al. (1995) using Nobeyama data found an excellent correspondence between the 17 GHz and SXR brightenings, both spatially and temporally. However, this study found that both the SXR and microwave emissions could be satisfactorily explained as purely thermal emission, and a search of BATSE hard X-ray data showed no nonthermal counterpart. White et al. (1995) were forced to conclude that the events may be different from flares. A more sensitive search for nonthermal emission was needed, in particular using lower frequency microwaves where the influence of nonthermal electrons would be more easily detected. Gary, Hartl and Shimizu (1997) found 34 SXR transient brightenings over a 10-day period in May 1992, for which OVRO (1-18 GHz) total power data were available. A comparison of the data showed a number of clear nonthermal signatures. In addition, one of the events was seen in the lowest energy (6-9.3 keV) channel of the BATSE SPEC detector, suggesting a connection between the microflares discovered in hard X-rays by Lin et al. (1984). The evidence that SXR transient brightenings are microflares is reviewed in this talk. We also attempt to place other small-scale brightenings in context with regard to SXR transient brightenings and microflares.

  10. ATLAS discoveries of optical transients

    NASA Astrophysics Data System (ADS)

    Tonry, J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Rest, A.; Smith, K. W.; Smartt, S. J.

    2016-06-01

    We report the following transients found by the ATLAS survey (see Tonry et al. ATel #8680). ATLAS is a twin 0.5m telescope system on Haleakala and Mauna Loa. The first unit is operational on Haleakala is robotically surveying the sky. Two filters are used, cyan and orange (denoted c and o, all mags in AB system), more information is on http://www.fallingstar.com.

  11. ATLAS discoveries of optical transients

    NASA Astrophysics Data System (ADS)

    Tonry, J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Rest, A.; Smith, K. W.; Smartt, S. J.

    2016-03-01

    We report the following transients found by the ATLAS survey (see Tonry et al. ATel #8680). ATLAS is a twin 0.5m telescope system on Haleakala and Mauna Loa. The first unit is operational on Haleakala is robotically surveying the sky. Two filters are used, cyan and orange (denoted c and o, all mags in AB system), more information is on http://www.fallingstar.com.

  12. ATLAS discoveries of optical transients

    NASA Astrophysics Data System (ADS)

    Tonry, J.; Denneau, L.; Stalder, B.; Heinze, A.; Sherstyuk, A.; Rest, A.; Smith, K. W.; Smartt, S. J.

    2016-08-01

    We report the following transients found by the ATLAS survey (see Tonry et al. ATel #8680). ATLAS is a twin 0.5m telescope system on Haleakala and Mauna Loa. The first unit is operational on Haleakala is robotically surveying the sky. Two filters are used, cyan and orange (denoted c and o, all mags in AB system), more information is on http://www.fallingstar.com.

  13. Transient virulence of emerging pathogens.

    PubMed

    Bolker, Benjamin M; Nanda, Arjun; Shah, Dharmini

    2010-05-01

    Should emerging pathogens be unusually virulent? If so, why? Existing theories of virulence evolution based on a tradeoff between high transmission rates and long infectious periods imply that epidemic growth conditions will select for higher virulence, possibly leading to a transient peak in virulence near the beginning of an epidemic. This transient selection could lead to high virulence in emerging pathogens. Using a simple model of the epidemiological and evolutionary dynamics of emerging pathogens, along with rough estimates of parameters for pathogens such as severe acute respiratory syndrome, West Nile virus and myxomatosis, we estimated the potential magnitude and timing of such transient virulence peaks. Pathogens that are moderately evolvable, highly transmissible, and highly virulent at equilibrium could briefly double their virulence during an epidemic; thus, epidemic-phase selection could contribute significantly to the virulence of emerging pathogens. In order to further assess the potential significance of this mechanism, we bring together data from the literature for the shapes of tradeoff curves for several pathogens (myxomatosis, HIV, and a parasite of Daphnia) and the level of genetic variation for virulence for one (myxomatosis). We discuss the need for better data on tradeoff curves and genetic variance in order to evaluate the plausibility of various scenarios of virulence evolution. PMID:19864267

  14. Transient Response in LMFBR System.

    1999-04-26

    SSC-L (the Super System Code) calculates the thermohydraulic response of loop-type liquid metal fast breeder reactor (LMFBR) systems during operational, incidental, and accidental transients, especially natural circulation events. Modules simulated and parameters calculated include: core flow rates and temperatures, loop flow rates and temperatures, pump performance, and heat exchanger operation. Additionally, SSC-L accounts for all plant protection and plant control systems. Although the primary emphasis is on transients for safety analysis, SSC-L can be usedmore » for many other applications, such as scoping analysis for plant design and specification of various components. Any number of user-specified loops, pipes, and nodes are permitted. Both single- and two-phase thermal-hydraulics are used in a multi-channel core representation. Inter-assembly flow redistribution is accounted for using a detailed fuel pin model. The heat transport system geometry is user-specified. SSC-L provides steady-state and transient options and a restart capability. Input is free format in a modular structure that makes use of abstract data management techniques.« less

  15. Spectral signatures of penumbral transients

    SciTech Connect

    Reardon, K.; Tritschler, A.

    2013-12-20

    In this work we investigate the properties of penumbral transients observed in the upper photospheric and chromospheric region above a sunspot penumbra using two-dimensional spectroscopic observations of the Ca II 854.21 nm line with a 5 s cadence. In our 30 minutes of observations, we identify several penumbral-micro jets (PMJs) with cotemporal observations from Dunn Solar Telescope/IBIS and Hinode/SOT. We find that the line profiles of these PMJ events show emission in the two wings of the line (±0.05 nm), but little modification of the line core. These are reminiscent of the line profiles of Ellerman bombs observed in plage and network regions. Furthermore, we find evidence that some PMJ events have a precursor phase starting 1 minute prior to the main brightening that might indicate initial heating of the plasma prior to an acoustic or bow shock event. With the IBIS data, we also find several other types of transient brightenings with timescales of less than 1 minute that are not clearly seen in the Hinode/SOT data. The spectral profiles and other characteristics of these events are significantly different from those of PMJs. The different appearances of all these transients are an indicator of the general complexity of the chromospheric magnetic field and underscore the highly dynamic behavior above sunspots. It also highlights the care that is needed in interpreting broadband filter images of chromospheric lines, which may conceal very different spectral profiles, and the underlying physical mechanisms at work.

  16. Symptoms of transient ischemic attack.

    PubMed

    Kim, Jong S

    2014-01-01

    Transient ischemic attack (TIA) is a cerebrovascular disease with temporary (<24 h) neurological symptoms. The symptoms of TIA patients are largely similar to those of ischemic stroke patients and include unilateral limb weakness, speech disturbances, sensory symptoms, visual disturbances, and gait difficulties. As these symptoms are transient, they are frequently evaluated based on patients' subjective reports, which are less precise than those of patients with stroke whose longer-lasting symptoms and signs can be reliably assessed by physicians. Some symptoms, such as monocular blindness, are much more common in TIA than in stroke, and limb shaking occurs almost exclusively in TIA patients. On the other hand, symptoms like hemivisual field defects or limb ataxia are underappreciated in TIA patients. These transient neurological symptoms are not necessarily caused by cerebrovascular diseases, but can be produced by a variety of non-vascular diseases. Careful history taking, examination, and appropriate imaging tests are needed to differentiate these TIA mimics from TIA. Each TIA symptom has a different specificity and sensitivity, and there has been an effort to assess the outcome of the patients through the use of specific clinical features. On top of this, recent developments in imaging techniques have greatly enhanced our ability to predict the outcomes of TIA patients. Perception or recognition of TIA symptoms may differ according to the race, sex, education, and specialty of physicians. Appropriate education of both the general population and physicians with regard to TIA symptoms is important as TIAs need emergent evaluation and treatment. PMID:24157558

  17. High level expression of Acidothermus cellulolyticus β-1, 4-endoglucanase in transgenic rice enhances the hydrolysis of its straw by cultured cow gastric fluid

    PubMed Central

    2011-01-01

    Background Large-scale production of effective cellulose hydrolytic enzymes is the key to the bioconversion of agricultural residues to ethanol. The goal of this study was to develop a rice plant as a bioreactor for the large-scale production of cellulose hydrolytic enzymes via genetic transformation, and to simultaneously improve rice straw as an efficient biomass feedstock for conversion of cellulose to glucose. Results In this study, the cellulose hydrolytic enzyme β-1, 4-endoglucanase (E1) gene, from the thermophilic bacterium Acidothermus cellulolyticus, was overexpressed in rice through Agrobacterium-mediated transformation. The expression of the bacterial E1 gene in rice was driven by the constitutive Mac promoter, a hybrid promoter of Ti plasmid mannopine synthetase promoter and cauliflower mosaic virus 35S promoter enhancer, with the signal peptide of tobacco pathogenesis-related protein for targeting the E1 protein to the apoplastic compartment for storage. A total of 52 transgenic rice plants from six independent lines expressing the bacterial E1 enzyme were obtained that expressed the gene at high levels without severely impairing plant growth and development. However, some transgenic plants exhibited a shorter stature and flowered earlier than the wild type plants. The E1 specific activities in the leaves of the highest expressing transgenic rice lines were about 20-fold higher than those of various transgenic plants obtained in previous studies and the protein amounts accounted for up to 6.1% of the total leaf soluble protein. A zymogram and temperature-dependent activity analyses demonstrated the thermostability of the E1 enzyme and its substrate specificity against cellulose, and a simple heat treatment can be used to purify the protein. In addition, hydrolysis of transgenic rice straw with cultured cow gastric fluid for one hour at 39°C and another hour at 81°C yielded 43% more reducing sugars than wild type rice straw. Conclusion Taken together

  18. Transient combustion in hybrid rockets

    NASA Astrophysics Data System (ADS)

    Karabeyoglu, Mustafa Arif

    1998-09-01

    Hybrid rockets regained interest recently as an alternative chemical propulsion system due to their advantages over the solid and liquid systems that are currently in use. Development efforts on hybrids revealed two important problem areas: (1) low frequency instabilities and (2) slow transient response. Both of these are closely related to the transient behavior which is a poorly understood aspect of hybrid operation. This thesis is mainly involved with a theoretical study of transient combustion in hybrid rockets. We follow the methodology of identifying and modeling the subsystems of the motor such as the thermal lags in the solid, boundary layer combustion and chamber gasdynamics from a dynamic point of view. We begin with the thermal lag in the solid which yield the regression rate for any given wall heat flux variation. Interesting phenomena such as overshooting during throttling and the amplification and phase lead regions in the frequency domain are discovered. Later we develop a quasi-steady transient hybrid combustion model supported with time delays for the boundary layer processes. This is integrated with the thermal lag system to obtain the thermal combustion (TC) coupled response. The TC coupled system with positive delays generated low frequency instabilities. The scaling of the instabilities are in good agreement with actual motor test data. Finally, we formulate a gasdynamic model for the hybrid chamber which successfully resolves the filling/emptying and longitudinal acoustic behavior of the motor. The TC coupled system is later integrated to the gasdynamic model to obtain the overall response (TCG coupled system) of gaseous oxidizer motors with stiff feed systems. Low frequency instabilities were also encountered for the TCG coupled system. Apart from the transient investigations, the regression rate behavior of liquefying hybrid propellants such as solid cryogenic materials are also studied. The theory is based on the possibility of enhancement

  19. Transient midventricular ballooning syndrome: a new variant.

    PubMed

    Hurst, R Todd; Askew, J Wells; Reuss, Christina S; Lee, Richard W; Sweeney, John P; Fortuin, F David; Oh, Jae K; Tajik, A Jamil

    2006-08-01

    We describe a new variant of transient left ventricular (LV) ballooning in North American Caucasian patients in which only the midventricle is affected. The patients described in this case series initially presented with emotional or physical stress and had similarities to transient apical ballooning syndrome; however, this variant is unique in that the transient ballooning involves the midventricle with hypercontractility of the apical and basal segments. The presentation, clinical features, and transient nature of the reported cases in this series are similar to transient LV apical ballooning and suggest a shared pathophysiologic etiology. Sparing of the apical segment with involvement of midventricle only supports etiologies not related to an epicardial coronary artery distribution. Although the pathophysiologic mechanism of the transient ventricular ballooning syndromes and other cases of catecholamine-associated transient ventricular dysfunction are not well understood, the emergence of this new variant raises further questions in the understanding of the "brain-heart" relationship. PMID:16875987

  20. Transient response of a circular cylinder to an electromagnetic pulse

    NASA Astrophysics Data System (ADS)

    Ma, J.; Ciric, I. R.

    1992-10-01

    The transient response of a perfectly conducting circular cylinder to an electromagnetic pulse of a double exponential type has been investigated, and a new analytical expression has been derived for the induced current density on the cylinder surface. The analysis performed in this paper is based on the frequency domain eigenfunction solution of the surface current density and yields an analytical expression which is theoretically valid for both the shadow and illuminated regions and for all the time ranges. Numerical calculations show that results for the entire time range after the instant the initial incident wave front has passed the cylinder axis can easily be obtained from this single analytical expression. For very large time values, only the first few terms in the expression are needed to give a satisfactory accuracy.

  1. Transient Receptor Potential (TRP) channels in T cells.

    PubMed

    Bertin, Samuel; Raz, Eyal

    2016-05-01

    The transient receptor potential (TRP) family of ion channels is widely expressed in many cell types and plays various physiological roles. Growing evidence suggests that certain TRP channels are functionally expressed in the immune system. Indeed, an increasing number of reports have demonstrated the functional expression of several TRP channels in innate and adaptive immune cells and have highlighted their critical role in the activation and function of these cells. However, very few reviews have been entirely dedicated to this subject. Here, we will summarize the recent findings with regards to TRP channel expression in T cells and discuss their emerging role as regulators of T cell activation and functions. Moreover, these studies suggest that beyond their pharmaceutical interest in pain management, certain TRP channels may represent potential novel therapeutic targets for various immune-related diseases. PMID:26468011

  2. Transient radiation-induced absorption in the materials for a GSGG laser

    NASA Astrophysics Data System (ADS)

    Brannon, P. J.

    1993-11-01

    Materials used in the optical elements of a 1,061 m GSGG (gadolinium scandium gallium garnet) laser have been tested for transient radiation-induced absorption. The transient radiation-induced absorption in KK1, Schott S7005 and S7010, and M382 glasses have been determined for discrete wavelengths in the range 440-750 nm. Also, the transient radiation-induced absorption in 'pure' and MgO doped LiNbO3 has been measured at 1,061 nm. Mathematical expressions composed of exponentials are fitted to the data.

  3. Transient-induced climate drift

    SciTech Connect

    Johansson, A.; Reinhold, B.; Saha, S. )

    1993-05-01

    The climate drift of various quantities associated with deep, planetary-scale, equilibrated, transient Rossby waves are estimated for the Southern Hemisphere extratropical summer as revealed by the DERF II (Dynamical Extended Range Forecasting) dataset. It is found that the vertical structures of these waves systematically become too baroclinic during the course of integration. There are two time scales associated with this climate drift. There is one very short time scale, estimated to be of the order of one day, when the waves become more barotropic. It is followed by a period when the wave baroclinicity monotonically increases, and after roughly 10 days the model structures appear to have reached their statistically equilibrated state. In the meantime, the kinetic energy of the transient waves decreases substantially to roughly half the observed value. After this initial drop, however, the transient kinetic energy increases again, and it is not clear if an equilibrium value has been reached after 30 days, which is the limit of the DERF II dataset. This third time scale is not found in the quantities directly associated with the vertical structures per se, but it is hypothesized to be a consequence of these errors. A theory is utilized that in a simplified way takes into account the processes that determine the vertical structure of baroclinic waves as well as their robustness as a means of understanding the processes leading to these errors. The implications from this theory are that the formulation and magnitude of the dissipative and diffusive processes in the model are the most likely problem, but there are other possibilities. 37 refs., 10 figs.

  4. Transient pool boiling in microgravity

    NASA Technical Reports Server (NTRS)

    Ervin, J. S.; Merte, H., Jr.; Keller, R. B.; Kirk, K.

    1992-01-01

    Transient nucleate pool boiling experiments using R113 are conducted for short times in microgravity and in earth gravity with different heater surface orientations and subcoolings. The heating surface is a transparent gold film sputtered on a quartz substrate, which simultaneously provides surface temperature measurements and permits viewing of the boiling process from beneath. For the microgravity experiments, which have uniform initial temperatures and no fluid motion, the temperature distribution in the R 113 at the moment of boiling inception is known. High speed cameras with views both across and through the heating surface record the boiling spread across the heater surface, which is classified into six distinct categories.

  5. Observations of Solar Radio Transients

    NASA Astrophysics Data System (ADS)

    Paige, Giorla

    2011-05-01

    A low frequency radio telescope has been recently been constructed on the campus of the The College of New Jersey (TCNJ) and has begun conducting observations at 20MHz as part of NASA'a Radio Jove program. This instrument is capable of observations of solar radio emission including strong prompt radio emission associated with solar burst events. We will discuss solar observations conducted with this instrument as well as an effort to conduct coincident observations with the Eight-meter-wavelength Transient Array (ETA) and the Long Wavelength Array (LWA).

  6. Light Echoes of Historic Transients

    NASA Astrophysics Data System (ADS)

    Rest, Armin; Sinnott, B.; Welch, D. L.; Prieto, J. L.; Bianco, F.

    2014-01-01

    Light echoes, light from a variable source scattered off dust, have been observed for over a century. The recent discovery of light echoes around centuries-old supernovae in the Milky Way and the Large Magellanic Cloud have allowed the spectroscopic characterization of these events, even without contemporaneous photometry and spectroscopy using modern instrumentation. Here we review the recent scientific advances using light echoes of ancient and historic transients, and focus on our latest work on SN 1987A's and Eta Carinae's light echoes.

  7. Transient Wellbore Fluid Flow Model

    1982-04-06

    WELBORE is a code to solve transient, one-dimensional two-phase or single-phase non-isothermal fluid flow in a wellbore. The primary thermodynamic variables used in solving the equations are the pressure and specific energy. An equation of state subroutine provides the density, quality, and temperature. The heat loss out of the wellbore is calculated by solving a radial diffusion equation for the temperature changes outside the bore. The calculation is done at each node point in themore » wellbore.« less

  8. Transient eddy current flow metering

    NASA Astrophysics Data System (ADS)

    Forbriger, J.; Stefani, F.

    2015-10-01

    Measuring local velocities or entire flow rates in liquid metals or semiconductor melts is a notorious problem in many industrial applications, including metal casting and silicon crystal growth. We present a new variant of an old technique which relies on the continuous tracking of a flow-advected transient eddy current that is induced by a pulsed external magnetic field. This calibration-free method is validated by applying it to the velocity of a spinning disk made of aluminum. First tests at a rig with a flow of liquid GaInSn are also presented.

  9. Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.

    PubMed

    Tucker, Torry A; Varga, Karoly; Bebok, Zsuzsa; Zsembery, Akos; McCarty, Nael A; Collawn, James F; Schwiebert, Erik M; Schwiebert, Lisa M

    2003-03-01

    Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function. PMID:12421695

  10. Transient simulation of absorption machines

    SciTech Connect

    Anand, D.K.; Allen, R.W.; Kumar, B.

    1982-08-01

    This paper presents a model for a water-cooled Lithium-Bromide/water absorption chiller and predicts its transient response both during the start-up phase and during the shutoff period. The simulation model incorporates such influencing factors as the thermodynamic properties of the working fluid, the absorbent, the heat-transfer configuration of different components of the chiller and related physical data. The time constants of different components are controlled by a set of key parameters that have been identified in this study. The results show a variable but at times significant amount of time delay before the chiller capacity gets close to its steadystate value. The model is intended to provide an insight into the mechanism of build-up to steady-state performance. By recognizing the significant factors contributing to transient degradation, steps can be taken to reduce such degradation. The evaluation of the residual capacity in the shut-off period will yield more realistic estimates of chiller COP for a chiller satisfying dynamic space cooling load.

  11. PRISMATIC CORE COUPLED TRANSIENT BENCHMARK

    SciTech Connect

    J. Ortensi; M.A. Pope; G. Strydom; R.S. Sen; M.D. DeHart; H.D. Gougar; C. Ellis; A. Baxter; V. Seker; T.J. Downar; K. Vierow; K. Ivanov

    2011-06-01

    The Prismatic Modular Reactor (PMR) is one of the High Temperature Reactor (HTR) design concepts that have existed for some time. Several prismatic units have operated in the world (DRAGON, Fort St. Vrain, Peach Bottom) and one unit is still in operation (HTTR). The deterministic neutronics and thermal-fluids transient analysis tools and methods currently available for the design and analysis of PMRs have lagged behind the state of the art compared to LWR reactor technologies. This has motivated the development of more accurate and efficient tools for the design and safety evaluations of the PMR. In addition to the work invested in new methods, it is essential to develop appropriate benchmarks to verify and validate the new methods in computer codes. The purpose of this benchmark is to establish a well-defined problem, based on a common given set of data, to compare methods and tools in core simulation and thermal hydraulics analysis with a specific focus on transient events. The benchmark-working group is currently seeking OECD/NEA sponsorship. This benchmark is being pursued and is heavily based on the success of the PBMR-400 exercise.

  12. Transient simulation of absorption machines

    NASA Astrophysics Data System (ADS)

    Anand, D. K.; Allen, R. W.; Kumar, B.

    1982-08-01

    This paper presents a model for a water-cooled Lithium-Bromide/water absorption chiller and predicts its transient response both during the start-up phase and during the shutoff period. The simulation model incorporates such influencing factors as the thermodynamic properties of the working fluid, the absorbent, the heat-transfer configuration of different components of the chiller and related physical data. The time constants of different components are controlled by a set of key parameters that have been identified in this study. The results show a variable but at times significant amount of time delay before the chiller capacity gets close to its steady-state value. The model is intended to provide an insight into the mechanism of build-up to steady-state performance. By recognizing the significant factors contributing to transient degradation, steps can be taken to reduce such degradation. The evaluation of the residual capacity in the shut-off period will yield more realistic estimates of chiller COP for a chiller satisfying dynamic space cooling load.

  13. [Acute and transient psychotic disorders].

    PubMed

    Marneros, A; Pillmann, F; Haring, A; Balzuweit, S

    2000-04-01

    Psychotic disorders with acute onset, a dramatic and polymorphous symptomatology and rapid resolution have been described in different countries and by different psychiatric schools. They have been called cycloid psychosis, bouffée délirante, psychogenic psychosis or good prognosis schizophrenia. ICD-10 has given an operational definition under the name "acute and transient psychotic disorders" (F23). Their nosological status is unclear. The Halle-Study of acute and transient psychotic disorders (ATPD) has investigated in a prospective manner clinical, para-clinical features and course of illness in 42 patients with ATPD and matched controls with positive schizophrenia, bipolar schizoaffective disorders as well as mentally healthy patients with acute surgical conditions. First results of our study show that ATPD amount to 4% of psychotic in-patients, prefer female sex, show short prodromi, marked affective disturbances within the episode and much better outcome as schizophrenic psychoses according to psychopathological, social, psychological and biographical criteria. Though ATPD may still be an inhomogeneous group, their clinical delineation from schizophrenia seems justified. PMID:10907609

  14. Postseismic relaxation and transient creep

    USGS Publications Warehouse

    Savage, J.C.; Svarc, J.L.; Yu, S.-B.

    2005-01-01

    Postseismic deformation has been observed in the epicentral area following the 1992 Landers (M = 7.3), 1999 Chi-Chi (M = 7.6), 1999 Hector Mine (M = 7.1), 2002 Denali (M = 7.9), 2003 San Simeon (M = 6.5), and 2004 Parkfield (M = 6.0) earthquakes. The observations consist of repeated GPS measurements of the position of one monument relative to another (separation ???100 km). The early observations (t < 0.1 year) are well fit by the function a' + c'log(t), where t is the time after the earthquake and a' and c' are constants chosen to fit the data. Because a log(t) time dependence is characteristic of transient (primary) creep, the early postseismic response may be governed by transient creep as Benioff proposed in 1951. That inference is provisional as the stress conditions prevailing in postseismic relaxation are not identical to the constant stress condition in creep experiments. The observed logarithmic time dependence includes no characteristic time that might aid in identifying the micromechanical cause.

  15. Transient Anion States of Biomolecules

    NASA Astrophysics Data System (ADS)

    Varella, Marcio

    2012-10-01

    Much of the interest on electron interactions with biomolecules is related to radiation damage [Gohlke and Illenberger, Europhys. News 33, 207 (2002)]. The high energy photons employed in radiology and radiotherapy generate a large number of fast electrons in living cells. These electrons thermalize in a picosecond scale, eventually forming dissociative matestable anions with water and biomolecules. In this work, we employ the parallel version of Schwinger Multichannel Method with Pseudopotentials [Bettega et al., Phys. Rev. A 47, 1111 (1993); Santos et al., J. Phys. Chem. 136, 084307 (2012)] to investigate transient anion states of protein and nucleic acid precursors. We address glycine in both neutral and zwitterionic forms, as well as glycine-water clusters and disulfide bonds. The interest on the two forms of glycine (and other amino acids) relies on the fact that only the neutral form is stable in the gas phase, while the zwitterion is more stable in solution, pointing out limitations of standard gas-phase studies. Electron attachment to disulfide bonds also has potential impact on protein stability. Finally we address transient anion states of substituted uracil molecules in the gas phase. [4pt] In collaboration with M. H. F. Bettega, S. d'A. Sanchez, R. F. da Costa, M. A. P. Lima, J. S. dos Santos, and F. Kossoski.

  16. Special issue on transient plasmas

    NASA Astrophysics Data System (ADS)

    Bailey, James; Hoarty, David; Mancini, Roberto; Yoneda, Hitoki

    2015-11-01

    This special issue of Journal of Physics B: Atomic, Molecular and Optical Physics is dedicated to the "spectroscopy of transient plasmas" covering plasma conditions produced by a range of pulsed laboratory sources including short and long pulse lasers, pulsed power devices, and free electron lasers (FELs). The full range of plasma spectroscopy up to high energy bremsstrahlung radiation, including line broadening analysis for application to data recorded with the ChemCam instrument on the Mars Science Laboratory rover Curiosity, is covered. This issue is timely as advances in optical lasers and x-ray FELs (XFEL) are enabling transient plasma to be probed at higher energies and shorter durations than ever before. New XFEL facilities being commissioned in Europe and Asia are adding to those operating in the US and Japan and the ELI high power laser project in Europe, due to open this year, will provide short pulse lasers of unprecedented power. This special issue represents a snapshot of the theoretical and experimental research in dense plasmas, electron kinetics, laser-induced breakdown spectroscopy of low temperature plasmas, inertial confinement fusion and non-equilibrium atomic physics using spectroscopy to diagnose plasmas produced by optical lasers, XFELs and pulsed-power machines.

  17. Thermal lens spectrometry: Optimizing amplitude and shortening the transient time

    NASA Astrophysics Data System (ADS)

    Silva, Rubens; de Araújo, Marcos A. C.; Jali, Pedro; Moreira, Sanclayton G. C.; Alcantara, Petrus; de Oliveira, Paulo C.

    2011-06-01

    Based on a model introduced by Shen et al. for cw laser induced mode-mismatched dual-beam thermal lens spectrometry (TLS), we explore the parameters related with the geometry of the laser beams and the experimental apparatus that influence the amplitude and time evolution of the transient thermal lens (TL) signal. By keeping the sample cell at the minimum waist of the excitation beam, our results show that high amplitude TL signals, very close to the optimized value, combined with short transient times may be obtained by reducing the curvature radius of the probe beam and the distance between the sample cell and the detector. We also derive an expression for the thermal diffusivity which is independent of the excitation laser beam waist, considerably improving the accuracy of the measurements. The sample used in the experiments was oleic acid, which is present in most of the vegetable oils and is very transparent in the visible spectral range.

  18. Thermal Diffusivity Measurements in Edible Oils using Transient Thermal Lens

    NASA Astrophysics Data System (ADS)

    Valdez, R. Carbajal.; Pérez, J. L. Jiménez.; Cruz-Orea, A.; Martín-Martínez, E. San.

    2006-11-01

    Time resolved thermal lens (TL) spectrometry is applied to the study of the thermal diffusivity of edible oils such as olive, and refined and thermally treated avocado oils. A two laser mismatched-mode experimental configuration was used, with a He Ne laser as a probe beam and an Ar+ laser as the excitation one. The characteristic time constant of the transient thermal lens was obtained by fitting the experimental data to the theoretical expression for a transient thermal lens. The results showed that virgin olive oil has a higher thermal diffusivity than for refined and thermally treated avocado oils. This measured thermal property may contribute to a better understanding of the quality of edible oils, which is very important in the food industry. The thermal diffusivity results for virgin olive oil, obtained from this technique, agree with those reported in the literature.

  19. Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans.

    PubMed

    Lim, Seungmo; Nam, Moon; Kim, Kil Hyun; Lee, Su-Heon; Moon, Jung-Kyung; Lim, Hyoun-Sub; Choung, Myoung-Gun; Kim, Sang-Mok; Moon, Jae Sun

    2016-02-01

    A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C

  20. Sucrose Represses the Developmentally Controlled Transient Activation of the Plastocyanin Gene in Arabidopsis thaliana Seedlings.

    PubMed Central

    Dijkwel, P. P.; Kock, PAM.; Bezemer, R.; Weisbeek, P. J.; Smeekens, SCM.

    1996-01-01

    The plastocyanin (PC) gene of Arabidopsis thaliana is activated independently of light during early seedling development. In etiolated seedlings, PC mRNA levels increase transiently and a maximum dark level is reached after 2 d of growth in darkness. In etiolated transgenic seedlings carrying a chimeric PC-promoter: luciferase fusion gene, luciferase activity is similarly increased after 2 d of growth. The transient increase in PC mRNA and luciferase activity levels can be repressed by sucrose. Nonmetabolizable sugars and polyethylene glycol do not have a major effect on PC gene expression. Also, light-grown seedlings show a similar transient and sucrose-sensitive increase in PC mRNA levels and luciferase activity, as in dark-grown seedlings, but here expression levels are 15- fold higher. These findings suggest the presence of a sucrose-sensitive, developmentally controlled expression mechanism that operates independently of light. PMID:12226197

  1. Discrete wavelet analysis of power system transients

    SciTech Connect

    Wilkinson, W.A.; Cox, M.D.

    1996-11-01

    Wavelet analysis is a new method for studying power system transients. Through wavelet analysis, transients are decomposed into a series of wavelet components, each of which is a time-domain signal that covers a specific octave frequency band. This paper presents the basic ideas of discrete wavelet analysis. A variety of actual and simulated transient signals are then analyzed using the discrete wavelet transform that help demonstrate the power of wavelet analysis.

  2. Transient hypoxia reprograms differentiating adipocytes for enhanced insulin sensitivity and triglyceride accumulation

    PubMed Central

    Lu, Hongyun; Gao, Zhanguo; Zhao, Zhiyun; Weng, Jianping; Ye, Jianping

    2015-01-01

    Objective To investigate the impact of transient (2-4 h) hypoxia on metabolic reprogramming of adipocytes. Methods The impact of transient hypoxia on metabolic reprogramming was investigated in 3T3-L1 cells before and after differentiation. Glucose uptake, fatty acid oxidation, lipolysis, and mitochondria were examined to determine the hypoxia effects. Preadipocytes were exposed to transient hypoxia (4h/day) in the course of differentiation. Insulin sensitivity and TG accumulation was examined in the cells at the end of differentiation to determine the reprogramming effects. AMPK activity and gene expression were determined by quantitative RT-PCR and Western blotting in search for mechanism of the reprogramming. Results In acute response to hypoxia, adipocytes exhibited an increase in insulin-dependent and -independent glucose uptake. Fatty acid β-oxidation and pyruvate dehydrogenase (PDH) activity were decreased. Multiple exposures of differentiating adipocytes to transient hypoxia enhanced insulin signaling, TG accumulation, expression of antioxidant genes in differentiated adipocytes in the absence of hypoxia. The metabolic memory was associated with elevated AMPK activity and gene expression (GLUT1, PGC-1α, PPARγ, SREBP, NRF-1, ESRRα, LPL). The enhanced insulin sensitivity was blocked by an AMPK inhibitor. Conclusions Repeated exposure of differentiating adipocytes to transient hypoxia is able to reprogram the cells for increased TG accumulation and enhanced insulin sensitivity. The metabolic alterations were observed in post-differentiated cells under normoxia. The reprogramming involves AMPK activation and gene expression in the metabolic pathways in cytosol and mitochondria. PMID:26219415

  3. Transient Hypoxia Improves Matrix Properties in Tissue Engineered Cartilage

    PubMed Central

    Yodmuang, Supansa; Gadjanski, Ivana; Chao, Pen-hsiu Grace; Vunjak-Novakovic, Gordana

    2014-01-01

    Adult articular cartilage is a hypoxic tissue, with oxygen tension ranging from <10% at the cartilage surface to <1% in the deepest layers. In addition to spatial gradients, cartilage development is also associated with temporal changes in oxygen tension. However, a vast majority of cartilage tissue engineering protocols involves cultivation of chondrocytes or their progenitors under ambient oxygen concentration (21% O2), that is, significantly above physiological levels in either developing or adult cartilage. Our study was designed to test the hypothesis that transient hypoxia followed by normoxic conditions results in improved quality of engineered cartilaginous ECM. To this end, we systematically compared the effects of normoxia (21% O2 for 28 days), hypoxia (5% O2 for 28 days) and transient hypoxia—reoxygenation (5% O2 for 7 days and 21% O2 for 21 days) on the matrix composition and expression of the chondrogenic genes in cartilage constructs engineered in vitro. We demonstrated that reoxygenation had the most effect on the expression of cartilaginous genes including COL2A1, ACAN, and SOX9 and increased tissue concentrations of amounts of glycosaminoglycans and type II collagen. The equilibrium Young’s moduli of tissues grown under transient hypoxia (510.01 ± 28.15 kPa) and under normoxic conditions (417.60 ± 68.46 kPa) were significantly higher than those measured under hypoxic conditions (279.61 ± 20.52 kPa). These data suggest that the cultivation protocols utilizing transient hypoxia with reoxygenation have high potential for efficient cartilage tissue engineering, but need further optimization in order to achieve higher mechanical functionality of engineered constructs. PMID:23203946

  4. A Case of Transient Global Amnesia

    PubMed Central

    Mccroskey, Aidan L.; Deyerle, Branden A.

    2016-01-01

    Transient global amnesia is a clinical syndrome characterized by the sudden onset of anterograde amnesia, accompanied by repetitive questioning, sometimes with a retrograde component, lasting up to 24 hours, without compromise of other neurologic function. Neuroimaging after an acutetransient global amnesia event often shows transient perturbation of specific hippocampal circuits that are involved in memory processing. Critical clinical distinctions, such as between transient global amnesia and other forms of transient amnesic episodes, as well as important clues to the underlying pathophysiologies are herein reviewed. Finally, we discuss the role of hippocampal insufficiency in the neurobiology of delusions. PMID:27354927

  5. Transient electromagnetic interference in substations

    SciTech Connect

    Wiggins, C.M.; Thomas, D.E.; Nickel, F.S.; Salas, T.M. ); Wright, S.E. )

    1994-10-01

    Electromagnetic interference levels on sensitive electronic equipment are quantified experimentally and theoretically in air and gas insulated substations of different voltages. Measurement techniques for recording interference voltages and currents and electric and magnetic fields are reviewed and actual interference data are summarized. Conducted and radiated interference coupling mechanisms and levels in substation control wiring are described using both measurement results and electromagnetic models validated against measurements. The nominal maximum field and control wire interference levels expected in the switchyard and inside the control house from switching operations, faults, and an average lightning strike are estimated using high frequency transient coupling models. Comparisons with standards are made and recommendations given concerning equipment shielding and surge protection.

  6. Regulatory Analysis of Reactivity Transients

    SciTech Connect

    Beyer, Carl E.; Clifford, Paul M.; Geelhood, Kenneth J.; Voglewede, John C.

    2009-08-01

    This paper will describe modifications made to the FRAPCON-3 and FRAPTRAN fuel performance codes and models that impact reactivity initiated accident (RIA) analyses. The modified models include an upper bound empirical and best estimate release models for fast transients, and a revised fuel failure model that accounts for ductile and brittle failure. Because experimental data exists for discrete test conditions, the codes and models are used to interpolate and to some extent, to extrapolate these test conditions. An upper bound empirical model for release is used to establish new recommended release fractions for long-lived and short lived (radioactive) isotopes for RIA events in Regulatory Guide 1.183. A best estimate release model is used in FRAPTRAN 1.4 based on grain boundary gas concentrations from FRAPCON-3.4 to predict release for RIA events. Code and model predictions will be compared to failure and release data from RIA tests to demonstrate accuracy.

  7. Transient Dendritic Solidification Experiment (TDSE)