Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.

PubMed

Weil, M; Rausch, T

1990-12-01

The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink.

Untargeted metabolomics analysis reveals dynamic changes in azelaic acid- and salicylic acid derivatives in LPS-treated Nicotiana tabacum cells.

PubMed

Mhlongo, M I; Tugizimana, F; Piater, L A; Steenkamp, P A; Madala, N E; Dubery, I A

2017-01-22

To counteract biotic stress factors, plants employ multilayered defense mechanisms responsive to pathogen-derived elicitor molecules, and regulated by different phytohormones and signaling molecules. Here, lipopolysaccharide (LPS), a microbe-associated molecular pattern (MAMP) molecule, was used to induce defense responses in Nicotiana tabacum cell suspensions. Intracellular metabolites were extracted with methanol and analyzed using a liquid chromatography-mass spectrometry (UHPLC-qTOF-MS/MS) platform. The generated data were processed and examined with multivariate and univariate statistical tools. The results show time-dependent dynamic changes and accumulation of glycosylated signaling molecules, specifically those of azelaic acid, salicylic acid and methyl-salicylate as contributors to the altered metabolomic state in LPS-treated cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Growth modulation effects of CBM2a under the control of AtEXP4 and CaMV35S promoters in Arabidopsis thaliana, Nicotiana tabacum and Eucalyptus camaldulensis.

PubMed

Keadtidumrongkul, Pornthep; Suttangkakul, Anongpat; Pinmanee, Phitsanu; Pattana, Kanokwan; Kittiwongwattana, Chokchai; Apisitwanich, Somsak; Vuttipongchaikij, Supachai

2017-08-01

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 (AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement.

Production, partial purification and characterization of xylanase using Nicotiana tabacum leaf dust as substrate.

PubMed

Acharya, Komal P; Shilpkar, Prateek

2016-03-01

Isolated Bacillus sp. was used in the present study for production of xylanase from Nicotiana tabacum leaf dust. The strain was able to give a maximum of 1.77 Uml⁻¹ xylanase activity under optimized fermentation conditions which was further increased upto 2.77 Uml⁻¹ after extraction and partial purification of enzyme. After partial purification, the enzyme was characterized and it gave the highest xylanase activity at pH 7.0, when 0.2 ml enzyme was incubated with 2.0% substrate (Nicotiana tabacum leaf dust) for 60 min at 60°C. Saccharification study of Nicotiana tabacum leaf dust with partially purified enzyme revealed that 18.4% reducing sugar was released in 20 hrs incubation, and TLC and HPTLC analysis showed that xylose and glucose sugars were obtained after hydrolysis of substrate. FTIR analysis confirmed decomposition of substrate.

Efficiency of Particle-Bombardment-Mediated Transformation Is Influenced by Cell Cycle Stage in Synchronized Cultured Cells of Tobacco 1

PubMed Central

Iida, Asako; Yamashita, Toshiya; Yamada, Yasuyuki; Morikawa, Hiromichi

1991-01-01

Plasmid DNA pB1221 harboring β-glucuronidase gene was delivered to synchronized cultured tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells of different cell cycle stages by a pneumatic particle gun. The cells bombarded at M and G2 phases gave 4 to 6 times higher transformation efficiency than those bombarded at the S and G1 phases. ImagesFigure 2 PMID:16668589

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

PubMed Central

Koshiba, Taichi; Kobayashi, Masaru; Matoh, Toru

2009-01-01

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD. PMID:19054807

Uptake of NO, NO 2 and O 3 by sunflower ( Helianthus annuus L.) and tobacco plants ( Nicotiana tabacum L.): dependence on stomatal conductivity

NASA Astrophysics Data System (ADS)

Neubert, A.; Kley, D.; Wildt, J.; Segschneider, H. J.; Förstel, H.

The uptake of NO, NO 2 and O 3 by sunflowers ( Helianthus annuus L. var. giganteus) and tobacco plants ( Nicotiana tabacum L. var. Bel W3), using concentrations representative for moderately polluted air, has been determined by gas exchange experiments. Conductivities for these trace gases were measured at different light fluxes ranging from 820 μEm -2s -1 to darkness. The conductivities to water vapor and the trace gases are highly correlated. It is concluded that the uptake of NO, NO 2 and O 3 by sunflowers and tobacco plants is linearly dependent on stomatal opening. While the uptake of NO is limited by the mesophyll resistance, the uptake of NO 2 is only by diffusion through the stomata. Loss processes by deposition to the leaf surfaces are more pronounced for O 3 than for NO and NO 2.

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

PubMed

Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

2014-08-01

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

Phenylpropanoid Defences in Nicotiana tabacum Cells: Overlapping Metabolomes Indicate Common Aspects to Priming Responses Induced by Lipopolysaccharides, Chitosan and Flagellin-22

PubMed Central

Mhlongo, Msizi I.; Piater, Lizelle A.; Madala, Ntakadzeni E.; Steenkamp, Paul A.; Dubery, Ian A.

2016-01-01

Plants have evolved both constitutive and inducible defence strategies to cope with different biotic stimuli and stresses. Exposure of a plant to a challenging stress can lead to a primed state that allows it to launch a more rapid and stronger defence. Here we applied a metabolomic approach to study and compare the responses induced in Nicotiana tabacum cells by microbe-associated molecular pattern (MAMP) molecules, namely lipopolysaccharides (LPS), chitosan (CHT) and flagellin-22 (FLG22). Early response metabolites, extracted with methanol, were analysed by UHPLC-MS/MS. Using multivariate statistical tools the metabolic profiles induced by these elicitors were analysed. In the metabolic fingerprint of these agents a total of 19 cinnamic acid derivatives conjugated to quinic acids (chlorogenic acids), shikimic acid, tyramine, polyamines or glucose were found as discriminant biomarkers. In addition, treatment with the phytohormones salicylic acid (SA), methyljasmonic acid (MJ) and abscisic acid (ABA) resulted in differentially-induced phenylpropanoid pathway metabolites. The results indicate that the phenylpropanoid pathway is activated by these elicitors while hydroxycinnamic acid derivatives are commonly associated with the metabolic response to the MAMPs, and that the activated responses are modulated by both SA and MJ, with ABA not playing a role. PMID:26978774

Metabolism of methoxychlor by the P450-monooxygenase CYP6G1 involved in insecticide resistance of Drosophila melanogaster after expression in cell cultures of Nicotiana tabacum.

PubMed

Joussen, Nicole; Schuphan, Ingolf; Schmidt, Burkhard

2010-03-01

Cytochrome P450 monooxygenase CYP6G1 of Drosophila melanogaster was heterologously expressed in a cell suspension culture of Nicotiana tabacum. This in vitro system was used to study the capability of CYP6G1 to metabolize the insecticide methoxychlor (=1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane, 1) against the background of endogenous enzymes of the corresponding non-transgenic culture. The Cyp6g1-transgenic cell culture metabolized 96% of applied methoxychlor (45.8 microg per assay) within 24 h by demethylation and hydroxylation mainly to trishydroxy and catechol methoxychlor (16 and 17%, resp.). About 34% of the metabolism and the distinct formation of trishydroxy and catechol methoxychlor were due to foreign enzyme CYP6G1. Furthermore, methoxychlor metabolism was inhibited by 43% after simultaneous addition of piperonyl butoxide (458 microg), whereas inhibition in the non-transgenic culture amounted to 92%. Additionally, the rate of glycosylation was reduced in both cultures. These results were supported by the inhibition of the metabolism of the insecticide imidacloprid (6; 20 microg, 24 h) in the Cyp6g1-transgenic culture by 82% in the presence of piperonyl butoxide (200 microg). Due to CYP6G1 being responsible for imidacloprid resistance of Drosophila or being involved in DDT resistance, it is likely that CYP6G1 conveys resistance to methoxychlor (1). Furthermore, treating Drosophila with piperonyl butoxide could weaken the observed resistance phenomena.

Evolutionary diversification of type-2 HDAC structure, function and regulation in Nicotiana tabacum.

PubMed

Nicolas-Francès, Valérie; Grandperret, Vincent; Liegard, Benjamin; Jeandroz, Sylvain; Vasselon, Damien; Aimé, Sébastien; Klinguer, Agnès; Lamotte, Olivier; Julio, Emilie; de Borne, François Dorlhac; Wendehenne, David; Bourque, Stéphane

2018-04-01

Type-2 HDACs (HD2s) are plant-specific histone deacetylases that play diverse roles during development and in responses to biotic and abiotic stresses. In this study we characterized the six tobacco genes encoding HD2s that mainly differ by the presence or the absence of a typical zinc finger in their C-terminal part. Of particular interest, these HD2 genes exhibit a highly conserved intron/exon structure. We then further investigated the phylogenetic relationships among the HD2 gene family, and proposed a model of the genetic events that led to the organization of the HD2 family in Solanaceae. Absolute quantification of HD2 mRNAs in N. tabacum and in its precursors, N. tomentosiformis and N. sylvestris, did not reveal any pseudogenization of any of the HD2 genes, but rather specific regulation of HD2 expression in these three species. Functional complementation approaches in Arabidopsis thaliana demonstrated that the four zinc finger-containing HD2 proteins exhibit the same biological function in response to salt stress, whereas the two HD2 proteins without zinc finger have different biological function. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric oxide modulates cadmium influx during cadmium-induced programmed cell death in tobacco BY-2 cells.

PubMed

Ma, Wenwen; Xu, Wenzhong; Xu, Hua; Chen, Yanshan; He, Zhenyan; Ma, Mi

2010-07-01

Nitric oxide (NO) is a bioactive gas and functions as a signaling molecule in plants exposed to diverse biotic and abiotic stresses including cadmium (Cd(2+)). Cd(2+) is a non-essential and toxic heavy metal, which has been reported to induce programmed cell death (PCD) in plants. Here, we investigated the role of NO in Cd(2+)-induced PCD in tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2). In this work, BY-2 cells exposed to 150 microM CdCl(2) underwent PCD with TUNEL-positive nuclei, significant chromatin condensation and the increasing expression of a PCD-related gene Hsr203J. Accompanied with the occurring of PCD, the production of NO increased significantly. The supplement of NO by sodium nitroprusside (SNP) had accelerated the PCD, whereas the NO synthase inhibitor Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME) and NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) alleviated this toxicity. To investigate the mechanism by which NO exerted its function, Cd(2+) concentration was measured subsequently. SNP led more Cd(2+) content than Cd(2+) treatment alone. By contrast, the prevention of NO by L-NAME decreased Cd(2+) accumulation. Using the scanning ion-selective electrode technique, we analyzed the pattern and rate of Cd(2+) fluxes. This analysis revealed the promotion of Cd(2+) influxes into cells by application of SNP, while L-NAME and cPTIO reduced the rate of Cd(2+) uptake or even resulted in net Cd(2+) efflux. Based on these founding, we concluded that NO played a positive role in CdCl(2)-induced PCD by modulating Cd(2+) uptake and thus promoting Cd(2+) accumulation in BY-2 cells.

Coniferyl alcohol hinders the growth of tobacco BY-2 cells and Nicotiana benthamiana seedlings.

PubMed

Väisänen, Enni E; Smeds, Annika I; Fagerstedt, Kurt V; Teeri, Teemu H; Willför, Stefan M; Kärkönen, Anna

2015-09-01

Externally added coniferyl alcohol at high concentrations reduces the growth of Nicotiana cells and seedlings. Coniferyl alcohol is metabolized by BY-2 cells to several compounds. Coniferyl alcohol (CA) is a common monolignol and a building block of lignin. The toxicity of monolignol alcohols has been stated in the literature, but there are only few studies suggesting that this is true. We investigated the physiological effects of CA on living plant cells in more detail. Tobacco (Nicotiana tabacum) Bright yellow-2 cells (BY-2) and Nicotiana benthamiana seedlings both showed concentration-dependent growth retardation in response to 0.5-5 mM CA treatment. In some cases, CA addition caused cell death in BY-2 cultures, but this response was dependent on the growth stage of the cells. Based on LC-MS/MS analysis, BY-2 cells did not accumulate the externally supplemented CA, but metabolized it to ferulic acid, ferulic acid glycoside, coniferin, and to some other phenolic compounds. In addition to growth inhibition, CA caused the formation of a lignin-like compound detected by phloroglucinol staining in N. benthamiana roots and occasionally in BY-2 cells. To prevent this, we added potassium iodide (KI, at 5 mM) to overcome the peroxidase-mediated CA polymerization to lignin. KI had, however, toxic effects on its own: in N. benthamiana seedlings, it caused reduction in growth; in BY-2 cells, reduction in growth and cell viability. Surprisingly, CA restored the growth of KI-treated BY-2 cells and N. benthamiana seedlings. Our results suggest that CA at high concentrations is toxic to plant cells.

Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?

PubMed

Shoji, Tsubasa; Hashimoto, Takashi

2008-08-01

Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.

Auxin Deprivation Induces Synchronous Golgi Differentiation in Suspension-Cultured Tobacco BY-2 Cells1

PubMed Central

Winicur, Zev M.; Feng Zhang, Guo; Andrew Staehelin, L.

1998-01-01

To date, the lack of a method for inducing plant cells and their Golgi stacks to differentiate in a synchronous manner has made it difficult to characterize the nature and extent of Golgi retailoring in biochemical terms. Here we report that auxin deprivation can be used to induce a uniform population of suspension-cultured tobacco (Nicotiana tabacum cv BY-2) cells to differentiate synchronously during a 4-d period. Upon removal of auxin, the cells stop dividing, undergo elongation, and differentiate in a manner that mimics the formation of slime-secreting epidermal and peripheral root-cap cells. The morphological changes to the Golgi apparatus include a proportional increase in the number of trans-Golgi cisternae, a switch to larger-sized secretory vesicles that bud from the trans-Golgi cisternae, and an increase in osmium staining of the secretory products. Biochemical alterations include an increase in large, fucosylated, mucin-type glycoproteins, changes in the types of secreted arabinogalactan proteins, and an increase in the amounts and types of molecules containing the peripheral root-cap-cell-specific epitope JIM 13. Taken together, these findings support the hypothesis that auxin deprivation can be used to induce tobacco BY-2 cells to differentiate synchronously into mucilage-secreting cells. PMID:9625703

Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).

PubMed

Xu, Bingfang; Copolla, Michael; Herr, John C; Timko, Michael P

2007-01-01

The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant

Moss is a key nurse plant for reintroduction of the endangered herb, Primulina tabacum Hance.

Treesearch

Hai Ren; Guohua Ma; Qianmei Zhang; Qinfeng Guo; Jun Wang; Zhengfeng Wang

2010-01-01

The rare and endangered plant Primulina tabacum is a calciphilous perennial herb found only at the entrances of a small number of karst cave drainages in southern China. In a conservation effort, we identified potentially suitable habitats and reintroduced P. tabacum plantlets (propagated in vitro) to one historical and two new cave entrances. The transplanted...

Species Origin of Genomic Factors in Nicotiana nudicaulis Watson Controlling Hybrid Lethality in Interspecific Hybrids between N. nudicaulis Watson and N. tabacum L

PubMed Central

Liu, Hongshuo; Marubashi, Wataru

2014-01-01

Hybrid lethality is expressed at 28°C in the cross Nicotiana nudicaulis×N. tabacum. The S subgenome of N. tabacum has been identified as controlling this hybrid lethality. To clarify the responsible genomic factor(s) of N. nudicaulis, we crossed N. trigonophylla (paternal progenitor of N. nudicaulis) with N. tabacum, because hybrids between N. sylvestris (maternal progenitor of N. nudicaulis) and N. tabacum are viable when grown in a greenhouse. In the cross N. trigonophylla×N. tabacum, approximately 50% of hybrids were vitrified, 20% were viable, and 20% were nonviable at 28°C. To reveal which subgenome of N. tabacum was responsible for these phenotypes, we crossed N. trigonophylla with two progenitors of N. tabacum, N. sylvestris (SS) and N. tomentosiformis (TT). In the cross N. sylvestris×N. trigonophylla, we confirmed that over half of hybrids of N. sylvestris×N. trigonophylla were vitrified, and none of the hybrids of N. trigonophylla×N. tomentosiformis were. The results imply that the S subgenome, encoding a gene or genes inducing hybrid lethality in the cross between N. nudicaulis and N. tabacum, has one or more genomic factors that induce vitrification. Furthermore, in vitrified hybrids of N. trigonophylla×N. tabacum and N. sylvestris×N. trigonophylla, we found that nuclear fragmentation, which progresses during expression of hybrid lethality, was accompanied by vitrification. This observation suggests that vitrification has a relationship to hybrid lethality. Based on these results, we speculate that when N. nudicaulis was formed approximately 5 million years ago, several causative genomic factors determining phenotypes of hybrid seedlings were inherited from N. trigonophylla. Subsequently, genome downsizing and various recombination-based processes took place. Some of the causative genomic factors were lost and some became genomic factor(s) controlling hybrid lethality in extant N. nudicaulis. PMID:24806486

Cadmium and zinc activate adaptive mechanisms in Nicotiana tabacum similar to those observed in metal tolerant plants.

PubMed

Vera-Estrella, Rosario; Gómez-Méndez, María F; Amezcua-Romero, Julio C; Barkla, Bronwyn J; Rosas-Santiago, Paul; Pantoja, Omar

2017-09-01

Tobacco germinated and grew in the presence of high concentrations of cadmium and zinc without toxic symptoms. Evidence suggests that these ions are sequestered into the vacuole by heavy metal/H + exchanger mechanisms. Heavy metal hyperaccumulation and hypertolerance are traits shared by a small set of plants which show specialized physiological and molecular adaptations allowing them to accumulate and sequester toxic metal ions. Nicotiana tabacum was used to test its potential as a metal-accumulator in a glass house experiment. Seed germination was not affected in the presence of increasing concentrations of zinc and cadmium. Juvenile and adult plants could concentrate CdCl 2 and ZnSO 4 to levels exceeding those in the hydroponic growth medium and maintained or increased their leaf dry weight when treated with 0.5- or 1-mM CdCl 2 or 1-mM ZnSO 4 for 5 days. Accumulation of heavy metals did not affect the chlorophyll and carotenoid levels, while variable effects were observed in cell sap osmolarity. Heavy metal-dependent H + transport across the vacuole membrane was monitored using quinacrine fluorescence quenching. Cadmium- or zinc-dependent fluorescence recovery revealed that increasing concentrations of heavy metals stimulated the activities of the tonoplast Cd 2+ or Zn 2+ /H + exchangers. Immunodetection of the V-ATPase subunits showed that the increased proton transport by zinc was not due to changes in protein amount. MTP1 and MTP4 immunodetection and semiquantitative RT-PCR of NtMTP1, NtNRAMP1, and NtZIP1 helped to identify the genes that are likely involved in sequestration of cadmium and zinc in the leaf and root tissue. Finally, we demonstrated that cadmium and zinc treatments induced an accumulation of zinc in leaf tissues. This study shows that N. tabacum possesses a hyperaccumulation response, and thus could be used for phytoremediation purposes.

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells.

PubMed

Nemoto, Keiichirou; Hara, Masamitsu; Goto, Shingo; Kasai, Kouji; Seki, Hikaru; Suzuki, Masashi; Oka, Atsuhiro; Muranaka, Toshiya; Mano, Yoshihiro

2009-05-01

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.

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

PubMed

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

2015-09-01

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

1,8-cineole inhibits both proliferation and elongation of BY-2 cultured tobacco cells.

PubMed

Yoshimura, Hiroko; Sawai, Yu; Tamotsu, Satoshi; Sakai, Atsushi

2011-03-01

Volatile monoterpenes such as 1,8-cineole inhibit the growth of Brassica campestris seedlings in a dose-dependent manner, and the growth-inhibitory effects are more severe for roots than hypocotyls. The preferential inhibition of root growth may be explained if the compounds inhibit cell proliferation more severely than cell elongation because root growth requires both elongation and proliferation of the constituent cells, whereas hypocotyl growth depends exclusively on elongation of existing cells. In order to examine this possibility, BY-2 suspension-cultured tobacco (Nicotiana tabacum) cells were treated with 1,8-cineole, and the inhibitory effects on cell proliferation and on cell elongation were assessed quantitatively. Treatment with 1,8-cineole lowered both the mitotic index and elongation of the cells in a dose-dependent manner, and the half-maximal inhibitory concentration (IC₅₀) for cell elongation was lower than that for cell proliferation. Moreover, 1,8-cineole also inhibited starch synthesis, with IC₅₀ lower than that for cell proliferation. Thus, the inhibitory effects of 1,8-cineole were not specific to cell proliferation; rather, 1,8-cineole seemed inhibitory to a variety of physiological activities when it was in direct contact with target cells. Based on these results, possible mechanisms for the mode of action of 1,8-cineole and for its preferential inhibition on root growth are discussed.

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

PubMed Central

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

2015-01-01

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

Auxin-Dependent Cell Division and Cell Elongation. 1-Naphthaleneacetic Acid and 2,4-Dichlorophenoxyacetic Acid Activate Different Pathways1

PubMed Central

Campanoni, Prisca; Nick, Peter

2005-01-01

During exponential phase, the tobacco (Nicotiana tabacum) cell line cv Virginia Bright Italia-0 divides axially to produce linear cell files of distinct polarity. This axial division is controlled by exogenous auxin. We used exponential tobacco cv Virginia Bright Italia-0 cells to dissect early auxin signaling, with cell division and cell elongation as physiological markers. Experiments with 1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated that these 2 auxin species affect cell division and cell elongation differentially; NAA stimulates cell elongation at concentrations that are much lower than those required to stimulate cell division. In contrast, 2,4-D promotes cell division but not cell elongation. Pertussis toxin, a blocker of heterotrimeric G-proteins, inhibits the stimulation of cell division by 2,4-D but does not affect cell elongation. Aluminum tetrafluoride, an activator of the G-proteins, can induce cell division at NAA concentrations that are not permissive for division and even in the absence of any exogenous auxin. The data are discussed in a model where the two different auxins activate two different pathways for the control of cell division and cell elongation. PMID:15734918

The plastidial 2-C-methyl-D-erythritol 4-phosphate pathway provides the isoprenyl moiety for protein geranylgeranylation in tobacco BY-2 cells.

PubMed

Gerber, Esther; Hemmerlin, Andréa; Hartmann, Michael; Heintz, Dimitri; Hartmann, Marie-Andrée; Mutterer, Jérôme; Rodríguez-Concepción, Manuel; Boronat, Albert; Van Dorsselaer, Alain; Rohmer, Michel; Crowell, Dring N; Bach, Thomas J

2009-01-01

Protein farnesylation and geranylgeranylation are important posttranslational modifications in eukaryotic cells. We visualized in transformed Nicotiana tabacum Bright Yellow-2 (BY-2) cells the geranylgeranylation and plasma membrane localization of GFP-BD-CVIL, which consists of green fluorescent protein (GFP) fused to the C-terminal polybasic domain (BD) and CVIL isoprenylation motif from the Oryza sativa calmodulin, CaM61. Treatment with fosmidomycin (Fos) or oxoclomazone (OC), inhibitors of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, caused mislocalization of the protein to the nucleus, whereas treatment with mevinolin, an inhibitor of the cytosolic mevalonate pathway, did not. The nuclear localization of GFP-BD-CVIL in the presence of MEP pathway inhibitors was completely reversed by all-trans-geranylgeraniol (GGol). Furthermore, 1-deoxy-d-xylulose (DX) reversed the effects of OC, but not Fos, consistent with the hypothesis that OC blocks 1-deoxy-d-xylulose 5-phosphate synthesis, whereas Fos inhibits its conversion to 2-C-methyl-d-erythritol 4-phosphate. By contrast, GGol and DX did not rescue the nuclear mislocalization of GFP-BD-CVIL in the presence of a protein geranylgeranyltransferase type 1 inhibitor. Thus, the MEP pathway has an essential role in geranylgeranyl diphosphate (GGPP) biosynthesis and protein geranylgeranylation in BY-2 cells. GFP-BD-CVIL is a versatile tool for identifying pharmaceuticals and herbicides that interfere either with GGPP biosynthesis or with protein geranylgeranylation.

Difference in the distribution and speciation of cellular nickel between nickel-tolerant and non-tolerant Nicotiana tabacum L. cv. BY-2 cells.

PubMed

Saito, Akihiro; Saito, Misa; Ichikawa, Yusuke; Yoshiba, Masaaki; Tadano, Toshiaki; Miwa, Eitaro; Higuchi, Kyoko

2010-02-01

To evaluate Ni dynamics at the subcellular level, the distribution and speciation of Ni were determined in wild-type (WT) and Ni-tolerant (NIT) tobacco BY-2 cell lines. When exposed to low but toxic levels of Ni, NIT cells were found to contain 2.5-fold more Ni (14% of whole-cell Ni values) in their cell walls than WT cells (6% of whole-cell Ni values). In addition to higher levels of Ni in the apoplast, a higher proportion (94%) of symplastic Ni was localized in the vacuoles of NIT cells than in the vacuoles of WT cells (81%). The concentration of cytosolic Ni in the NIT cells was significantly lower (18 nmol g(-1) FW) than that in the WT cells (85 nmol g(-1) FW). In silico simulation showed that 95% of vacuolar Ni was in the form of Ni-citrate complexes, and that free Ni(2+) was virtually absent in the NIT cells. On the other hand, the amount of free metal ions was markedly increased in WT cells because free citrate was depleted by chelation of Ni. A protoplast viability assay using BCECF-AM further demonstrated that the main mechanism that confers strong Ni tolerance was present in the symplast as opposed to the cell wall.

Phytodetoxification of TNT by transplastomic tobacco (Nicotiana tabacum) expressing a bacterial nitroreductase.

PubMed

Zhang, Long; Rylott, Elizabeth L; Bruce, Neil C; Strand, Stuart E

2017-09-01

Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT. The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.

PubMed

Ducos, Eric; Fraysse, Staffan; Boutry, Marc

2005-12-19

In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family.

Manganese toxicity to chlorophyll synthesis in tobacco callus. [Nicotiana tabacum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clairmont, K.B.; Hagar, W.G.; Davis, E.A.

1986-01-01

Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectivity inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant.

NtLTP4, a lipid transfer protein that enhances salt and drought stresses tolerance in Nicotiana tabacum.

PubMed

Xu, Yang; Zheng, Xinxin; Song, Yunzhi; Zhu, Lifei; Yu, Zipeng; Gan, Liming; Zhou, Shumei; Liu, Hongmei; Wen, Fujiang; Zhu, Changxiang

2018-06-11

Lipid transfer proteins (LTPs), a class of small, ubiquitous proteins, play critical roles in various environmental stresses. However, their precise biological functions remain unknown. Here we isolated an extracellular matrix-localised LTP, NtLTP4, from Nicotiana tabacum. The overexpression of NtLTP4 in N. tabacum enhanced resistance to salt and drought stresses. Upon exposure to high salinity, NtLTP4-overexpressing lines (OE lines) accumulated low Na + levels. Salt-responsive genes, including Na + /H + exchangers (NHX1) and high-affinity K + transporter1 (HKT1), were dramatically higher in OE lines than in wild-type lines. NtLTP4 might regulate transcription levels of NHX1 and HKT1 to alleviate the toxicity of Na + . Interestingly, OE lines enhanced the tolerance of N. tabacum to drought stress by reducing the transpiration rate. Moreover, NtLTP4 could increase reactive oxygen species (ROS)-scavenging enzyme activity and expression levels to scavenge excess ROS under drought and high salinity conditions. We used a two-hybrid yeast system and screened seven putative proteins that interact with NtLTP4 in tobacco. An MAPK member, wound-induced protein kinase, was confirmed to interact with NtLTP4 via co-immunoprecipitation and a firefly luciferase complementation imaging assay. Taken together, this is the first functional analysis of NtLTP4, and proves that NtLTP4 positively regulates salt and drought stresses in N. tabacum.

GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture

PubMed Central

Tripathi, Prateek; Rabara, Roel C.; Lin, Jun; Rushton, Paul J.

2013-01-01

Drought is the major cause of crop losses worldwide. Water stress-inducible promoters are important for understanding the mechanisms of water stress responses in crop plants. Here we utilized tobacco (Nicotiana tabacum L.) Bright Yellow 2 (BY-2) cell system in presence of polyethylene glycol, salt and phytohormones. Extension of the system to 85 mM NaCl led to inducibility of up to 10-fold with the water stress and salt responsive soybean GmWRKY53 promoter. Upon ABA and JA treatment fold inducibility was up to 5-fold and 14-fold, respectively. Thus, we hypothesize that GmWRKY53 could be used as potential model candidate for dissecting drought regulatory elements as well as understanding crosstalk utilizing a rapid heterologous system of BY-2 culture. PMID:23511199

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Hypergravity Leads to the Redistribution of Calcium Ions in Plant Cell

NASA Astrophysics Data System (ADS)

Nedukha, Olena M.

2008-06-01

The study of hypergravity influence on calcium ions distribution and on the relative amount of Ca2+ in cells of Nicotiana tabacum callus was carried out using the centrifuge. 15-day-old N. tabacum callus grown in a Murashige and Scoog agar medium was exposed to hypergravity at 6.5 g and 14 g for 15 and 60 min. The control samples and the centrifuged callus were loaded with Fluo-4 and then studied by the confocal laser-scanning microscopy. The visible redistribution of Ca2+ in the investigated cells and the appearance of calcium-microdomains in cytoplasm have been established under influence of hypergravity. Readaptation of Ca2+ distribution in the cells occurred in 2-4 h after hypergravity ending. It is suggested that influence of hypergravity lead to change of ionic transport of plasmalemma and endomembranes, and also to efflux of Ca2+ from apoplast.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

PubMed Central

Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

2016-01-01

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Response of antioxidant enzymes in Nicotiana tabacum clones during phytoextraction of heavy metals.

PubMed

Lyubenova, Lyudmila; Nehnevajova, Erika; Herzig, Rolf; Schröder, Peter

2009-07-01

Tobacco, Nicotiana tabacum, is a widely used model plant for growth on heavy-metal-contaminated sites. Its high biomass and deep rooting system make it interesting for phytoextraction. In the present study, we investigated the antioxidative activities and glutathione-dependent enzymes of different tobacco clones optimized for better Cd and Zn accumulation in order to characterize their performance in the field. The improved heavy metal resistance also makes the investigated tobacco clones interesting for understanding the plant defense enzyme system in general. Freshly harvested plant material (N. tabacum leaves) was used to investigate the antioxidative cascade in plants grown on heavy metal contaminated sites with and without amendments of different ammonium nitrate and ammonium sulfate fertilizers. Plants were grown on heavily polluted soils in north-east Switzerland. Leaves were harvested at the field site and directly deep frozen in liquid N(2). Studies were concentrated on the antioxidative enzymes of the Halliwell-Asada cycle, and spectrophotometric measurements of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), superoxide dismutase (SOD, EC 1.15.1.1), glutathione peroxidase (GPX, EC 1.11.1.9), glutathione reductase (GR, EC 1.6.4.2), glutathione S-transferase (GST, EC 2.5.1.18) were performed. We tried to explain the relationship between fertilizer amendments and the activity of the enzymatic defense systems. When tobacco (N. tabacum) plants originating from different mutants were grown under field conditions with varying fertilizer application, the uptake of cadmium and zinc from soil increased with increasing biomass. Depending on Cd and Zn uptake, several antioxidant enzymes showed significantly different activities. Whereas SOD and CAT were usually elevated, several other enzymes, and isoforms of GST were strongly inhibited. Heavy metal uptake represents severe stress to plants, and specific antioxidative enzymes are induced at the

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

PubMed

Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

2016-04-01

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cell-to-cell movement of plastids in plants.

PubMed

Thyssen, Gregory; Svab, Zora; Maliga, Pal

2012-02-14

Our objective was to test whether or not plastids and mitochondria, the two DNA-containing organelles, move between cells in plants. As our experimental approach, we grafted two different species of tobacco, Nicotiana tabacum and Nicotiana sylvestris. Grafting triggers formation of new cell-to-cell contacts, creating an opportunity to detect cell-to-cell organelle movement between the genetically distinct plants. We initiated tissue culture from sliced graft junctions and selected for clonal lines in which gentamycin resistance encoded in the N. tabacum nucleus was combined with spectinomycin resistance encoded in N. sylvestris plastids. Here, we present evidence for cell-to-cell movement of the entire 161-kb plastid genome in these plants, most likely in intact plastids. We also found that the related mitochondria were absent, suggesting independent movement of the two DNA-containing organelles. Acquisition of plastids from neighboring cells provides a mechanism by which cells may be repopulated with functioning organelles. Our finding supports the universality of intercellular organelle trafficking and may enable development of future biotechnological applications.

Characterization of secretory phospholipase A₂ with phospholipase A₁ activity in tobacco, Nicotiana tabacum (L.).

PubMed

Fujikawa, Yukichi; Fujikawa, Ritsuko; Iijima, Noriaki; Esaka, Muneharu

2012-03-01

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.

Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells

PubMed Central

Voitsekhovskaja, Olga V.; Schiermeyer, Andreas; Reumann, Sigrun

2014-01-01

Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism. PMID:25477890

Plant peroxisomes are degraded by starvation-induced and constitutive autophagy in tobacco BY-2 suspension-cultured cells.

PubMed

Voitsekhovskaja, Olga V; Schiermeyer, Andreas; Reumann, Sigrun

2014-01-01

Very recently, autophagy has been recognized as an important degradation pathway for quality control of peroxisomes in Arabidopsis plants. To further characterize the role of autophagy in plant peroxisome degradation, we generated stable transgenic suspension-cultured cell lines of heterotrophic Nicotiana tabacum L. cv. Bright Yellow 2 expressing a peroxisome-targeted version of enhanced yellow fluorescent protein. Indeed, this cell line model system proved advantageous for detailed cytological analyses of autophagy stages and for quantification of cellular peroxisome pools under different culturing conditions and upon inhibitor applications. Complementary biochemical, cytological, and pharmacological analyses provided convincing evidence for peroxisome degradation by bulk autophagy during carbohydrate starvation. This degradation was slowed down by the inhibitor of autophagy, 3-methyladenine (3-MA), but the 3-MA effect ceased at advanced stages of starvation, indicating that another degradation mechanism for peroxisomes might have taken over. 3-MA also caused an increase particularly in peroxisomal proteins and cellular peroxisome numbers when applied under nutrient-rich conditions in the logarithmic growth phase, suggesting a high turnover rate for peroxisomes by basal autophagy under non-stress conditions. Together, our data demonstrate that a great fraction of the peroxisome pool is subject to extensive autophagy-mediated turnover under both nutrient starvation and optimal growth conditions. Our analyses of the cellular pool size of peroxisomes provide a new tool for quantitative investigations of the role of plant peroxisomes in reactive oxygen species metabolism.

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

PubMed

Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C

1994-01-01

We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

Conservation and possible reintroduction of an endangered plant based on an analysis of community ecology: a case study of Primulina tabacum Hance in China

Treesearch

Hai Ren; Qianmei Zhang; Zhengfeng Wang; Qinfeng Guo; June Wang; Nan Liu; Kaiming Liang

2010-01-01

The distribution of the rare and endangered perennial herb Primulina tabacum Hance is restricted to eight karst caves in southern China. To conserve P. tabacum and to evaluate possible reintroduction, we studied its historical distribution and conducted field surveys of both its biotic and physical environment. We used detrended...

Soft material-based microculture system having air permeable cover sheet for the protoplast culture of Nicotiana tabacum.

PubMed

Ju, Jong Il; Ko, Jung-Moon; Kim, So Hyeon; Baek, Ju Yeoul; Cha, Hyeon-Cheol; Lee, Sang Hoon

2006-08-01

In plant cell culture, the delivery of nutrition and gas (mainly oxygen) to the cells is the most important factor for viability. In this paper, we propose a polydimethylsiloxane (PDMS)-based microculture system that is designed to have good aeration. PDMS is known to have excellent air permeability, and through the experimental method, we investigated the relation between the degree of air delivery and the thickness of the PDMS sheet covering the culture chamber. We determined the proper thickness of the cover sheet, and cultured protoplasts of Nicotiana tabacum in a culture chamber covered with a PDMS sheet having thickness of 400 microm. The cells were successfully divided, and lived well inside the culture chamber for 10 days. In addition, protoplasts were cultured inside the culture chambers covered with the cover glass and the PDMS sheet, respectively, and the microcolonies were formed well inside the PDMS covered chamber after 10 days.

The grapevine VvWRKY2 gene enhances salt and osmotic stress tolerance in transgenic Nicotiana tabacum.

PubMed

Mzid, Rim; Zorrig, Walid; Ben Ayed, Rayda; Ben Hamed, Karim; Ayadi, Mariem; Damak, Yosra; Lauvergeat, Virginie; Hanana, Mohsen

2018-06-01

Our study aims to assess the implication of WRKY transcription factor in the molecular mechanisms of grapevine adaptation to salt and water stresses. In this respect, a full-length VvWRKY2 cDNA, isolated from a Vitis vinifera grape berry cDNA library, was constitutively over-expressed in Nicotiana tabacum seedlings. Our results showed that transgenic tobacco plants exhibited higher seed germination rates and better growth, under both salt and osmotic stress treatments, when compared to wild type plants. Furthermore, our analyses demonstrated that, under stress conditions, transgenic plants accumulated more osmolytes, such as soluble sugars and free proline, while no changes were observed regarding electrolyte leakage, H 2 O 2 , and malondialdehyde contents. The improvement of osmotic adjustment may be an important mechanism underlying the role of VvWRKY 2 in promoting tolerance and adaptation to abiotic stresses. Principal component analysis of our results highlighted a clear partition of plant response to stress. On the other hand, we observed a significant adaptation behaviour response for transgenic lines under stress. Taken together, all our findings suggest that over-expression of VvWRKY2 gene has a compelling role in abiotic stress tolerance and, therefore, would provide a useful strategy to promote abiotic stress tolerance in grape via molecular-assisted breeding and/or new biotechnology tools.

Biologically active recombinant human erythropoietin expressed in hairy root cultures and regenerated plantlets of Nicotiana tabacum L.

PubMed Central

Schäfer, Holger; Ramamoorthy, Siva; Wink, Michael

2017-01-01

Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells. Two versions of rhEPO clones, with or without the N-terminal calreticulin (cal) fusion sequence, were produced by cloning the amplified construct into gateway binary vector pK7WG2D. Following Agrobacterium rhizogenes mediated transformation of tobacco explants; integration and expression of constructs in hairy roots were confirmed by several tests at DNA, RNA and protein levels. The amount of intracellular rhEPO from hairy root cultures with cal signal peptide was measured up to 66.75 ng g-1 of total soluble protein. The presence of the ER signal peptide (cal) was essential for the secretion of rhEPO into the spent medium; no protein was detected from hairy root cultures without ER signal peptide. The addition of polyvinylpyrrolidone enhanced the stabilization of secreted rhEPO leading to a 5.6 fold increase to a maximum concentration of 185.48 pg rhEPOHR g-1 FW hairy root cultures. The rhizo-secreted rhEPO was separated by HPLC and its biological activity was confirmed by testing distinct parameters for proliferation and survival in retinal pigment epithelial cells (ARPE). In addition, the rhEPO was detected to an amount 14.8 ng g-1 of total soluble leaf protein in transgenic T0 generation plantlets regenerated from hairy root cultures with cal signal peptide. PMID:28800637

Phosphorus acquisition by citrate- and phytase-exuding Nicotiana tabacum plant mixtures depends on soil phosphorus availability and root intermingling.

PubMed

Giles, Courtney D; Richardson, Alan E; Cade-Menun, Barbara J; Mezeli, Malika M; Brown, Lawrie K; Menezes-Blackburn, Daniel; Darch, Tegan; Blackwell, Martin Sa; Shand, Charles A; Stutter, Marc I; Wendler, Renate; Cooper, Patricia; Lumsdon, David G; Wearing, Catherine; Zhang, Hao; Haygarth, Philip M; George, Timothy S

2018-03-02

Citrate and phytase root exudates contribute to improved phosphorus (P) acquisition efficiency in Nicotiana tabacum (tobacco) when both exudates are produced in a P deficient soil. To test the importance of root intermingling in the interaction of citrate and phytase exudates, Nicotiana tabacum plant-lines with constitutive expression of heterologous citrate (Cit) or fungal phytase (Phy) exudation traits were grown under two root treatments (roots separated or intermingled) and in two soils with contrasting soil P availability. Complementarity of plant mixtures varying in citrate efflux rate and mobility of the expressed phytase in soil was determined based on plant biomass and P accumulation. Soil P composition was evaluated using solution 31 P NMR spectroscopy. In the soil with limited available P, positive complementarity occurred in Cit+Phy mixtures with roots intermingled. Root separation eliminated positive interactions in mixtures expressing the less mobile phytase (Aspergillus niger PhyA) whereas positive complementarity persisted in mixtures that expressed the more mobile phytase (Peniophora lycii PhyA). Soils from Cit+Phy mixtures contained less inorganic P and more organic P compared to monocultures. Exudate-specific strategies for the acquisition of soil P were most effective in P-limited soil and depended on citrate efflux rate and the relative mobility of the expressed phytase in soil. Plant growth and soil P utilization in plant systems with complementary exudation strategies are expected to be greatest where exudates persist in soil and are expressed synchronously in space and time. This article is protected by copyright. All rights reserved.

Root-specific expression of opine genes and opine accumulation in some cultivars of the naturally occurring genetically modified organism Nicotiana tabacum.

PubMed

Chen, Ke; de Borne, François Dorlhac; Julio, Emilie; Obszynski, Julie; Pale, Patrick; Otten, Léon

2016-08-01

Previous studies have shown that Nicotiana tabacum contains three Agrobacterium-derived T-DNA sequences inherited from its paternal ancestor Nicotiana tomentosiformis. Among these, the TB locus carries an intact mannopine synthase 2' gene (TB-mas2'). This gene is similar to the Agrobacterium rhizogenes A4-mas2' gene that encodes the synthesis of the Amadori compound deoxyfructosyl-glutamine (DFG or santhopine). In this study we show that TB-mas2' is expressed at very low levels in N. tomentosiformis and in most N. tabacum cultivars; however, some cultivars show high TB-mas2' expression levels. The TB-mas2' promoter sequences of low- and high-expressing cultivars are identical. The low/high level of expression segregates as a single Mendelian factor in a cross between a low- and a high-expression cultivar. pTB-mas2'-GUS and pA4-mas2'-GUS reporter genes were stably introduced in N. benthamiana. Both were mainly expressed in the root expansion zone and leaf vasculature. Roots of tobacco cultivars with high TB-mas2' expression contain detectable levels of DFG. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

PubMed

Lamotte, Olivier; Courtois, Cécile; Dobrowolska, Grazyna; Besson, Angélique; Pugin, Alain; Wendehenne, David

2006-04-15

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE PAGES

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; ...

2017-01-18

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. We tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids inmore » leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. Furthermore, when expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ERvesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. Our results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.« less

Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

USDA-ARS?s Scientific Manuscript database

Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with

Cell Wall Structure in Cells Adapted to Growth on the Cellulose-Synthesis Inhibitor 2,6-Dichlorobenzonitrile 1

PubMed Central

Shedletzky, Esther; Shmuel, Miri; Trainin, Tali; Kalman, Sara; Delmer, Deborah

1992-01-01

Our previous work (E. Shedletzky, M. Shmuel, D.P. Delmer, D.T.A. Lamport [1990] Plant Physiol 94:980-987) showed that suspension-cultured tomato cells adapted to growth on the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a markedly altered cell wall composition, most notably a markedly reduced level of the cellulose-xyloglucan network. This study compares the adaptation to DCB of two cell lines from dicots (tomato [Lycopersicon esculentum] and tobacco [Nicotiana tabacum]) and a Graminaceous monocot (barley [Hordeum bulbosum] endosperm). The difference in wall structures between the dicots and the monocot is reflected in the very different types of wall modifications induced by growth on DCB. The dicots, having reduced levels of cellulose and xyloglucan, possess walls the major integrity of which is provided by Ca2+-bridged pectates because protoplasts can be prepared from these cells simply by treatment with divalent cation chelator and a purified endopolygalacturonase. The tensile strength of these walls is considerably less than walls from nonadapted cells, but wall porosity is not altered. In contrast, walls from adapted barley cells contain very little pectic material and normal to elevated levels of noncellulosic polysaccharides compared with walls from nonadapted cells. Surprisingly, they have tensile strengths higher than their nonadapted counterpart, although cellulose levels are reduced by 70%. Evidence is presented that these walls obtain their additional strength by an altered pattern of cross-linking of polymers involving phenolic components. Such cross-linking may also explain the observation that the porosity of these walls is also considerably reduced. Cells of adapted lines of both the dicots and barley are resistant to plasmolysis, suggesting that they possess very strong connections between the wall and the plasma membrane. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:16652933

Mouse fat storage-inducing transmembrane protein 2 (FIT2) promotes lipid droplet accumulation in plants.

PubMed

Cai, Yingqi; McClinchie, Elizabeth; Price, Ann; Nguyen, Thuy N; Gidda, Satinder K; Watt, Samantha C; Yurchenko, Olga; Park, Sunjung; Sturtevant, Drew; Mullen, Robert T; Dyer, John M; Chapman, Kent D

2017-07-01

Fat storage-inducing transmembrane protein 2 (FIT2) is an endoplasmic reticulum (ER)-localized protein that plays an important role in lipid droplet (LD) formation in animal cells. However, no obvious homologue of FIT2 is found in plants. Here, we tested the function of FIT2 in plant cells by ectopically expressing mouse (Mus musculus) FIT2 in Nicotiana tabacum suspension-cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT2 dramatically increased the number and size of LDs in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis. When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum, FIT2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER-LD junction sites. FIT2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER-vesicle exit sites. Collectively, these results demonstrate that mouse FIT2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT2 homologues in higher plants. These results suggest also that FIT2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. [Nicotiana tabacum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Constantin, M.J.; Henke, R.R.; Mansur, M.A.

1977-01-01

Incorporating activated charcoal (AC) in culture media has been shown to affect growth and development of various organisms. Since AC stimulates the development of tobacco haploid plantlets from cultured anthers, research was conducted to determine the effect of activated charcoal on pith-derived callus growth and shoot development in Nicotiana tabacum cv. Wisconsin 38. Our results indicate that the hormones required for callus growth and shoot development in Wisconsin-38 tobacco are adsorbed by AC, thereby inhibiting callus growth and prohibiting shoot development. This effect was observed even when AC was removed from the medium by filtration prior to culturing the callus.

Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response.

PubMed

Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé; Faber, Anne-Marie; Morsomme, Pierre; Boutry, Marc

2011-02-01

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of AgMaT2, a Plasma Membrane Mannitol Transporter from Celery, Expressed in Phloem Cells, Including Phloem Parenchyma Cells[OA

PubMed Central

Juchaux-Cachau, Marjorie; Landouar-Arsivaud, Lucie; Pichaut, Jean-Philippe; Campion, Claire; Porcheron, Benoit; Jeauffre, Julien; Noiraud-Romy, Nathalie; Simoneau, Philippe; Maurousset, Laurence; Lemoine, Rémi

2007-01-01

A second mannitol transporter, AgMaT2, was identified in celery (Apium graveolens L. var. dulce), a species that synthesizes and transports mannitol. This transporter was successfully expressed in two different heterologous expression systems: baker's yeast (Saccharomyces cerevisiae) cells and tobacco (Nicotiana tabacum) plants (a non-mannitol-producing species). Data indicated that AgMaT2 works as an H+/mannitol cotransporter with a weak selectivity toward other polyol molecules. When expressed in tobacco, AgMaT2 decreased the sensitivity to the mannitol-secreting pathogenic fungi Alternaria longipes, suggesting a role for polyol transporters in defense mechanisms. In celery, in situ hybridization showed that AgMaT2 was expressed in the phloem of leaflets, petioles from young and mature leaves, floral stems, and roots. In the phloem of petioles and leaflets, AgMaT2, as localized with specific antibodies, was present in the plasma membrane of three ontologically related cell types: sieve elements, companion cells, and phloem parenchyma cells. These new data are discussed in relation to the physiological role of AgMaT2 in regulating mannitol fluxes in celery petioles. PMID:17631523

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells.

PubMed

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-05-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.

Production and characterization of anti-(mucin MUC1) single-domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi).

PubMed

Ismaili, Ahmad; Jalali-Javaran, Mokhtar; Rasaee, Mohammad J; Rahbarizadeh, Fatemeh; Forouzandeh-Moghadam, Mehdi; Memari, Hamid Rajabi

2007-05-01

Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CH1s (constant heavy-chain domains). The antigen-specific binding fragments of these heavy-chain antibodies therefore comprise one single domain (the so-called 'VHH') and are of great importance in biotechnological applications. To evaluate the expression and biological activity of sdAbs (single-domain antibodies) in plants, which, on account of their small size and antigen-recognition properties, would have a major impact on antibody-engineering strategies, we constructed a pBI121-VHH gene encoding the recombinant sdAb fragments with specificity for a cancer-associated mucin, MUC1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC1 antigens and immunocytochemistry with cancerous cell lines confirmed that the activity of these molecules compared favourably with that of the parent recombinant antibodies. Protein purification was achieved by using sequential (NH4)2SO4 precipitation, gel filtration and immunoaffinity chromatography. Analysis of the purified VHH by ELISA indicated that the purified antibody fragments were able to react successfully with a MUC1-related peptide. These results reaffirm that the tobacco plant is a suitable host for the production of correctly folded VHH antibody fragments with diagnostic and therapeutic applications.

Cytoskeletal mechanisms in positioning of the second-division spindles and meiotic restitution in tobacco (Nicotiana tabacum L.) microsporogenesis.

PubMed

Sidorchuk, Yuriy Vladimirovich; Deineko, Elena Victorovna

2017-06-01

Microsporogenesis patterns of the polyploid (2n = 4x = 96) and diploid (2n = 2x = 48) Nicotiana tabacum L. (cv. Havana Petit line SR1) plants have been analyzed and compared. Four types of abnormal positions of the second-division spindles-tripolar, parallel, proximal, and fused-have been observed. Of these abnormalities, only tripolar (2.4%) and parallel (1.4%) spindles are observable in diploid plants. As for polyploids, the increased ploidy is accompanied by an increase in the incidence of tripolar (22.8%) and parallel (8.1%) spindle orientations and emergence of two remaining abnormalities (proximal and fused spindles, 3.3%). As has been shown, the spindle position abnormalities in diploid plants have no effect on the meiotic products, whereas both dyads and triads are detectable among the tetrads in polyploid plants. Analysis of cytoskeletal remodeling has allowed for the insight into the role of interzonal radial microtubule system in spindle positioning during the second division. The reason underlying the change in spindle positioning is disturbed polymerization-depolymerization processes and interdigitation of microtubule plus ends within the interzonal cytoskeleton system in late telophase I-interkinesis and prophase II. As has been demonstrated, fused second-division spindles are formed as a result of fused cytoskeletal structures in prophase-prometaphase II in the case when the nuclei are drawn abnormally close to one another. © 2017 International Federation for Cell Biology.

AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca(2+) increase.

PubMed

Francia, Doriana; Chiltz, Annick; Lo Schiavo, Fiorella; Pugin, Alain; Bonfante, Paola; Cardinale, Francesca

2011-09-01

The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapid and transient increase in free cytosolic Ca(2+) in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca(2+)-channel blocker (La(3+)) showed that in these cells, MAPK activation was dependent on the cytosolic Ca(2+) increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca(2+) transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Measuring the mechanical properties of plant cells by combining micro-indentation with osmotic treatments.

PubMed

Weber, Alain; Braybrook, Siobhan; Huflejt, Michal; Mosca, Gabriella; Routier-Kierzkowska, Anne-Lise; Smith, Richard S

2015-06-01

Growth in plants results from the interaction between genetic and signalling networks and the mechanical properties of cells and tissues. There has been a recent resurgence in research directed at understanding the mechanical aspects of growth, and their feedback on genetic regulation. This has been driven in part by the development of new micro-indentation techniques to measure the mechanical properties of plant cells in vivo. However, the interpretation of indentation experiments remains a challenge, since the force measures results from a combination of turgor pressure, cell wall stiffness, and cell and indenter geometry. In order to interpret the measurements, an accurate mechanical model of the experiment is required. Here, we used a plant cell system with a simple geometry, Nicotiana tabacum Bright Yellow-2 (BY-2) cells, to examine the sensitivity of micro-indentation to a variety of mechanical and experimental parameters. Using a finite-element mechanical model, we found that, for indentations of a few microns on turgid cells, the measurements were mostly sensitive to turgor pressure and the radius of the cell, and not to the exact indenter shape or elastic properties of the cell wall. By complementing indentation experiments with osmotic experiments to measure the elastic strain in turgid cells, we could fit the model to both turgor pressure and cell wall elasticity. This allowed us to interpret apparent stiffness values in terms of meaningful physical parameters that are relevant for morphogenesis. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Effects of arbuscular mycorrhizal fungi inoculation on arsenic accumulation by tobacco (Nicotiana tabacum L.).

PubMed

Hua, Jianfeng; Lin, Xiangui; Yin, Rui; Jiang, Qian; Shao, Yufang

2009-01-01

A pot experiment was conducted to study the effects of arbuscular mycorrhizal (AM) fungi (from contaminated or uncontaminated soils) on arsenic (As) uptake of tobacco (Nicotiana tabacum L.) in As-contaminated soil. Mycorrhizal colonization rate, dry weight, As and P uptake by plants, concentrations of water-extractable As and As fractions were determined. A low mycorrhizal colonization rate (< 25%) was detected. Our research indicated that AM fungi isolated from polluted soils were no more effective than those from unpolluted soils when grown in symbiosis with tobacco. No significant differences were observed in roots and stalks dry weights among all treatments. Leaves and total plant dry weights were much higher in Glomus versiforme treatment than that in control treatment. As contents in roots and stalks from mycorrhizal treatments were much lower than that from control treatment. Total plant As content exhibited the same trend. P concentrations in tobacco were not affected by colonization, nor were stalks, leaves and total plant P contents. Roots P contents were remarkably lower in HN treatments than in other treatments. Meanwhile, decreased soil pH and lower water-extractable As concentrations and higher levels of As fraction bound to well-crystallized hydrous oxides of Fe and Al were found in mycorrhizal treatments than in controls. The protective effect of mycorrhiza against plant As uptake may be associated with changes in As solubility mediated by changing soil pH. These results indicated that under As stress, proper mechanisms employed by AM fungi can protect tobacco against As uptake. Results confirmed that AM fungi can play an important role in food quality and safety.

Potency of Nicotiana tabacum as anti - microfouling

NASA Astrophysics Data System (ADS)

Aunurohim, Nurilma, Dian Ahmada; Kuswytasari, Nengah Dwianita

2017-06-01

In general, the attachment and growth of organisms on the surface of the object or material immersed in the sea are called Biofouling. Biofouling microscopic called microfouling, where it acts as a precursor of the next engaging organisms that are generally larger in size (called macrofouling). In recent time, biofouling control over the use of chemicals in antifouling paints. Usage tributyltin polishing copolymer paints (TBT - SPC paint) containing TBT has adverse effects on non-target marine organisms. This study uses tobacco dust as an anti-microfouling, which is the waste generated during the process of tobacco leaves. Five of bacterial isolates have been founded at Surabaya coastal in a preliminary test with iron plates. Then those isolates tested to detect and visualized of biofilm. The activity of anti-bacteria had been done and the result is known that more high of tobacco extract given, a diameter of zone inhibit high too. The extract of tobacco dust can be applied most effectively to be anti-microfouling is a concentration of 40%, isolates 3 and 4 are a type of bacteria that is most inhibited growth. So, in this result, Nicotiana tabacum is potential as an anti-microfouling.

Acid-growth response and alpha-expansins in suspension cultures of bright yellow 2 tobacco

NASA Technical Reports Server (NTRS)

Link, B. M.; Cosgrove, D. J.

1998-01-01

The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of alpha-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple alpha-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.

Genome-Wide Identification and Expression Profiling Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family in Tobacco (Nicotiana tabacum L.).

PubMed

Wang, Meng; Xu, Zongchang; Ding, Anming; Kong, Yingzhen

2018-05-24

Xyloglucan endotransglucosylase/hydrolase genes ( XTHs ) encode enzymes required for the reconstruction and modification of xyloglucan backbones, which will result in changes of cell wall extensibility during growth. A total of 56 NtXTH genes were identified from common tobacco, and 50 cDNA fragments were verified by PCR amplification. The 56 NtXTH genes could be classified into two subfamilies: Group I/II and Group III according to their phylogenetic relationships. The gene structure, chromosomal localization, conserved protein domains prediction, sub-cellular localization of NtXTH proteins and evolutionary relationships among Nicotiana tabacum , Nicotiana sylvestrisis , Nicotiana tomentosiformis , Arabidopsis , and rice were also analyzed. The NtXTHs expression profiles analyzed by the TobEA database and qRT-PCR revealed that NtXTHs display different expression patterns in different tissues. Notably, the expression patterns of 12 NtXTHs responding to environment stresses, including salinity, alkali, heat, chilling, and plant hormones, including IAA and brassinolide, were characterized. All the results would be useful for the function study of NtXTHs during different growth cycles and stresses.

Changes in the Antioxidant Systems as Part of the Signaling Pathway Responsible for the Programmed Cell Death Activated by Nitric Oxide and Reactive Oxygen Species in Tobacco Bright-Yellow 2 Cells1

PubMed Central

de Pinto, Maria Concetta; Tommasi, Franca; De Gara, Laura

2002-01-01

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for the activation of the hypersensitive reaction, a defense response induced in the noncompatible plant-pathogen interaction. However, its involvement in activating programmed cell death (PCD) in plant cells has been questioned. In this paper, the involvement of the cellular antioxidant metabolism in the signal transduction triggered by these bioactive molecules has been investigated. NO and ROS levels were singularly or simultaneously increased in tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells by the addition to the culture medium of NO and/or ROS generators. The individual increase in NO or ROS had different effects on the studied parameters than the simultaneous increase in the two reactive species. NO generation did not cause an increase in phenylalanine ammonia-lyase (PAL) activity or induction of cellular death. It only induced minor changes in ascorbate (ASC) and glutathione (GSH) metabolisms. An increase in ROS induced oxidative stress in the cells, causing an oxidation of the ASC and GSH redox pairs; however, it had no effect on PAL activity and did not induce cell death when it was generated at low concentrations. In contrast, the simultaneous increase of NO and ROS activated a process of death with the typical cytological and biochemical features of hypersensitive PCD and a remarkable rise in PAL activity. Under the simultaneous generation of NO and ROS, the cellular antioxidant capabilities were also suppressed. The involvement of ASC and GSH as part of the transduction pathway leading to PCD is discussed. PMID:12376637

Myosin XI-Dependent Formation of Tubular Structures from Endoplasmic Reticulum Isolated from Tobacco Cultured BY-2 Cells1[W][OA

PubMed Central

Yokota, Etsuo; Ueda, Haruko; Hashimoto, Kohsuke; Orii, Hidefumi; Shimada, Tomoo; Hara-Nishimura, Ikuko; Shimmen, Teruo

2011-01-01

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER. PMID:21427277

Short Exogenous Peptides Regulate Expression of CLE, KNOX1, and GRF Family Genes in Nicotiana tabacum.

PubMed

Fedoreyeva, L I; Dilovarova, T A; Ashapkin, V V; Martirosyan, Yu Ts; Khavinson, V Kh; Kharchenko, P N; Vanyushin, B F

2017-04-01

Exogenous short biologically active peptides epitalon (Ala-Glu-Asp-Gly), bronchogen (Ala-Glu-Asp-Leu), and vilon (Lys-Glu) at concentrations 10 -7 -10 -9  M significantly influence growth, development, and differentiation of tobacco (Nicotiana tabacum) callus cultures. Epitalon and bronchogen, in particular, both increase growth of calluses and stimulate formation and growth of leaves in plant regenerants. Because the regulatory activity of the short peptides appears at low peptide concentrations, their action to some extent is like that of the activity of phytohormones, and it seems to have signaling character and epigenetic nature. The investigated peptides modulate in tobacco cells the expression of genes including genes responsible for tissue formation and cell differentiation. These peptides differently modulate expression of CLE family genes coding for known endogenous regulatory peptides, the KNOX1 genes (transcription factor genes) and GRF (growth regulatory factor) genes coding for respective DNA-binding proteins such as topoisomerases, nucleases, and others. Thus, at the level of transcription, plants have a system of short peptide regulation of formation of long-known peptide regulators of growth and development. The peptides studied here may be related to a new generation of plant growth regulators. They can be used in the experimental botany, plant molecular biology, biotechnology, and practical agronomy.

Sensory role of actin in auxin-dependent responses of tobacco BY-2.

PubMed

Huang, Xiang; Maisch, Jan; Nick, Peter

2017-11-01

Polar auxin transport depends on the polar localization of auxin-efflux carriers. The cycling of these carriers between cell interior and plasma membrane depends on actin. The dynamic of actin not only affects auxin transport, but also changes the auxin-responsiveness. To study the potential link between auxin responsiveness and actin dynamics, we investigated developmental responses of the non-transformed BY-2 (Nicotiana tabacum L. cv Bright Yellow 2) cell line and the transgenic BY-2 strain GF11 (stably transformed BY-2 cells with a GFP-fimbrin actin-binding domain 2 construct). The developmental process was divided into three distinct stages: cell cycling, cell elongation and file disintegration. Several phenotypes were measured to monitor the cellular responses to different concentrations of exogenous natural auxin (Indole-3-acetic acid, IAA). We found that auxin stimulated and prolonged the mitotic activity, and delayed the exit from the proliferation phase. However, both responses were suppressed in the GF11 line. At the stationary phase of the cultivation cycle, auxin strongly accelerated the cell file disintegration. Interestingly, it was not suppressed but progressed to a more complete disintegration in the GF11 line. During the cultivation cycle, we also followed the organization of actin in the GF11 line and did not detect any significant difference in actin organization from untreated control or exogenous IAA treatment. Therefore, our findings indicate that the specific differences observed in the GF11 line must be linked with a function of actin that is not structural. It means that there is a sensory role of actin for auxin signaling. Copyright © 2017 Elsevier GmbH. All rights reserved.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, A.; Manzara, T.; Lurquin, P.F.

1984-09-17

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciensmore » T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.« less

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals.

PubMed

Colgan, Richard; Atkinson, Christopher J; Paul, Matthew; Hassan, Sally; Drake, Pascal M W; Sexton, Amy L; Santa-Cruz, Simon; James, David; Hamp, Keith; Gutteridge, Colin; Ma, Julian K-C

2010-04-01

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth

Nicotiana tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway.

PubMed

Ghanta, Srijani; Bhattacharyya, Dipto; Sinha, Ragini; Banerjee, Anindita; Chattopadhyay, Sharmila

2011-05-01

The elaborate networks and the crosstalk of established signaling molecules like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), abscisic acid (ABA), reactive oxygen species (ROS) and glutathione (GSH) play key role in plant defense response. To obtain further insight into the mechanism through which GSH is involved in this crosstalk to mitigate biotic stress, transgenic Nicotiana tabacum overexpressing Lycopersicon esculentum gamma-glutamylcysteine synthetase (LeECS) gene (NtGB lines) were generated with enhanced level of GSH in comparison with wild-type plants exhibiting resistance to pathogenesis as well. The expression levels of non-expressor of pathogenesis-related genes 1 (NPR1)-dependent genes like pathogenesis-related gene 1 (NtPR1), mitogen-activated protein kinase kinase (NtMAPKK), glutamine synthetase (NtGLS) were significantly enhanced along with NtNPR1. However, the expression levels of NPR1-independent genes like NtPR2, NtPR5 and short-chain dehydrogenase/reductase family protein (NtSDRLP) were either insignificant or were downregulated. Additionally, increase in expression of thioredoxin (NtTRXh), S-nitrosoglutathione reductase 1 (NtGSNOR1) and suppression of isochorismate synthase 1 (NtICS1) was noted. Comprehensive analysis of GSH-fed tobacco BY2 cell line in a time-dependent manner reciprocated the in planta results. Better tolerance of NtGB lines against biotrophic Pseudomonas syringae pv. tabaci was noted as compared to necrotrophic Alternaria alternata. Through two-dimensional gel electrophoresis (2-DE) and image analysis, 48 differentially expressed spots were identified and through identification as well as functional categorization, ten proteins were found to be SA-related. Collectively, our results suggest GSH to be a member in cross-communication with other signaling molecules in mitigating biotic stress likely through NPR1-dependent SA-mediated pathway.

The relative absorption cross-sections of photosystem I and photosystem II in chloroplasts from three types of Nicotiana tabacum.

PubMed

Melis, A; Thielen, A P

1980-02-08

In the present study we used three types of Nicotiana tabacum, cv John William's Broad Leaf (the wild type and two mutants, the yellow-green Su/su and the yellow Su/su var. Aurea) in order to correlat functional properties of Photosystem II and Photosystem I with the structural organization of their chloroplasts. The effective absorption cross-section of Photosystem II and Photosystem I centers was measured by means of the rate constant of their photoconversion under light-limiting conditions. In agreement with earlier results (Okabe, K., Schmid, G.H. and Straub, J. (1977) Plant Physiol. 60, 150--156) the photosynthetic unit size for both System II and System I in the two mutants was considerably smaller as compared to the wild type. We observed biphasic kinetics in the photoconversion of System II in all three types of N. tabacum. However, the photoconversion of System I occurred with monophasic and exponential kinetics. Under our experimental conditions, the effective cross-section of Photosystem I was comparable to that of the fast System II component (alpha centers). The relative amplitude of the slow System II component (beta centers) varied between 30% in the wild type to 70% in the Su/su var. Aurea mutant. The increased fraction of beta centers is correlated with the decreased fraction of appressed photosynthetic membranes in the chloroplasts of the two mutants. As a working hypothesis, it is suggested that beta centers are located on photosynthetic membranes directly exposed to the stroma medium.

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

NASA Astrophysics Data System (ADS)

Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

2006-06-01

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Molecular variability and evolution of the pectate lyase (pel-2) parasitism gene in cyst nematodes parasitizing different solanaceous plants.

PubMed

Geric Stare, Barbara; Fouville, Didier; Širca, Saša; Gallot, Aurore; Urek, Gregor; Grenier, Eric

2011-02-01

While pectate lyases are major parasitism factors in plant-parasitic nematodes, there is little information on the variability of these genes within species and their utility as pathotype or host range molecular markers. We have analysed polymorphisms of pectate lyase 2 (pel-2) gene, which degrades the unesterified polygalacturonate (pectate) of the host cell-wall, in the genus Globodera. Molecular variability of the pel-2 gene and the predicted protein was evaluated in populations of G. rostochiensis, G. pallida, G. "mexicana" and G. tabacum. Seventy eight pel-2 sequences were obtained and aligned. Point mutations were observed at 373 positions, 57% of these affect the coding part of the gene and produce 129 aa replacements. The observed polymorphism does not correlate either to the pathotypes proposed in potato cyst nematodes (PCN) or the subspecies described in tobacco cyst nematodes. The trees reveal a topology different from the admitted species topology as G. rostochiensis and G. pallida sequences are more similar to each other than to G. tabacum. Species-specific sites, potentially applicable for identification, and sites distinguishing PCN from tobacco cyst nematodes, were identified. As both G. rostochiensis and G. pallida display the same host range, but distinct from G. tabacum, which cannot parasitize potato plants, it is tempting to speculate that pel-2 genes polymorphism may be implicated in this adaptation, a view supported by the fact that no active pectate lyase 2 was found in G. "mexicana", a close relative of G. pallida that is unable to develop on cultivated potato varieties.

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions

PubMed Central

Thai, Long; Rush, Jeffrey S.; Maul, Jude E.; Devarenne, Timothy; Rodgers, Dana L.; Chappell, Joseph; Waechter, Charles J.

1999-01-01

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [3H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [3H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [3H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [3H]F-OH or [3H]geranylgeraniol ([3H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [3H]F-P and [3H]F-P-P were synthesized when exogenous [3H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [3H]F-P and [3H]F-P-P. In agreement with this kinetic pattern of labeling, [32P]F-P and [32P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ-32P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [3H]GG-OH to [3H]geranylgeranyl monophosphate and [3H]geranylgeranyl pyrophosphate ([3H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [3H]CTP was formed when microsomes were incubated with [3H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation. PMID:10557276

Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions.

PubMed

Thai, L; Rush, J S; Maul, J E; Devarenne, T; Rodgers, D L; Chappell, J; Waechter, C J

1999-11-09

The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [(3)H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [(3)H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [(3)H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [(3)H]F-OH or [(3)H]geranylgeraniol ([(3)H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [(3)H]F-P and [(3)H]F-P-P were synthesized when exogenous [(3)H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor-product relationship between [(3)H]F-P and [(3)H]F-P-P. In agreement with this kinetic pattern of labeling, [(32)P]F-P and [(32)P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [gamma-(32)P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [(3)H]GG-OH to [(3)H]geranylgeranyl monophosphate and [(3)H]geranylgeranyl pyrophosphate ([(3)H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [(3)H]CTP was formed when microsomes were incubated with [(3)H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein

The Role of Sink Strength and Nitrogen Availability in the Down-Regulation of Photosynthetic Capacity in Field-Grown Nicotiana tabacum L. at Elevated CO2 Concentration.

PubMed

Ruiz-Vera, Ursula M; De Souza, Amanda P; Long, Stephen P; Ort, Donald R

2017-01-01

Down-regulation of photosynthesis is among the most common responses observed in C 3 plants grown under elevated atmospheric CO 2 concentration ([CO 2 ]). Down-regulation is often attributed to an insufficient capacity of sink organs to use or store the increased carbohydrate production that results from the stimulation of photosynthesis by elevated [CO 2 ]. Down-regulation can be accentuated by inadequate nitrogen (N) supply, which may limit sink development. While there is strong evidence for down-regulation of photosynthesis at elevated [CO 2 ] in enclosure studies most often involving potted plants, there is little evidence for this when [CO 2 ] is elevated fully under open-air field treatment conditions. To assess the importance of sink strength on the down-regulation of photosynthesis and on the potential of N to mitigate this down-regulation under agriculturally relevant field conditions, two tobacco cultivars ( Nicotiana tabacum L. cv. Petit Havana; cv. Mammoth) of strongly contrasting ability to produce the major sink of this crop, leaves, were grown under ambient and elevated [CO 2 ] and with two different N additions in a free air [CO 2 ] (FACE) facility. Photosynthetic down-regulation at elevated [CO 2 ] reached only 9% in cv. Mammoth late in the season likely reflecting sustained sink strength of the rapidly growing plant whereas down-regulation in cv. Petit Havana reached 25%. Increased N supply partially mitigated down-regulation of photosynthesis in cv. Petit Havana and this mitigation was dependent on plant developmental stage. Overall, these field results were consistent with the hypothesis that sustained sink strength, that is the ability to utilize photosynthate, and adequate N supply will allow C 3 crops in the field to maintain enhanced photosynthesis and therefore productivity as [CO 2 ] continues to rise.

Transcriptome analysis of resistant and susceptible tobacco (Nicotiana tabacum) in response to root-knot nematode Meloidogyne incognita infection.

PubMed

Xing, Xuexia; Li, Xiaohui; Zhang, Mingzhen; Wang, Yuan; Liu, Bingyang; Xi, Qiliang; Zhao, Ke; Wu, Yunjie; Yang, Tiezhao

2017-01-22

The root-knot nematode (RKN) Meloidogyne incognita reproduces on the roots of tobacco (Nicotiana tabacum), damaging crops, reducing crop yield, and causing economic losses annually. The development of resistant genotypes is an alternative strategy to effectively control these losses. However, the molecular mechanism responsible for host pathogenesis and defense responses in tobacco specifically against RKNs remain poorly understood. Here, root transcriptome analysis of resistant (Yuyan12) and susceptible (Changbohuang) tobacco varieties infected with RKNs was performed. Moreover, 2623 and 545 differentially expressed genes (DEGs) in RKN-infected roots were observed in Yuyan12 and Changbohuang, respectively, compared to those in non-infected roots, including 289 DEGs commonly expressed in the two genotypes. Among these DEGs, genes encoding cell wall modifying proteins, auxin-related proteins, the ROS scavenging system, and transcription factors involved in various biological and physiochemical processes were significantly expressed in both the resistant and susceptible genotypes. This work is thus the first report on the relationships in the RKN-tobacco interaction using transcriptome analysis, and the results provide important information on the mechanism of RKN resistance in tobacco. Copyright © 2016 Elsevier Inc. All rights reserved.

In Vitro Assay of Ethanolic Heat Reflux Extract of Nicotiana tabacum L. var Virginia Against Nosocomial Bacteria Pathogen

NASA Astrophysics Data System (ADS)

Pramono, Andri; Fauzantoro, Ahmad; Rizki Hidayati, Irma; Hygea, Arina; Puspita, Oktaviani Sandra; Muktamiroh, Hikmah; Simanjuntak, Kristina; Gozan, Misri

2018-03-01

Tobacco plays an important role in international trade as one of the export commodities. Indonesia is one of the good quality export contributors of tobacco leaves in the world. Nevertheless, tobacco is used only as a raw material for the cigarette industries, and the rise on anti-cigarette regulations prompted the exploration of alternative product from tobacco plants. The content of alkaloids, flavonoids, terpenoids and steroids in tobacco leaves were reported in literatures as antibacterial. Therefore, this study proposed in vitro assay of the ethanolic heat reflux extract (EHRE) of Nicotiana tabacum var. Virginia against nosocomial bacteria pathogen ((Pseudomonas aeruginosa (ATCC 27853), Eschericia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212)). Kirby-bauer diffusion method was used for this assay. The concentration of the EHRE for Kirby-bauer assay were 20; 40; 60; 80; and 100%. The presence of clear zones on Kirby-bauer test, against the growth of each nosocomial bacteria pathogen show that tobacco extract has antibacterial effect. Statistical analysis result showed that each extract concentration had significant difference value (p <0,05). This study indicated that the content (alkaloids, flavonoids, terpenoids and steroids) of tobacco leaf extracts (N. tabacum) has potential as antibacterial against nosocomial bacteria pathogen. Nevertheless, optimization of tobacco leaf extract to obtain maximum active ingredient still needs to be done. This study is important for further development of the tobacco leaf extract as antibacterial

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

NASA Astrophysics Data System (ADS)

Wang, Quan; Serban, Andrew J.; Wachter, Rebekka M.; Moerner, W. E.

2018-03-01

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ˜0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase.

PubMed

Wang, Quan; Serban, Andrew J; Wachter, Rebekka M; Moerner, W E

2018-03-28

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 μM range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s -1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.

Melatonin redirects carbohydrates metabolism during sugar starvation in plant cells.

PubMed

Kobylińska, Agnieszka; Borek, Sławomir; Posmyk, Małgorzata M

2018-05-01

Recent studies have shown that melatonin is an important molecule in plant physiology. It seems that the most important is that melatonin efficacy eliminates oxidative stress (direct and indirect antioxidant) and moreover induce plant stress reaction and switch on different defence strategies (preventively and interventively actions). In this report, the impact of exogenous melatonin on carbohydrate metabolism in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells during sugar starvation was examined. We analysed starch concentration, α-amylase and PEPCK activity as well as proteolytic activity in culture media. It has been shown that BY-2 cell treatment with 200 nM of melatonin improved viability of sugar-starved cells. It was correlated with higher starch content and phosphoenolpyruvate carboxykinase (PEPCK) activity. The obtained results revealed that exogenous melatonin under specific conditions (stress) can play regulatory role in sugar metabolism, and it may modulate carbohydrate concentration in etiolated BY-2 cells. Moreover, our results confirmed the hypothesis that if the starch is synthesised even in sugar-starved cells, it is highly probable that melatonin shifts the BY-2 cell metabolism on gluconeogenesis pathway and allows for synthesis of carbohydrates from nonsugar precursors, that is amino acids. These points to another defence strategy that was induced by exogenous melatonin applied in plants to overcome adverse environmental conditions. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Effect of ectopic expression of NtEXPA5 gene on cell size and growth of organs of transgenic tobacco plants].

PubMed

Kuluev, B R; Safiullina, M G; Kniazev, A V; Chemeris, A V

2013-01-01

We obtained transgenic tobacco plants demonstrating overexpression of NtEXPA5 gene that encodes alpha-expansin of Nicotiana tabacum. The transgenic plants were characterized by increased size of leaves and stems. However, size of flowers remained almost unchanged. The increase of organ sizes was induced by cell stretching only. Moreover, the number of cell divisions was even decreased. The obtained data suggest tight interaction between cell stretching regulation and cell division, which together provide the basic mechanism aimed at the controlling of plant organ sizes.

Expression of programmed cell death1 in T follicular helper cells is regulated by prostaglandin E2 secreted by HBV-infected HepG2.2.1.5 cells.

PubMed

Sui, Zhefeng; Shi, Ying; Gao, Zhiling; Yang, Deguang; Wang, Zhihao

2017-06-01

The present study aimed to investigate the distribution of T follicular helper (Tfh)-cell subsets in patients with hepatitis B virus (HBV) and determine the underlying mechanism of HBV regulation of Tfh cells. The frequency of peripheral blood Tfh subsets was analyzed using flow cytometry. The expression level of programmed cell death‑1 (PD‑1) and prostaglandin E2 (PGE2) was quantified using reverse transcription‑quantitative polymerase chain reaction and western blotting. The PGE2 level in culture supernatant was detected using enzyme‑linked immunosorbent assay. A Transwell chamber was used to co‑culture Tfh cells with HepG2 and HepG2.2.1.5. The percentage of inducible T‑cell costimulator (ICOS)+ and total Tfh cells was high at the immune activation (IA) group; however, it was reduced in the immune tolerance (IT), responders with HBsAg seroconversion (RP) and healthy control (HC) groups. The percentage of PD‑1+ Tfh cells was significantly higher in IA and IT compared with RP and HC. The ratio of PD‑1+/total Tfh cells was positively correlated with the load of HBV DNA; therefore, this ratio may act as an indicator for HBV replication. The expression level of PD‑1 in Tfh cells was higher in the HepG2.2.1.5 co‑cultured group compared with the HepG2 group, this may be due to the high PGE2 expression level in HBV‑infected HepG2.2.1.5 cells. The findings of the present study revealed an imbalanced distribution of PD‑1+ Tfh cells in patients with HBV at different immune phases. Additionally, HBV may upregulate the expression of PD‑1 in Tfh cells by promoting HepG2.2.1.5 to secret PGE2. Identifying the effect of HBV on Tfh‑cell subsets is crucial for improving immuno-based therapy for HBV.

Methylation-sensitive amplified polymorphism-based genome-wide analysis of cytosine methylation profiles in Nicotiana tabacum cultivars.

PubMed

Jiao, J; Wu, J; Lv, Z; Sun, C; Gao, L; Yan, X; Cui, L; Tang, Z; Yan, B; Jia, Y

2015-11-26

This study aimed to investigate cytosine methylation profiles in different tobacco (Nicotiana tabacum) cultivars grown in China. Methylation-sensitive amplified polymorphism was used to analyze genome-wide global methylation profiles in four tobacco cultivars (Yunyan 85, NC89, K326, and Yunyan 87). Amplicons with methylated C motifs were cloned by reamplified polymerase chain reaction, sequenced, and analyzed. The results show that geographical location had a greater effect on methylation patterns in the tobacco genome than did sampling time. Analysis of the CG dinucleotide distribution in methylation-sensitive polymorphic restriction fragments suggested that a CpG dinucleotide cluster-enriched area is a possible site of cytosine methylation in the tobacco genome. The sequence alignments of the Nia1 gene (that encodes nitrate reductase) in Yunyan 87 in different regions indicate that a C-T transition might be responsible for the tobacco phenotype. T-C nucleotide replacement might also be responsible for the tobacco phenotype and may be influenced by geographical location.

Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves

DOE Office of Scientific and Technical Information (OSTI.GOV)

Howard, Amanda R.; Heppler, Marty L.; Ju, Ho-Jong

Experiments were conducted to compare the plasmodesmal transport activities of Potato virus X (PVX) TGBp1 and coat protein (CP) in several plant species. Microinjection experiments indicated that TGBp1 gates plasmodesmata in Nicotiana tabacum leaves. These results support previous microinjection studies indicating that TGBp1 gates plasmodesmata in Nicotiana benthamiana and Nicotiana clevelandii leaves. To study protein movement, plasmids expressing the green fluorescent protein (GFP) gene fused to the PVX TGBp1 or CP genes were biolistically bombarded to leaves taken from four different PVX host species. GFP/TGBp1 moved between adjacent cells in N. tabacum, N. clevelandii, N. benthamiana, and Lycopersicon esculentum, whereasmore » GFP/CP moved only in N. benthamiana leaves. Mutations m12 and m13 were introduced into the TGBp1 gene and both mutations eliminated TGBp1 ATPase active site motifs, inhibited PVX movement, reduced GFP/TGBp1 cell-to-cell movement in N. benthamiana leaves, and eliminated GFP/TGBp1 movement in N. tabacum, N. clevelandii, and L. esculentum leaves. GFP/TGBp1m13 formed aggregates in tobacco cells. The ability of GFP/CP and mutant GFP/TGBp1 fusion proteins to move in N. benthamiana and not in the other PVX host species suggests that N. benthamiana plants have a unique ability to promote protein intercellular movement.« less

The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

NASA Technical Reports Server (NTRS)

Krishnamurthy, Konduru; Heppler, Marty; Mitra, Ruchira; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

2003-01-01

Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

Impact of mitochondrial alternative oxidase expression on the response of Nicotiana tabacum to cold temperature.

PubMed

Wang, Jia; Rajakulendran, Nirusan; Amirsadeghi, Sasan; Vanlerberghe, Greg C

2011-08-01

The plant mitochondrial electron transport chain (ETC) includes a non-energy conserving alternative oxidase (AOX) thought to dampen reactive oxygen species (ROS) generation by the ETC and/or facilitate carbon metabolism by uncoupling it from ATP turnover. When wild-type (WT) Nicotiana tabacum grown at 28°C/22°C (light/dark) were transferred to 12°C/5°C, they showed a large induction of leaf Aox1a mRNA and AOX protein within 24 h. Transfer to cold also resulted in a large accumulation of monosaccharides, an increase in transcript level of genes encoding important ROS-scavenging enzymes and a moderate increase in lipid peroxidation. Transgenic plants with suppressed AOX level showed less cold-induced sugar accumulation than WT while transgenic plants with enhanced AOX levels showed enhanced sugar accumulation. This is inconsistent with the hypothesis that AOX acts to burn excess carbohydrate, but rather suggests a role for AOX to aid sugar accumulation, at least during cold stress. At 28°C/22°C, plants with suppressed AOX had elevated levels of lipid peroxidation compared with WT, while plants with enhanced AOX had reduced lipid peroxidation. This is consistent with the hypothesis that AOX dampens ROS generation and oxidative damage. However, this inverse relationship between AOX level and lipid peroxidation did not hold upon shift to cold. Under this stress condition, plants with strong suppression of AOX show enhanced induction of ROS-scavenging enzymes compared with WT and decline in lipid peroxidation. These data suggest that, under stress conditions, the lack of AOX enhances a mitochondrial stress-signaling pathway able to increase the ROS-scavenging capacity of the cell. Copyright © Physiologia Plantarum 2011.

Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

PubMed

Buschmann, Henrik

2016-01-01

The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

PubMed

Oliver, J E; Sefick, S A; Parker, J K; Arnold, T; Cobine, P A; De La Fuente, L

2014-10-01

Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.

PubMed

Nambo, Masakazu; Kurihara, Daisuke; Yamada, Tomomi; Nishiwaki-Ohkawa, Taeko; Kadofusa, Naoya; Kimata, Yusuke; Kuwata, Keiko; Umeda, Masaaki; Ueda, Minako

2016-11-01

Cell proliferation is crucial to the growth of multicellular organisms, and thus the proper control of cell division is important to prevent developmental arrest or overgrowth. Nevertheless, tools for controlling cell proliferation are still poor in plant. To develop novel tools, we focused on a specific compound family, triarylmethanes, whose members show various antiproliferative activities in animals. By combining organic chemistry to create novel and diverse compounds containing the triarylmethyl moiety and biological screens based on live-cell imaging of a fluorescently labeled tobacco Bright Yellow-2 (BY-2) culture cell line (Nicotiana tabacum), we isolated (3-furyl)diphenylmethane as a strong but partially reversible inhibitor of plant cell division. We also found that this agent had efficient antiproliferative activity in developing organs of Arabidopsis thaliana without causing secondary defects in cell morphology, and induced rapid cell division arrest independent of the cell cycle stage. Given that (3-furyl)diphenylmethane did not affect the growth of a human cell line (HeLa) and a budding yeast (Saccharomyces cerevisiae), it should act specifically on plants. Taking our results together, we propose that the combination of desired chemical synthesis and detailed biological analysis is an effective tool to create novel drugs, and that (3-furyl)diphenylmethane is a specific antiproliferative agent for plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Activity and Accumulation of Cell Division-Promoting Phenolics in Tobacco Tissue Cultures 1

PubMed Central

Teutonico, Rita A.; Dudley, Matthew W.; Orr, John D.; Lynn, David G.; Binns, Andrew N.

1991-01-01

Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division. ImagesFigure 2 PMID:16668384

Effects of a petunia scaffold/matrix attachment region on copy number dependency and stability of transgene expression in Nicotiana tabacum.

PubMed

Dietz-Pfeilstetter, Antje; Arndt, Nicola; Manske, Ulrike

2016-04-01

Transgenes in genetically modified plants are often not reliably expressed during development or in subsequent generations. Transcriptional gene silencing (TGS) as well as post-transcriptional gene silencing (PTGS) have been shown to occur in transgenic plants depending on integration pattern, copy number and integration site. In an effort to reduce position effects, to prevent read-through transcription and to provide a more accessible chromatin structure, a P35S-ß-glucuronidase (P35S-gus) transgene flanked by a scaffold/matrix attachment region from petunia (Petun-SAR), was introduced in Nicotiana tabacum plants by Agrobacterium tumefaciens mediated transformation. It was found that Petun-SAR mediates enhanced expression and copy number dependency up to 2 gene copies, but did not prevent gene silencing in transformants with multiple and rearranged gene copies. However, in contrast to the non-SAR transformants where silencing was irreversible and proceeded during long-term vegetative propagation and in progeny plants, gus expression in Petun-SAR plants was re-established in the course of development. Gene silencing was not necessarily accompanied by DNA methylation, while the gus transgene could still be expressed despite considerable CG methylation within the coding region.

TET2 mutations in B cells of patients affected by angioimmunoblastic T-cell lymphoma.

PubMed

Schwartz, Friederike H; Cai, Qian; Fellmann, Eva; Hartmann, Sylvia; Mäyränpää, Mikko I; Karjalainen-Lindsberg, Marja-Liisa; Sundström, Christer; Scholtysik, René; Hansmann, Martin-Leo; Küppers, Ralf

2017-06-01

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Structural and functional similarities between osmotin from Nicotiana tabacum seeds and human adiponectin.

PubMed

Miele, Marco; Costantini, Susan; Colonna, Giovanni

2011-02-02

Osmotin, a plant protein, specifically binds a seven transmembrane domain receptor-like protein to exert its biological activity via a RAS2/cAMP signaling pathway. The receptor protein is encoded in the gene ORE20/PHO36 and the mammalian homolog of PHO36 is a receptor for the human hormone adiponectin (ADIPOR1). Moreover it is known that the osmotin domain I can be overlapped to the β-barrel domain of adiponectin. Therefore, these observations and some already existing structural and biological data open a window on a possible use of the osmotin or of its derivative as adiponectin agonist. We have modelled the three-dimensional structure of the adiponectin trimer (ADIPOQ), and two ADIPOR1 and PHO36 receptors. Moreover, we have also modelled the following complexes: ADIPOQ/ADIPOR1, osmotin/PHO36 and osmotin/ADIPOR1. We have then shown the structural determinants of these interactions and their physico-chemical features and analyzed the related interaction residues involved in the formation of the complexes. The stability of the modelled structures and their complexes was always evaluated and controlled by molecular dynamics. On the basis of these results a 9 residues osmotin peptide was selected and its interaction with ADIPOR1 and PHO36 was modelled and analysed in term of energetic stability by molecular dynamics. To confirm in vivo the molecular modelling data, osmotin has been purified from nicotiana tabacum seeds and its nine residues peptide synthesized. We have used cultured human synovial fibroblasts that respond to adiponectin by increasing the expression of IL-6, TNF-alpha and IL-1beta via ADIPOR1. The biological effect on fibroblasts of osmotin and its peptide derivative has been found similar to that of adiponectin confirming the results found in silico.

Antigen recognition by H-2-restricted T cells. I. Cell-free antigen processing

PubMed Central

1983-01-01

We examined the ability of a set of cloned chicken ovalbumin (cOVA)- specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I- region molecules by T cells. PMID:6193218

Comparison of the biotransformation of the 14C-labelled insecticide carbaryl by non-transformed and human CYP1A1-, CYP1A2-, and CYP3A4-transgenic cell cultures of Nicotiana tabacum.

PubMed

Schmidt, Burkhard; Faymonville, Tanja; Gembé, Eva; Joussen, Nicole; Schuphan, Ingolf

2006-08-01

Transgenic tobacco-cell-suspension cultures expressing separately the human cytochrome P450 monooxygenases CYP1A1, CYP1A2, and CYP3A4 were utilized to study the biotransformation of the 14C-labelled insecticide carbaryl (=naphthalen-1-yl methylcarbamate). The resulting data were compared to similar data from the corresponding non-transformed (NT) tobacco-cell culture and commercially available membrane preparations (Bactosomes) of genetically modified bacteria separately containing the same human P450s. A rapid conversion rate of carbaryl was observed with the CYP1A1 and CYP1A2 cells, where only 49.7 and 0.2% of applied carbaryl (1 mg/l), respectively, remained after 24 h, as compared to 77.7% in the non-transformed culture. Unexpectedly, the corresponding results obtained from the CYP3A4 cultures were not definite. With 25 mg/l of carbaryl and 96 h of incubation, it was proven that the insecticide is also substrate of CYP3A4. This finding was supported by GC/EI-MS analysis of the primary metabolite pattern produced by the isozyme. This consisted of naphthalene-1-ol, N-(hydroxymethyl)carbaryl, 4-hydroxycarbaryl, and 5-hydroxycarbaryl, whereas the main product in non-transformed cells was N-(hydroxymethyl)carbaryl. Data obtained from the CYP1A1, CYP1A2, or CYP3A4 Bactosomes agreed with those of the P450-transgenic tobacco cells. Problems with GC/EI-MS analysis of carbaryl and its metabolites are discussed.

Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

PubMed Central

Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

2014-01-01

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses. PMID:25153631

Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

PubMed

Shi, Yanmei; Wang, Ran; Luo, Zhaopeng; Jin, Lifeng; Liu, Pingping; Chen, Qiansi; Li, Zefeng; Li, Feng; Wei, Chunyang; Wu, Mingzhu; Wei, Pan; Xie, He; Qu, Lingbo; Lin, Fucheng; Yang, Jun

2014-08-22

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Inhibition of HBV Replication in HepG2.2.15 Cells by Human Peripheral Blood Mononuclear Cell-Derived Dendritic Cells.

PubMed

Liu, Tao; Song, Hong-Li; Zheng, Wei-Ping; Shen, Zhong-Yang

2015-01-01

Anti-HBV therapy is essential for patients awaiting liver transplantation. This study aimed to explore the effects of dendritic cells (DCs) derived from the peripheral blood of hepatitis B patients on the replication of HBV in vivo and to evaluate the biosafety of DCs in clinical therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from HBV-infected patients and maturation-promoting factors and both HBsAg and HBcAg were used to induce DC maturation. Mature DCs and lymphocytes were co-cultured with human hepatocyte cell HL-7702 or HBV-producing human hepatocellular carcinoma cell HepG2.2.15. We found that mature lymphocytes exposed to DCs in vitro did not influence morphology or activities of HL-7702 and HepG2.2.15 cells. Liver function indexes and endotoxin levels in the cell supernatants did not change in these co-cultures. Additionally, supernatant and intracellular HBV DNA levels were reduced when HepG2.2.15 cells were co-cultured with mature lymphocytes that had been cultured with DCs, and HBV covalently closed circular DNA (cccDNA) levels in HepG2.2.15 cells also decreased. Importantly, DC-mediated immunotherapy had no mutagenic effect on HBV genomic DNA by gene sequencing of the P, S, X, and C regions of HBV genomic DNA. We conclude that PBMC-derived DCs from HBV-infected patients act on autologous lymphocytes to suppress HBV replication and these DC clusters showed favorable biosafety. © 2015 by the Association of Clinical Scientists, Inc.

Hybrid proline-rich proteins: novel players in plant cell elongation?

PubMed Central

Dvořáková, Lenka; Srba, Miroslav; Opatrny, Zdenek; Fischer, Lukas

2012-01-01

Background and Aims Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. Methods To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. Key Results In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. Conclusions Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion. PMID:22028464

A hyper-thermostable α-amylase from Pyrococcus furiosus accumulates in Nicotiana tabacum as functional aggregates.

PubMed

Zhu, Hong; Reynolds, L Bruce; Menassa, Rima

2017-06-19

Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces malto-oligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase.

Cyclic nucleotide content of tobacco BY-2 cells.

PubMed

Richards, Helen; Das, Swadipa; Smith, Christopher J; Pereira, Louisa; Geisbrecht, Alan; Devitt, Nicola J; Games, David E; van Geyschem, Jan; Gareth Brenton, A; Newton, Russell P

2002-11-01

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.

[In vitro regeneration and applications using vegetable cell and tissue culture].

PubMed

Jordán, M

1990-10-01

Plant cells by means of their totipotency and aided by in vitro culture techniques can be induced to perform morphogenesis leading to somatic embryoids and massive clonal multiplication; microspores or pollen can be triggered to recover haploid plants, then characters expressed via haploidy can be selected and fixed. Protoplasts from different species can lead to recombinations. We report here work done on Carica pubescens, where somatic embryoids were obtained from cells; in Prunus avium androgenesis leading to pollen calli was triggered, while plants were recovered from Nicotiana tabacum anthers. Fusion products were obtained using C. pubescens and C. papaya protoplasts, leading up to calli and shoots.

Regulation of bacteria population behaviors by AI-2 "consumer cells" and "supplier cells".

PubMed

Quan, Yufen; Meng, Fankang; Ma, Xinyu; Song, Xinhao; Liu, Xiao; Gao, Weixia; Dang, Yulei; Meng, Yao; Cao, Mingfeng; Song, Cunjiang

2017-09-19

Autoinducer-2 (AI-2) is a universal signal molecule and enables an individual bacteria to communicate with each other and ultimately control behaviors of the population. Harnessing the character of AI-2, two kinds of AI-2 "controller cells" ("consumer cells" and "supplier cells") were designed to "reprogram" the behaviors of entire population. For the consumer cells, genes associated with the uptake and processing of AI-2, which includes LsrACDB, LsrFG, LsrK, were overexpressed in varying combinations. Four consumer cell strains were constructed: Escherichia coli MG1655 pLsrACDB (NK-C1), MG1655 pLsrACDBK (NK-C2), MG1655 pLsrACDBFG (NK-C3) and MG1655 pLsrACDBFGK (NK-C4). The key enzymes responsible for production of AI-2, LuxS and Mtn, were also overexpressed, yielding strains MG1655 pLuxS (NK-SU1), and MG1655 pLuxS-Mtn (NK-SU2). All the consumer cells could decrease the environmental AI-2 concentration. NK-C2 and NK-C4 were most effective in AI-2 uptake and inhibited biofilm formation. While suppliers can increase the environmental AI-2 concentration and NK-SU2 was most effective in supplying AI-2 and facilitated biofilm formation. Further, reporter strain, MG1655 pLGFP was constructed. The expression of green fluorescent protein (GFP) in reporter cells was initiated and guided by AI-2. Mixture of consumer cells and reporter cells suggest that consumer cells can decrease the AI-2 concentration. And the supplier cells were co-cultured with reporter cells, indicating that supplier cells can provide more AI-2 compared to the control. The consumer cells and supplier cells could be used to regulate environmental AI-2 concentration and the biofilm formation. They can also modulate the AI-2 concentration when they were co-cultured with reporter cells. It can be envisioned that this system will become useful tools in synthetic biology and researching new antimicrobials.

The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants.

PubMed

Persson, S; Wyatt, S E; Love, J; Thompson, W F; Robertson, D; Boss, W F

2001-07-01

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

The Ca(2+) status of the endoplasmic reticulum is altered by induction of calreticulin expression in transgenic plants

NASA Technical Reports Server (NTRS)

Persson, S.; Wyatt, S. E.; Love, J.; Thompson, W. F.; Robertson, D.; Boss, W. F.; Brown, C. S. (Principal Investigator)

2001-01-01

To investigate the endoplasmic reticulum (ER) Ca(2+) stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca(2+)-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca(2+) uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent (45)Ca(2+) accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca(2+) ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of (45)Ca(2+) released, and a 2- to 3-fold increase in the amount of (45)Ca(2+) retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca(2+) pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca(2+)-containing medium to Ca(2+)-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca(2+) stores and thereby enhances the survival of plants grown in low Ca(2+) medium.

Characterization of the programmed cell death induced by metabolic products of Alternaria alternata in tobacco BY-2 cells.

PubMed

Cheng, Dan-Dan; Jia, Yu-Jiao; Gao, Hui-Yuan; Zhang, Li-Tao; Zhang, Zi-Shan; Xue, Zhong-Cai; Meng, Qing-Wei

2011-02-01

Alternaria alternata has received considerable attention in current literature and most of the studies are focused on its pathogenic effects on plant chloroplasts, but little is known about the characteristics of programmed cell death (PCD) induced by metabolic products (MP) of A. alternata, the effects of the MP on mitochondrial respiration and its relation to PCD. The purpose of this study was to explore the mechanism of MP-induced PCD in non-green tobacco BY-2 cells and to explore the role of mitochondrial inhibitory processes in the PCD of tobacco BY-2 cells. MP treatment led to significant cell death that was proven to be PCD by the concurrent cytoplasm shrinkage, chromatin condensation and DNA laddering observed in the cells. Moreover, MP treatment resulted in the overproduction of reactive oxygen species (ROS), rapid ATP depletion and a respiratory decline in the tobacco BY-2 cells. It was concluded that the direct inhibition of the mitochondrial electron transport chain (ETC), alternative pathway (AOX) capacity and catalase (CAT) activity by the MP might be the main contributors to the MP-induced ROS burst observed in tobacco BY-2 cells. The addition of adenosine together with the MP significantly inhibited ATP depletion without preventing PCD; however, when the cells were treated with the MP plus CAT, ROS overproduction was blocked and PCD did not occur. The data presented here demonstrate that the ROS burst played an important role in MP-induced PCD in the tobacco BY-2 cells.

IL-4 production by group 2 innate lymphoid cells promotes food allergy by blocking regulatory T-cell function.

PubMed

Noval Rivas, Magali; Burton, Oliver T; Oettgen, Hans C; Chatila, Talal

2016-09-01

Food allergy is a major health issue, but its pathogenesis remains obscure. Group 2 innate lymphoid cells (ILC2s) promote allergic inflammation. However their role in food allergy is largely unknown. We sought to investigate the role of ILC2s in food allergy. Food allergy-prone mice with a gain-of-function mutation in the IL-4 receptor α chain (Il4raF709) were orally sensitized with food allergens, and the ILC2 compartment was analyzed. The requirement for ILC2s in food allergy was investigated by using Il4raF709, IL-33 receptor-deficient (Il1rl1(-/-)), IL-13-deficient (Il13(-/-)), and IL-4-deficient (Il4(-/-)) mice and by adoptive transfer of in vitro-expanded ILC2s. Direct effects of ILC2s on regulatory T (Treg) cells and mast cells were analyzed in coculture experiments. Treg cell control of ILC2s was assessed in vitro and in vivo. Il4raF709 mice with food allergy exhibit increased numbers of ILC2s. IL-4 secretion by ILC2s contributes to the allergic response by reducing allergen-specific Treg cell and activating mast cell counts. IL-33 receptor deficiency in Il4raF709 Il1rl1(-/-) mice protects against allergen sensitization and anaphylaxis while reducing ILC2 induction. Adoptive transfer of wild-type and Il13(-/-) but not Il4(-/-) ILC2s restored sensitization in Il4raF709 Il1rl1(-/-) mice. Treg cells suppress ILC2s in vitro and in vivo. IL-4 production by IL-33-stimulated ILC2s blocks the generation of allergen-specific Treg cells and favors food allergy. Strategies to block ILC2 activation or the IL-33/IL-33 receptor pathway can lead to innovative therapies in the treatment of food allergy. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy.

PubMed

Jacobsen, Christine; Oechsle, Karin; Hauschild, Jessica; Steinemann, Gustav; Spath, Brigitte; Bokemeyer, Carsten; Ruf, Wolfram; Honecker, Friedemann; Langer, Florian

2015-09-01

Patients with germ cell tumors (GCTs) receiving cisplatin-based chemotherapy are at increased risk of thrombosis, but the underlying cellular and molecular mechanisms remain obscure. To study baseline tissue factor (TF) expression by GCT cell lines and its modulation by cisplatin treatment. TF expression was assessed by single-stage clotting and thrombin generation assay, flow cytometry, ELISA, and Western blot analysis. Cell cycle analysis and detection of phosphatidylserine (PS) membrane exposure were carried out by flow cytometry. TF mRNA was analyzed by quantitative RT-PCR. Significant expression of TF-specific procoagulant activity (PCA) was detected on three non-seminoma (NT2, 2102Ep, NCCIT) and one seminoma cell line (TCam-2). Treatment with 0.4μM cisplatin (corresponding to the IC50) for 48hrs increased TF PCA on NT2 cells 3-fold, an effect that was largely independent of PS exposure and that could not be explained by translocation of active TF from intracellular storage pools. Cisplatin-induced TF PCA expression in NT2 cells did not occur before 12hrs, but was steady thereafter and accompanied by a 2-fold increase in total and surface-located TF antigen. Importantly, increased TF gene transcription or production and release of an intermediate factor were not involved in this process. Cell cycle analysis suggested that cisplatin-induced G2/M arrest resulted in an accumulation of procoagulant TF on the membrane surface of NT2 cells. In addition to induction of apoptosis/necrosis with PS-mediated activation of preformed TF, cisplatin may alter the procoagulant phenotype of GCT cells through an increase in total cellular TF antigen. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Ca2+ Status of the Endoplasmic Reticulum Is Altered by Induction of Calreticulin Expression in Transgenic Plants1

PubMed Central

Persson, Staffan; Wyatt, Sarah E.; Love, John; Thompson, William F.; Robertson, Dominique; Boss, Wendy F.

2001-01-01

To investigate the endoplasmic reticulum (ER) Ca2+ stores in plant cells, we generated tobacco (Nicotiana tabacum; NT1) suspension cells and Arabidopsis plants with altered levels of calreticulin (CRT), an ER-localized Ca2+-binding protein. NT1 cells and Arabidopsis plants were transformed with a maize (Zea mays) CRT gene in both sense and antisense orientations under the control of an Arabidopsis heat shock promoter. ER-enriched membrane fractions from NT1 cells were used to examine how altered expression of CRT affects Ca2+ uptake and release. We found that a 2.5-fold increase in CRT led to a 2-fold increase in ATP-dependent 45Ca2+ accumulation in the ER-enriched fraction compared with heat-shocked wild-type controls. Furthermore, after treatment with the Ca2+ ionophore ionomycin, ER microsomes from NT1 cells overproducing CRT showed a 2-fold increase in the amount of 45Ca2+ released, and a 2- to 3-fold increase in the amount of 45Ca2+ retained compared with wild type. These data indicate that altering the production of CRT affects the ER Ca2+ pool. In addition, CRT transgenic Arabidopsis plants were used to determine if altered CRT levels had any physiological effects. We found that the level of CRT in heat shock-induced CRT transgenic plants correlated positively with the retention of chlorophyll when the plants were transferred from Ca2+-containing medium to Ca2+-depleted medium. Together these data are consistent with the hypothesis that increasing CRT in the ER increases the ER Ca2+ stores and thereby enhances the survival of plants grown in low Ca2+ medium. PMID:11457960

Isolation of Autolysosomes from Tobacco BY-2 Cells.

PubMed

Takatsuka, Chihiro; Inoue-Aono, Yuko; Moriyasu, Yuji

2017-01-01

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H + -ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Upregulation of Phosphatidylinositol 3-Kinase (PI3K) Enhances Ethylene Biosynthesis and Accelerates Flower Senescence in Transgenic Nicotiana tabacum L.

PubMed

Dek, Mohd Sabri Pak; Padmanabhan, Priya; Sherif, Sherif; Subramanian, Jayasankar; Paliyath, And Gopinadhan

2017-07-15

Phosphatidylinositol 3-kinase (PI3K) is a key enzyme that phosphorylates phosphatidylinositol at 3'-hydroxyl position of the inositol head group initiating the generation of several phosphorylated phosphatidylinositols, collectively referred to as phosphoinositides. The function of PI3K in plant senescence and ethylene signal transduction process was studied by expression of Solanum lycopersicum PI3K in transgenic Nicotiana tabacum , and delineating its effect on flower senescence. Detached flowers of transgenic tobacco plants with overexpressed Sl - PI3K (OX) displayed accelerated senescence and reduced longevity, when compared to the flowers of wild type plants. Flowers from PI3K-overexpressing plants showed enhanced ethylene production and upregulated expression of 1-aminocyclopropane-1-carboxylic acid oxidase 1 ( ACO1 ). Real time polymerase chain reaction (PCR) analysis showed that PI3K was expressed at a higher level in OX flowers than in the control. Seedlings of OX-lines also demonstrated a triple response phenotype with characteristic exaggerated apical hook, shorter hypocotyls and increased sensitivity to 1-aminocyclopropane-1-carboxylate than the control wild type seedlings. In floral tissue from OX-lines, Solanum lycopersicum phosphatidylinositol 3-kinase green fluorescent protein (PI3K-GFP) chimera protein was localized primarily in stomata, potentially in cytoplasm and membrane adjacent to stomatal pores in the guard cells. Immunoblot analysis of PI3K expression in OX lines demonstrated increased protein level compared to the control. Results of the present study suggest that PI3K plays a crucial role in senescence by enhancing ethylene biosynthesis and signaling.

Melatonin Protects Cultured Tobacco Cells against Lead-Induced Cell Death via Inhibition of Cytochrome c Translocation

PubMed Central

Kobylińska, Agnieszka; Reiter, Russel J.; Posmyk, Malgorzata M.

2017-01-01

Melatonin was discovered in plants more than two decades ago and, especially in the last decade, it has captured the interests of plant biologists. Beyond its possible participation in photoperiod processes and its role as a direct free radical scavenger as well as an indirect antioxidant, melatonin is also involved in plant defense strategies/reactions. However, the mechanisms that this indoleamine activates to improve plant stress tolerance still require identification and clarification. In the present report, the ability of exogenous melatonin to protect Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells against the toxic exposure to lead was examined. Studies related to cell proliferation and viability, DNA fragmentation, possible translocation of cytochrome c from mitochondria to cytosol, cell morphology after fluorescence staining and also the in situ accumulation of superoxide radicals measured via the nitro blue tetrazolium reducing test, were conducted. This work establishes a novel finding by correcting the inhibition of release of mitochondrial ctytocrome c in to the cytoplasm with the high accumulation of superoxide radicals. The results show that pretreatment with 200 nm of melatonin protected tobacco cells from DNA damage caused by lead. Melatonin, as an efficacious antioxidant, limited superoxide radical accumulation as well as cytochrome c release thereby, it likely prevents the activation of the cascade of processes leading to cell death. Fluorescence staining with acridine orange and ethidium bromide documented that lead-stressed cells additionally treated with melatonin displayed intact nuclei. The results revealed that melatonin at proper dosage could significantly increase BY-2 cell proliferation and protected them against death. It was proved that melatonin could function as an effective priming agent to promote survival of tobacco cells under harmful lead-induced stress conditions. PMID:28959267

Indoor and outdoor genotoxic load detected by the Comet assay in leaves of Nicotiana tabacum cultivars Bel B and Bel W3.

PubMed

Restivo, Francesco Maria; Laccone, Maria Concetta; Buschini, Annamaria; Rossi, Carlo; Poli, Paola

2002-03-01

Environmental pollution assessment and control are priority issues for both developed and developing countries of the world. The use of plant material for a more complete picture of environmental health appears to be particularly appealing. Here we validate a previous plant-adapted Comet assay on leaf tissues of Nicotiana tabacum cultivars Bel B and Bel W3. The effects of H(2)O(2) on DNA damage in Bel B and Bel W3 agree with the hypothesis that some component of the machinery that protects DNA integrity from oxidative stress may be impaired in cv. Bel W3. Exposure in the field on sunny summer days (peak ozone concentration >80 p.p.b.) showed significantly higher DNA damage in cv. Bel W3 if plants were collected and subjected to the Comet assay when the air ozone concentration was reaching its peak value, but not when plants were sampled early in the morning and hence after a period of low ozone concentration. The different results suggest that Bel W3 possesses a less efficient recovery apparatus that requires a longer period of activity to be effective and/or is less protected against reactive oxygen species production during exposure to ozone. However, it cannot be excluded that the increase in mean DNA damage is the result of the presence of a genotoxic agent(s) other than ozone. Interestingly, Bel W3 also appears to be more responsive, compared with Bel B, when exposed to ambient indoor pollutants. The use of cv. Bel W3 increases the sensitivity of the assay under both indoor and field conditions. However, different classes of mutagens should be tested to define the range of profitable utilization of this tobacco cultivar for environmental genotoxicity detection.

BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

PubMed

Liu, Jun; Ben, Qi-Wen; Yao, Wei-Yan; Zhang, Jian-Jun; Chen, Da-Fan; He, Xiang-Yi; Li, Lei; Yuan, Yao-Zong

2012-06-01

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

Activation of mixed glia by Abeta-specific Th1 and Th17 cells and its regulation by Th2 cells.

PubMed

McQuillan, K; Lynch, Marina A; Mills, Kingston H G

2010-05-01

Microglia are innate immune cells of the CNS, that act as antigen-presenting cells (APC) for antigen-specific T cells and respond to inflammatory stimuli, such as amyloid-beta (Abeta), resulting in the release of neurotoxic factors and pro-inflammatory cytokines. Astrocytes can also act as APC and modulate the function of microglia. However, the role of distinct T cell subtypes, in particular Th17 cells, in glial activation and subsequent modulatory effects of Th2 cells are poorly understood. Here, we generated Abeta-specific Th1, Th2, and Th17 cells and examined their role in modulating Abeta-induced activation of microglia in a mixed glial culture, a preparation which mimics the complex APC types in the brain. We demonstrated that mixed glia acted as an effective APC for Abeta-specific Th1 and Th17 cells. Addition of Abeta-specific Th2 cells suppressed the Abeta-induced IFN-gamma production by Th1 cells and IL-17 production by Th17 cells with glia as the APC. Co-culture of Abeta-specific Th1 or Th17 cells with glia markedly enhanced Abeta-induced pro-inflammatory cytokine production and expression of MHC class II and co-stimulatory molecules on the microglia. Addition of Abeta-specific Th2 cells inhibited Th17 cell-induced IL-1beta and IL-6 production by mixed glia and attenuated Th1 cell-induced CD86 and CD40 expression on microglia. The modest enhancement of MHC class II and CD86 expression on astrocytes by Abeta-specific Th1 and Th17 was not attenuated by Th2 cells. These data indicate that Abeta-specific Th1 and Th17 cells induce inflammatory activation of glia, and that this is in part regulated by Th2 cells. Copyright 2010 Elsevier Inc. All rights reserved.

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

PubMed

Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

2013-01-01

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Alteration of gene expression by restriction enzymes electroporated into plant cells.

PubMed

Ashraf, M; Altschuler, M; Galasinski, S; Griffiths, T D

1993-06-01

The alteration in the expression of a beta-glucuronidase (GUS) reporter gene was used to monitor the effect of restriction endonucleases electroporated into the tobacco (Nicotiana tabacum L.) protoplasts. Restriction enzyme (RE) Hind III which does not have a recognition site within the gene cassette, had little effect on enzyme activity. In contrast restriction endonucleases Hae III and Sau3A1 which possess 8 and 16 recognition sites in the GUS cassette, were found to reduce the enzyme activity by 89% and 94% respectively when compared to control electroporations. Restriction-site mutation analysis (RSM) and Southern blot analysis indicated the enzymatic degradation of GUS coding sequence by the REs Hae III and Sau3A1. Results of this study suggest that on electroporation, REs can enter into plant cells and alter the expression of the GUS gene. The alteration of gene expression is thus correlated with the digestion of GUS template DNA. Future applications of this technique could include addressing fundamental questions with regard to DNA repair, site-specific recombination, identifying mutations, insertional mutagenesis, enhancement of stable transformation and gene tagging in plants.

PGE2 maintains self-renewal of human adult stem cells via EP2-mediated autocrine signaling and its production is regulated by cell-to-cell contact.

PubMed

Lee, Byung-Chul; Kim, Hyung-Sik; Shin, Tae-Hoon; Kang, Insung; Lee, Jin Young; Kim, Jae-Jun; Kang, Hyun Kyoung; Seo, Yoojin; Lee, Seunghee; Yu, Kyung-Rok; Choi, Soon Won; Kang, Kyung-Sun

2016-05-27

Mesenchymal stem cells (MSCs) possess unique immunomodulatory abilities. Many studies have elucidated the clinical efficacy and underlying mechanisms of MSCs in immune disorders. Although immunoregulatory factors, such as Prostaglandin E2 (PGE2), and their mechanisms of action on immune cells have been revealed, their effects on MSCs and regulation of their production by the culture environment are less clear. Therefore, we investigated the autocrine effect of PGE2 on human adult stem cells from cord blood or adipose tissue, and the regulation of its production by cell-to-cell contact, followed by the determination of its immunomodulatory properties. MSCs were treated with specific inhibitors to suppress PGE2 secretion, and proliferation was assessed. PGE2 exerted an autocrine regulatory function in MSCs by triggering E-Prostanoid (EP) 2 receptor. Inhibiting PGE2 production led to growth arrest, whereas addition of MSC-derived PGE2 restored proliferation. The level of PGE2 production from an equivalent number of MSCs was down-regulated via gap junctional intercellular communication. This cell contact-mediated decrease in PGE2 secretion down-regulated the suppressive effect of MSCs on immune cells. In conclusion, PGE2 produced by MSCs contributes to maintenance of self-renewal capacity through EP2 in an autocrine manner, and PGE2 secretion is down-regulated by cell-to-cell contact, attenuating its immunomodulatory potency.

Leukemia cells demonstrate a different metabolic perturbation provoked by 2-deoxyglucose.

PubMed

Miwa, Hiroshi; Shikami, Masato; Goto, Mineaki; Mizuno, Shohei; Takahashi, Miyuki; Tsunekawa-Imai, Norikazu; Ishikawa, Takamasa; Mizutani, Motonori; Horio, Tomohiro; Gotou, Mayuko; Yamamoto, Hidesuke; Wakabayashi, Motohiro; Watarai, Masaya; Hanamura, Ichiro; Imamura, Akira; Mihara, Hidetsugu; Nitta, Masakazu

2013-05-01

The shift in energy metabolism from oxidative phosphorylation to glycolysis can serve as a target for the inhibition of cancer growth. Here, we examined the metabolic changes induced by 2-deoxyglucose (2-DG), a glycolysis inhibitor, in leukemia cells by metabolome analysis. NB4 cells mainly utilized glucose as an energy source by glycolysis and oxidative phosphorylation in mitochondria, since metabolites in the glycolytic pathway and in the tricarboxylic acid (TCA) cycle were significantly decreased by 2-DG. In THP-1 cells, metabolites in the TCA cycle were not decreased to the same extent by 2-DG as in NB4 cells, which indicates that THP-1 utilizes energy sources other than glucose. TCA cycle metabolites in THP-1 cells may be derived from acetyl-CoA by fatty acid β-oxidation, which was supported by abundant detection of carnitine and acetylcarnitine in THP-1 cells. 2-DG treatment increased the levels of pentose phosphate pathway (PPP) metabolites and augmented the generation of NADPH by glucose-6-phosphate dehydrogenase. An increase in NADPH and upregulation of glutathione synthetase expression resulted in the increase in the reduced form of glutathione by 2-DG in NB4 cells. We demonstrated that a combination of 2-DG and inhibition of PPP by dehydroepiandrosterone (DHEA) effectively suppressed the growth of NB4 cells. The replenishment of the TCA cycle by fatty acid oxidation by carnitine palmitoyltransferase in THP-1 cells, treated by 2-DG, might be regulated by AMPK, as the combination of 2-DG and inhibition of AMPK by compound C potently suppressed the growth of THP-1 cells. Although 2-DG has been effective in preclinical and clinical studies, this treatment has not been fully explored due to concerns related to potential toxicities such as brain toxicity at high doses. We demonstrated that a combination of 2-DG and DHEA or compound C at a relatively low concentration effectively inhibits the growth of NB4 and THP-1 cells, respectively. These observations may

Effects of aluminum oxide nanoparticles on the growth, development, and microRNA expression of tobacco (Nicotiana tabacum).

PubMed

Burklew, Caitlin E; Ashlock, Jordan; Winfrey, William B; Zhang, Baohong

2012-01-01

Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al(2)O(3) nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al(2)O(3) nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al(2)O(3) nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al(2)O(3) nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al(2)O(3) nanoparticles in the environment.

Effects of Aluminum Oxide Nanoparticles on the Growth, Development, and microRNA Expression of Tobacco (Nicotiana tabacum)

PubMed Central

Burklew, Caitlin E.; Ashlock, Jordan; Winfrey, William B.; Zhang, Baohong

2012-01-01

Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al2O3 nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al2O3 nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al2O3 nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al2O3 nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al2O3 nanoparticles in the environment. PMID:22606225

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

PubMed

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

2014-04-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

PubMed Central

Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

2014-01-01

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells.

PubMed

Da Silva, Daniel; Lachaud, Christophe; Cotelle, Valérie; Brière, Christian; Grat, Sabine; Mazars, Christian; Thuleau, Patrice

2011-05-01

Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction. 

Programmed cell death of tobacco BY-2 cells induced by still culture conditions is affected by the age of the culture under agitation.

PubMed

Hiraga, Asahi; Kaneta, Tsuyoshi; Sato, Yasushi; Sato, Seiichi

2010-01-25

Evans Blue staining indicated that actively growing tobacco BY-2 cells in the exponential phase died more rapidly than quiescent cells in the stationary phase when the cells cultured under agitation were placed under still conditions. Fifty percent cell death was induced at about 18, 26, 80 and 140 h for early, mid, late exponential- and stationary-phase cells, respectively. Actively growing cells became TUNEL (transferase-mediated dUTP nick end labelling)-positive more rapidly than quiescent cells, suggesting that the cell death evaluated by Evans Blue is accompanied by DNA cleavages. Electrophoresis of genomic DNA showed a typical 'DNA laddering' pattern formed by multiples of about 200 bp internucleosomal units. Chromatin condensation was first detected at least within 24 h by light microscopy, and then cell shrinkage followed. These findings suggest that the death of BY-2 cells induced by still conditions is PCD (programmed cell death).

Isoprene emission aids recovery of photosynthetic performance in transgenic Nicotiana tabacum following high intensity acute UV-B exposure.

PubMed

Centritto, Mauro; Haworth, Matthew; Marino, Giovanni; Pallozzi, Emanuele; Tsonev, Tsonko; Velikova, Violeta; Nogues, Isabel; Loreto, Francesco

2014-09-01

Isoprene emission by terrestrial plants is believed to play a role in mitigating the effects of abiotic stress on photosynthesis. Ultraviolet-B light (UV-B) induces damage to the photosynthetic apparatus of plants, but the role of isoprene in UV-B tolerance is poorly understood. To investigate this putative protective role, we exposed non-emitting (NE) control and transgenic isoprene emitting (IE) Nicotiana tabacum (tobacco) plants to high intensity UV-B exposure. Methanol emissions increased with UV-B intensity, indicating oxidative damage. However, isoprene emission was unaffected during exposure to UV-B radiation, but declined in the 48 h following UV-B treatment at the highest UV-B intensities of 9 and 15 Wm(-2). Photosynthesis and the performance of photosystem II (PSII) declined to similar extents in IE and NE plants following UV-B exposure, suggesting that isoprene emission did not ameliorate the immediate impact of UV-B on photosynthesis. However, after the stress, photosynthesis and PSII recovered in IE plants, which maintained isoprene formation, but not in NE plants. Recovery of IE plants was also associated with elevated antioxidant levels and cycling; suggesting that both isoprene formation and antioxidant systems contributed to reinstating the integrity and functionality of cellular membranes and photosynthesis following exposure to excessive levels of UV-B radiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

The Arabidopsis EIN2 restricts organ growth by retarding cell expansion

PubMed Central

Feng, Guanping; Liu, Gang; Xiao, Jianhua

2015-01-01

The growth of plant organ to its characteristic size is a fundamental developmental process, but the mechanism is still poorly understood. Plant hormones play a great role in organ size control by modulating cell division and/or cell expansion. ETHYLENE INSENSITVE 2 (EIN2) was first identified by a genetic screen for ethylene insensitivity and is regarded as a central component of ethylene signaling, but its role in cell growth has not been reported. Here we demonstrate that changed expression of EIN2 led to abnormity of cell expansion by morphological and cytological analyses of EIN2 loss-of-function mutants and the overexpressing transgenic plant. Our findings suggest that EIN2 controls final organ size by restricting cell expansion. PMID:26039475

Analyses of chlorogenic acids and related cinnamic acid derivatives from Nicotiana tabacum tissues with the aid of UPLC-QTOF-MS/MS based on the in-source collision-induced dissociation method.

PubMed

Ncube, Efficient N; Mhlongo, Msizi I; Piater, Lizelle A; Steenkamp, Paul A; Dubery, Ian A; Madala, Ntakadzeni E

2014-01-01

Chlorogenic acids (CGAs) are a class of phytochemicals that are formed as esters between different derivatives of cinnamic acid and quinic acid molecules. In plants, accumulation of these compounds has been linked to several physiological responses against various stress factors; however, biochemical synthesis differs from one plant to another. Although structurally simple, the analysis of CGA molecules with modern analytical platforms poses an analytical challenge. The objective of the study was to perform a comparison of the CGA profiles and related derivatives from differentiated tobacco leaf tissues and undifferentiated cell suspension cultures. Using an UHPLC-Q-TOF-MS/MS fingerprinting method based on the in-source collision induced dissociation (ISCID) approach, a total of 19 different metabolites with a cinnamic acid core moiety were identified. These metabolites were either present in both leaf tissue and cell suspension samples or in only one of the two plant systems. Profile differences point to underlying biochemical similarities or differences thereof. Using this method, the regio- and geometric-isomer profiles of chlorogenic acids of the two tissue types of Nicotiana tabacum were achieved. The method was also shown to be applicable for the detection of other related molecules containing a cinnamic acid core.

Aphidicolin-induced nuclear elongation in tobacco BY-2 cells.

PubMed

Yasuhara, Hiroki; Kitamoto, Kazuki

2014-05-01

Plant nuclei are known to differentiate into various shapes within a single plant. However, little is known about the mechanisms of nuclear morphogenesis. We found that nuclei of tobacco BY-2 cells were highly elongated on long-term treatment with 5 mg l⁻¹ aphidicolin, an inhibitor of DNA polymerase α. In aphidicolin-treated cells, the nuclear length was correlated with the cell length. During culture in the presence of aphidicolin, the nuclei were elongated in parallel with cell elongation. Nuclear elongation was inhibited by the inhibition of cell elongation with 2,6-dichlorobenzonitrile, a cellulose synthesis inhibitor. However, cell elongation induced in the auxin-depleted medium in the absence of aphidicolin did not cause nuclear elongation, indicating that cell elongation alone is not sufficient for nuclear elongation. Treatment with either latrunculin B or propyzamide inhibited the aphidicolin-induced nuclear elongation, indicating that both actin filaments and microtubules (MTs) are required for nuclear elongation. Observations using BY-YTHCLR2 cells, in which actin filaments, MTs and nuclei were simultaneously visualized, revealed that the longitudinally arranged MT bundles associated with the nucleus play an important role in nuclear elongation, and that actin filaments affect the formation of these MT bundles. In aphidicolin-treated cells, the nuclear DNA contents of the elongated nuclei exceeded 4C, and the nuclear length was highly correlated with the nuclear DNA content. In cells treated with 50 mg l⁻¹ aphidicolin, cells were elongated and nucleus-associated longitudinal MT bundles were formed, but the nuclear DNA contents did not exceed 4C and the nuclei did not elongate. These results indicate that an increase in the nuclear DNA content above 4C is also required for nuclear elongation.

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2

PubMed Central

Hodson, Daniel J.; Shaffer, Arthur L.; Xiao, Wenming; Wright, George W.; Schmitz, Roland; Phelan, James D.; Yang, Yandan; Webster, Daniel E.; Rui, Lixin; Kohlhammer, Holger; Nakagawa, Masao; Waldmann, Thomas A.; Staudt, Louis M.

2016-01-01

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3. Finally, by introducing mutations designed to disrupt the OCT2–OCA-B interface, we reveal a requirement for this protein–protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell–restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity. PMID:26993806

Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

PubMed

Dumont, Marie; Lehner, Arnaud; Bardor, Muriel; Burel, Carole; Vauzeilles, Boris; Lerouxel, Olivier; Anderson, Charles T; Mollet, Jean-Claude; Lerouge, Patrice

2015-12-01

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 μm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Tryptophan hydroxylase 2 (TPH2) in a neuronal cell line: modulation by cell differentiation and NRSF/rest activity.

PubMed

Gentile, Maria Teresa; Nawa, Yukino; Lunardi, Gianluigi; Florio, Tullio; Matsui, Hiroaki; Colucci-D'Amato, Luca

2012-12-01

Serotonin (5-HT) is a neurotransmitter involved in many aspects of the neuronal function. The synthesis of 5-HT is initiated by the hydroxylation of tryptophan, catalyzed by tryptophan hydroxylase (TPH). Two isoforms of TPH (TPH1 and TPH2) have been identified, with TPH2 almost exclusively expressed in the brain. Following TPH2 discovery, it was reported that polymorphisms of both gene and non-coding regions are associated with a spectrum of psychiatric disorders. Thus, insights into the mechanisms that specifically regulate TPH2 expression and its modulation by exogenous stimuli may represent a new therapeutic approach to modify serotonergic neurotransmission. To this aim, a CNS-originated cell line expressing TPH2 endogenously represents a valid model system. In this study, we report that TPH2 transcript and protein are modulated by neuronal differentiation in the cell line A1 mes-c-myc (A1). Moreover, we show luciferase activity driven by the human TPH2 promoter region and demonstrate that upon mutation of the NRSF/REST responsive element, the promoter activity strongly increases with cell differentiation. Our data suggest that A1 cells could represent a model system, allowing an insight into the mechanisms of regulation of TPH2 and to identify novel therapeutic targets in the development of drugs for the management of psychiatric disorders. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Sulfated lentinan induced mitochondrial dysfunction leads to programmed cell death of tobacco BY-2 cells.

PubMed

Wang, Jie; Wang, Yaofeng; Shen, Lili; Qian, Yumei; Yang, Jinguang; Wang, Fenglong

2017-04-01

Sulphated lentinan (sLTN) is known to act as a resistance inducer by causing programmed cell death (PCD) in tobacco suspension cells. However, the underlying mechanism of this effect is largely unknown. Using tobacco BY-2 cell model, morphological and biochemical studies revealed that mitochondrial reactive oxygen species (ROS) production and mitochondrial dysfunction contribute to sLNT induced PCD. Cell viability, and HO/PI fluorescence imaging and TUNEL assays confirmed a typical cell death process caused by sLNT. Acetylsalicylic acid (an ROS scavenger), diphenylene iodonium (an inhibitor of NADPH oxidases) and protonophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (a protonophore and an uncoupler of mitochondrial oxidative phosphorylation) inhibited sLNT-induced H 2 O 2 generation and cell death, suggesting that ROS generation linked, at least partly, to a mitochondrial dysfunction and caspase-like activation. This conclusion was further confirmed by double-stained cells with the mitochondria-specific marker MitoTracker RedCMXRos and the ROS probe H 2 DCFDA. Moreover, the sLNT-induced PCD of BY-2 cells required cellular metabolism as up-regulation of the AOX family gene transcripts and induction of the SA biosynthesis, the TCA cycle, and miETC related genes were observed. It is concluded that mitochondria play an essential role in the signaling pathway of sLNT-induced ROS generation, which possibly provided new insight into the sLNT-mediated antiviral response, including PCD. Copyright © 2016. Published by Elsevier Inc.

Direct targeting of MEK1/2 and RSK2 by silybin induces cell cycle arrest and inhibits melanoma cell growth

PubMed Central

Lee, Mee-Hyun; Huang, Zunnan; Kim, Dong Joon; Kim, Sung-Hyun; Kim, Myoung Ok; Lee, Sung-Young; Xie, Hua; Park, Si Jun; Kim, Jae Young; Kundu, Joydeb Kumar; Bode, Ann M.; Surh, Young-Joon; Dong, Zigang

2013-01-01

Abnormal functioning of multiple gene products underlies the neoplastic transformation of cells. Thus, chemopreventive and/or chemotherapeutic agents with multigene targets hold promise in the development of effective anticancer drugs. Silybin, a component of milk thistle, is a natural anticancer agent. In the present study, we investigated the effect of silybin on melanoma cell growth and elucidated its molecular targets. Our study revealed that silybin attenuated the growth of melanoma xenograft tumors in nude mice. Silybin inhibited the kinase activity of mitogen-activated protein kinase kinase (MEK)-1/2 and ribosomal S6 kinase (RSK)-2 in melanoma cells. The direct binding of silybin with MEK1/2 and RSK2 was explored using a computational docking model. Treatment of melanoma cells with silybin attenuated the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and RSK2, which are regulated by the upstream kinases MEK1/2. The blockade of MEK1/2-ERK1/2-RSK2 signaling by silybin resulted in a reduced activation of nuclear factor-kappaB, activator protein-1 and signal transducer and activator of transcription-3, which are transcriptional regulators of a variety of proliferative genes in melanomas. Silybin, by blocking the activation of these transcription factors, induced cell cycle arrest at the G1 phase and inhibited melanoma cell growth in vitro and in vivo. Taken together, silybin suppresses melanoma growth by directly targeting MEK- and RSK-mediated signaling pathways. PMID:23447564

Cell type-specific regulation of beta2-adrenoceptor mRNA by agonists.

PubMed

Danner, S; Lohse, M J

1997-07-16

Prolonged agonist stimulation of beta2-adrenoceptors results in receptor down-regulation which is often paralleled by a reduction of the corresponding mRNA. In this study, we investigated the agonist-dependent regulation of beta2-adrenoceptor mRNA in DDT1-MF2 smooth muscle cells and C6 glioma cells. In DDT1-MF2 cells the half-life of the mRNA was 12 h in monolayer compared to 2 h in suspension cultures. Under both conditions, the agonist isoproterenol reduced this half-life by a factor of 2. In contrast, in C6 glioma cells isoproterenol had no effect on the mRNA stability, even though it reduced mRNA levels by approximately 50%. Isoproterenol-induced downregulation of beta2-adrenoceptor mRNA was completely blocked in C6 cells by the presence of a protein synthesis inhibitor, while this was not so in DDT1-MF2-cells. These data show that beta2-adrenoceptor downregulation occurs via cell-type specific mechanisms.

Coatomer subunit beta 2 (COPB2), identified by label-free quantitative proteomics, regulates cell proliferation and apoptosis in human prostate carcinoma cells.

PubMed

Mi, Yuanyuan; Sun, Chuanyu; Wei, Bingbing; Sun, Feiyu; Guo, Yijun; Hu, Qingfeng; Ding, Weihong; Zhu, Lijie; Xia, Guowei

2018-01-01

Label-free quantitative proteomics has broad applications in the identification of differentially expressed proteins. Here, we applied this method to identify differentially expressed proteins (such as coatomer subunit beta 2 [COPB2]) and evaluated the functions and molecular mechanisms of these proteins in prostate cancer (PCA) cell proliferation. Proteins extracted from surgically resected PCA tissues and adjacent tissues of 3 patients were analyzed by label-free quantitative proteomics. The target protein was confirmed by bioinformatics and GEO dataset analyses. To investigate the role of the target protein in PCA, we used lentivirus-mediated small-interfering RNA (siRNA) to knockdown protein expression in the prostate carcinoma cell line, CWR22RV1 cells and assessed gene and protein expression by reverse transcription quantitative polymerase chain reaction and western blotting. CCK8 and colony formation assays were conducted to evaluate cell proliferation. Cell cycle distributions and apoptosis were assayed by flow cytometry. We selected the differentiation-related protein COPB2 as our target protein based on the results of label-free quantitative proteomics. High expression of COPB2 was found in PCA tissue and was related to poor overall survival based on a public dataset. Cell proliferation was significantly inhibited in COPB2-knockdown CWR22RV1 cells, as demonstrated by CCK8 and colony formation assays. Additionally, the apoptosis rate and percentage of cells in the G 1 phase were increased in COPB2-knockdown cells compared with those in control cells. CDK2, CDK4, and cyclin D1 were downregulated, whereas p21 Waf1/Cip1 and p27 Kip1 were upregulated, affecting the cell cycle signaling pathway. COPB2 significantly promoted CWR22RV1 cell proliferation through the cell cycle signaling pathway. Thus, silencing of COPB2 may have therapeutic applications in PCA. Copyright © 2017 Elsevier Inc. All rights reserved.

Dihydroartemisinin inhibits indoxyl sulfate (IS)-promoted cell cycle progression in mesangial cells by targeting COX-2/mPGES-1/PGE2 cascade.

PubMed

Mungun, Harr-Keshauve; Li, Shuzhen; Zhang, Yue; Huang, Songming; Jia, Zhanjun; Ding, Guixia; Zhang, Aihua

2018-01-01

Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has been used as an antimalarial drug. Recently, roles of artemisinin and its derivatives in treating diseases besides antimalarial effect were documented. Thus, this study was undertaken to investigate the role of DHA in indoxyl sulfate (IS)-promoted cell cycle progression in glomerular mesangial cells, as well as the potential mechanisms. Under the basal condition, DHA significantly retarded the cell cycle progression as shown by decreased cell percentage in S phase and increased cell percentage in G1/G0 phases in line with reduced cell cycle proteins cyclin A2 and cyclin D1. Interestingly, DHA also inactivated the COX-2/mPGES-1/PGE 2 cascade which has been shown to play a critical role in promoting the mesangial cell cycle progression by our previous studies. Next, we investigated the role of DHA in IS-triggered cell cycle progression in this mesangial cell line. As expected, DHA treatment significantly retarded IS-induced cell cycle progression and inhibited the activation of COX-2/mPGES-1/PGE 2 cascade induced by IS. In summary, these data indicated that DHA inhibited the cell cycle progression in glomerular mesangial cells under normal condition or IS challenge possibly through the inhibition of COX-2/mPGES-1/PGE 2 cascade, suggesting a potential of DHA in treating glomerular diseases with mesangial cell proliferation.

Microparticles released by vascular endothelial cells increase hypoxia inducible factor expression in human proximal tubular HK-2 cells.

PubMed

Fernandez-Martínez, Ana Belen; Torija, Ana Valdehita; Carracedo, Julia; Ramirez, Rafael; de Lucio-Cazaña, Francisco Javier

2014-08-01

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells1[W][OA

PubMed Central

Enami, Kazuhiko; Ozawa, Tomoki; Motohashi, Noriko; Nakamura, Masayuki; Tanaka, Kan; Hanaoka, Mitsumasa

2011-01-01

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates. PMID:21771917

Gab2 Phosphorylation by RSK Inhibits Shp2 Recruitment and Cell Motility

PubMed Central

Zhang, Xiaocui; Lavoie, Genevieve; Fort, Loic; Huttlin, Edward L.; Tcherkezian, Joseph; Galan, Jacob A.; Gu, Haihua; Gygi, Steven P.; Carreno, Sebastien

2013-01-01

The scaffolding adapter protein Gab2 (Grb2-associated binder) participates in the signaling response evoked by various growth factors and cytokines. Gab2 is overexpressed in several human malignancies, including breast cancer, and was shown to promote mammary epithelial cell migration. The role of Gab2 in the activation of different signaling pathways is well documented, but less is known regarding the feedback mechanisms responsible for its inactivation. We now demonstrate that activation of the Ras/mitogen-activated protein kinase (MAPK) pathway promotes Gab2 phosphorylation on basic consensus motifs. More specifically, we show that RSK (p90 ribosomal S6 kinase) phosphorylates Gab2 on three conserved residues, both in vivo and in vitro. Mutation of these phosphorylation sites does not alter Gab2 binding to Grb2, but instead, we show that Gab2 phosphorylation inhibits the recruitment of the tyrosine phosphatase Shp2 in response to growth factors. Expression of an unphosphorylatable Gab2 mutant in mammary epithelial cells promotes an invasion-like phenotype and increases cell motility. Taken together, these results suggest that RSK is part of a negative-feedback loop that restricts Gab2-dependent epithelial cell motility. On the basis of the widespread role of Gab2 in receptor signaling, these findings also suggest that RSK plays a regulatory function in diverse receptor systems. PMID:23401857

Nicotiana tabacum as model for ozone - plant surface reactions

NASA Astrophysics Data System (ADS)

Jud, Werner; Fischer, Lukas; Wohlfahrt, Georg; Tissier, Alain; Canaval, Eva; Hansel, Armin

2015-04-01

Elevated tropospheric ozone concentrations are considered a toxic threat to plants, responsible for global crop losses with associated economic costs of several billion dollars per year. The ensuing injuries have been related to the uptake of ozone through the stomatal pores and oxidative effects damaging the internal leaf tissue. A striking question of current research is the environment and plant specific partitioning of ozone loss between gas phase, stomatal or plant surface sink terms. Here we show results from ozone fumigation experiments using various Nicotiana Tabacum varieties, whose surfaces are covered with different amounts of unsaturated diterpenoids exuded by their glandular trichomes. Exposure to elevated ozone levels (50 to 150 ppbv) for 5 to 15 hours in an exceptionally clean cuvette system did neither result in a reduction of photosynthesis nor caused any visible leaf damage. Both these ozone induced stress effects have been observed previously in ozone fumigation experiments with the ozone sensitive tobacco line Bel-W3. In our case ozone fumigation was accompanied by a continuous release of oxygenated volatile organic compounds, which could be clearly associated to their condensed phase precursors for the first time. Gas phase reactions of ozone were avoided by choosing a high enough gas exchange rate of the plant cuvette system. In the case of the Ambalema variety, that is known to exude only the diterpenoid cis-abienol, ozone fumigation experiments yield the volatiles formaldehyde and methyl vinyl ketone (MVK). The latter could be unequivocally separated from isomeric methacrolein (MACR) by the aid of a Selective Reagent Ion Time-of-Flight Mass Spectrometer (SRI-ToF-MS), which was switched every six minutes from H3O+ to NO+ primary ion mode and vice versa. Consistent with the picture of an ozone protection mechanism caused by reactive diterpenoids at the leaf surface are the results from dark-light experiments. The ozone loss obtained from the

Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and endothelial cells

PubMed Central

Krieg, Carsten; Létourneau, Sven; Pantaleo, Giuseppe; Boyman, Onur

2010-01-01

IL-2 immunotherapy is an attractive treatment option for certain metastatic cancers. However, administration of IL-2 to patients can lead, by ill-defined mechanisms, to toxic adverse effects including severe pulmonary edema. Here, we show that IL-2–induced pulmonary edema is caused by direct interaction of IL-2 with functional IL-2 receptors (IL-2R) on lung endothelial cells in vivo. Treatment of mice with high-dose IL-2 led to efficient expansion of effector immune cells expressing high levels of IL-2Rβγ, including CD8+ T cells and natural killer cells, which resulted in a considerable antitumor response against s.c. and pulmonary B16 melanoma nodules. However, high-dose IL-2 treatment also affected immune cell lineage marker-negative CD31+ pulmonary endothelial cells via binding to functional αβγ IL-2Rs, expressed at low to intermediate levels on these cells, thus causing pulmonary edema. Notably, IL-2–mediated pulmonary edema was abrogated by a blocking antibody to IL-2Rα (CD25), genetic disruption of CD25, or the use of IL-2Rβγ–directed IL-2/anti-IL-2 antibody complexes, thereby interfering with IL-2 binding to IL-2Rαβγ+ pulmonary endothelial cells. Moreover, IL-2/anti-IL-2 antibody complexes led to vigorous activation of IL-2Rβγ+ effector immune cells, which generated a dramatic antitumor response. Thus, IL-2/anti-IL-2 antibody complexes might improve current strategies of IL-2–based tumor immunotherapy. PMID:20547866

Specific binding of tubeimoside-2 with proteins in hepatocarcinoma HepG2 cells: investigation by molecular spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Sun; Shi-Sheng, Sun; Ying-Yong, Zhao; Jun, Fan

2012-07-01

In this study, we compared different binding interactions of TBMS2 with proteins both in hepatocarcinoma HepG2 cells and in normal embryo hepatic L02 cells by using fluorescence, absorption, and CD spectroscopy. The fluorescence data revealed that the fluorescence intensity of proteins in the HepG2 and L02 cells decreased in the presence of TBMS2 by 30.79% and 12.01%, respectively. Binding constants and thermodynamic parameters were obtained for systems of TBMS2 with the two kinds of cell proteins. The results indicated that HepG2 cell proteins had a higher TBMS2 binding activity than those in the L02 cells. Analysis of the TBMS2 cytotoxic activities showed that TBMS2 could selectively induce apoptosis of HepG2 cells by binding to them, while its apoptotic effect on L02 cells was relatively weaker.

Silicon does not mitigate cell death in cultured tobacco BY-2 cells subjected to salinity without ethylene emission.

PubMed

Liang, Xiaolei; Wang, Huahua; Hu, Yanfeng; Mao, Lina; Sun, Lili; Dong, Tian; Nan, Wenbin; Bi, Yurong

2015-02-01

Silicon induces cell death when ethylene is suppressed in cultured tobacco BY-2 cells. There is a crosstalk between Si and ethylene signaling. Silicon (Si) is beneficial for plant growth. It alleviates both biotic and abiotic stresses in plants. How Si works in plants is still mysterious. This study investigates the mechanism of Si-induced cell death in tobacco BY-2 cell cultures when ethylene is suppressed. Results showed that K2SiO3 alleviated the damage of NaCl stress. Si treatment rapidly increased ethylene emission and the expression of ethylene biosynthesis genes. Treatments with Si + Ag and Si + aminooxyacetic acid (AOA, ethylene biosynthesis inhibitor) reduced the cell growth and increased cell damage. The treatment with Si + Ag induced hydrogen peroxide (H2O2) generation and ultimately cell death. Some nucleus of BY-2 cells treated with Si + Ag appeared TUNEL positive. The inhibition of H2O2 and nitric oxide (NO) production reduced the cell death rate induced by Si + Ag treatment. Si eliminated the up-regulation of alternative pathway by Ag. These data suggest that ethylene plays an important role in Si function in plants. Without ethylene, Si not only failed to enhance plant resistance, but also elevated H2O2 generation and further induced cell death in tobacco BY-2 cells.

Nitrogen Metabolism in Plant Cell Suspension Cultures

PubMed Central

Behrend, Josef; Mateles, Richard I.

1976-01-01

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from 14C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes. It appears that the role of organic acids is neither connected to ammonium transport nor to relief of ammonia toxicity, but may be related to the need for additional carbon skeletons for synthesis of amino acids. PMID:16659706

Phenotype-Based Screening of Small Molecules to Modify Plant Cell Walls Using BY-2 Cells.

PubMed

Okubo-Kurihara, Emiko; Matsui, Minami

2018-01-01

The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.

Glutaredoxin 2 prevents H(2)O(2)-induced cell apoptosis by protecting complex I activity in the mitochondria.

PubMed

Wu, Hongli; Xing, Kuiyi; Lou, Marjorie F

2010-10-01

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200muM hydrogen peroxide (H(2)O(2)) for 24h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H(2)O(2)-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H(2)O(2) treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H(2)O(2)-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H(2)O(2)-induced apoptosis is likely associated with its ability to preserve complex I. Published by Elsevier B.V.

Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells.

PubMed

Hisanaga, Etsuko; Nagasawa, Masahiro; Ueki, Kohjiro; Kulkarni, Rohit N; Mori, Masatomo; Kojima, Itaru

2009-01-01

Calcium-permeable cation channel TRPV2 is expressed in pancreatic beta-cells. We investigated regulation and function of TRPV2 in beta-cells. Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse beta-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence. In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a beta-cell line derived from islets obtained from a beta-cell-specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse beta-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose. TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on beta-cells.

Lipotoxicity in HepG2 cells triggered by free fatty acids

PubMed Central

Yao, Hong-Rui; Liu, Jun; Plumeri, Daniel; Cao, Yong-Bing; He, Ting; Lin, Ling; Li, Yu; Jiang, Yuan-Ying; Li, Ji; Shang, Jing

2011-01-01

The goal of this study was to investigate the lipid accumulation and lipotoxicity of free fatty acids (FFAs) induced in HepG2 cells. HepG2 cells were co-incubated with various concentrations of FFAs for 24h and the intracellular lipid contents were observed by Oil Red O and Nile Red staining methods. The lipotoxicity of HepG2 cells were then detected by Hoechest 33342/PI, Annexin V-FITC/PI double-staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) experiment tests. The experiments showed a lipid accumulation and lipotoxicity by increasing FFA concentration gradients. Through cell morphological observation and quantitative analysis, FFAs have shown to increase in a dose-dependent manner compared with the control group. The data collected from hoechst 33342/PI, annexin V-FITC/PI double staining and also MTT experiments showed that cell apoptosis and necrosis significantly increased with increasing FFA concentrations. Apoptosis was not obvious in the 1 mM FFAs-treated group compared to the other two groups. In a certain concentration range, FFAs induced intracellular lipid accumulation and lipotoxicity of HepG2 cells in a dose-dependent manner. PMID:21654881

Zinc-Dependent Protection of Tobacco and Rice Cells From Aluminum-Induced Superoxide-Mediated Cytotoxicity

PubMed Central

Lin, Cun; Hara, Ayaka; Comparini, Diego; Bouteau, François; Kawano, Tomonori

2015-01-01

Al3+ toxicity in growing plants is considered as one of the major factors limiting the production of crops on acidic soils worldwide. In the last 15 years, it has been proposed that Al3+ toxicity are mediated with distortion of the cellular signaling mechanisms such as calcium signaling pathways, and production of cytotoxic reactive oxygen species (ROS) causing oxidative damages. On the other hand, zinc is normally present in plants at high concentrations and its deficiency is one of the most widespread micronutrient deficiencies in plants. Earlier studies suggested that lack of zinc often results in ROS-mediated oxidative damage to plant cells. Previously, inhibitory action of Zn2+ against lanthanide-induced superoxide generation in tobacco cells have been reported, suggesting that Zn2+ interferes with the cation-induced ROS production via stimulation of NADPH oxidase. In the present study, the effect of Zn2+ on Al3+-induced superoxide generation in the cell suspension cultures of tobacco (Nicotiana tabacum L., cell-line, BY-2) and rice (Oryza sativa L., cv. Nipponbare), was examined. The Zn2+-dependent inhibition of the Al3+-induced oxidative burst was observed in both model cells selected from the monocots and dicots (rice and tobacco), suggesting that this phenomenon (Al3+/Zn2+ interaction) can be preserved in higher plants. Subsequently induced cell death in tobacco cells was analyzed by lethal cell staining with Evans blue. Obtained results indicated that presence of Zn2+ at physiological concentrations can protect the cells by preventing the Al3+-induced superoxide generation and cell death. Furthermore, the regulation of the Ca2+ signaling, i.e., change in the cytosolic Ca2+ ion concentration, and the cross-talks among the elements which participate in the pathway were further explored. PMID:26648960

Cytokinesis defect in BY-2 cells caused by ATP-competitive kinase inhibitors.

PubMed

Kozgunova, Elena; Higashiyama, Tetsuya; Kurihara, Daisuke

2016-10-02

Cytokinesis is last but not least in cell division as it completes the formation of the two cells. The main role in cell plate orientation and expansion have been assigned to microtubules and kinesin proteins. However, recently we reported severe cytokinesis defect in BY-2 cells not accompanied by changes in microtubules dynamics. Here we also confirmed that distribution of kinesin NACK1 is not the cause of cytokinesis defect. We further explored inhibition of the cell plate expansion by ATP-competitive inhibitors. Two different inhibitors, 5-Iodotubercidin and ML-7 resulted in a very similar phenotype, which indicates that they target same protein cascade. Interestingly, in our previous study we showed that 5-Iodotubercidin treatment affects concentration of actin filaments on the cell plate, while ML-7 is inhibitor of myosin light chain kinase. Although not directly, it indicates importance of actomyosin complex in plant cytokinesis.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells.

PubMed

Gocher, Angela M; Azabdaftari, Gissou; Euscher, Lindsey M; Dai, Shuhang; Karacosta, Loukia G; Franke, Thomas F; Edelman, Arthur M

2017-08-25

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca 2+ /calmodulin (CaM)-dependent protein kinase kinase 2 (β) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G 1 /S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca 2+ /CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca 2+ i chelation or CaM inhibition. Ionomycin-induced Ca 2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic and stoichiometric constraints determine the pathway of H2O2 consumption by red blood cells.

PubMed

Orrico, Florencia; Möller, Matías N; Cassina, Adriana; Denicola, Ana; Thomson, Leonor

2018-05-09

Red blood cells (RBC) are considered as a circulating sink of H 2 O 2 , but a significant debate remains over the role of the different intraerythocyte peroxidases. Herein we examined the kinetic of decomposition of exogenous H 2 O 2 by human RBC at different cell densities, using fluorescent and oxymetric methods, contrasting the results against a mathematical model. Fluorescent measurements as well as oxygen production experiments showed that catalase was responsible for most of the decomposition of H 2 O 2 at cell densities suitable for both experimental settings (0.1-10 × 10 10 cell L -1 ), since sodium azide but not N-ethylmaleimide (NEM) inhibited H 2 O 2 consumption. Oxygen production decreased at high cell densities until none was detected above 1.1 × 10 12 cell L- 1 , being recovered after inhibition of the thiol dependent systems by NEM. This result underlined that the consumption of H 2 O 2 by catalase prevail at RBC densities regularly used for research, while the thiol dependent systems predominate when the cell density increases, approaching the normal number in blood (5 × 10 12 cell L- 1 ). The mathematical model successfully reproduced experimental results and at low cell number it showed a time sequence involving Prx as the first line of defense, followed by catalase, with a minor role by Gpx. The turning points were given by the total consumption of reduced Prx in first place and reduced GSH after that. However, Prx alone was able to account for the added H 2 O 2 (50µM) at physiological RBC density, calling attention to the importance of cell density in defining the pathway of H 2 O 2 consumption and offering an explanation to current apparently conflicting results in the literature. Copyright © 2018. Published by Elsevier Inc.

Cell-Cell Communication Between Fibroblast and 3T3-L1 Cells Under Co-culturing in Oxidative Stress Condition Induced by H2O2.

PubMed

Subramaniyan, Sivakumar Allur; Kim, Sidong; Hwang, Inho

2016-10-01

The present study was carried out to understand the interaction between fibroblast and 3T3-L1 preadipocyte cells under H 2 O 2 -induced oxidative stress condition. H 2 O 2 (40 μM) was added in co-culture and monoculture of fibroblast and 3T3-L1 cell. The cells in the lower well were harvested for analysis and the process was carried out for both cells. The cell growth, oxidative stress markers, and antioxidant enzymes were analyzed. Additionally, the mRNA expressions of caspase-3 and caspase-7 were selected for analysis of apoptotic pathways and TNF-α and NF-κB were analyzed for inflammatory pathways. The adipogenic marker such as adiponectin and PPAR-γ and collagen synthesis markers such as LOX and BMP-1 were analyzed in the co-culture of fibroblast and 3T3-L1 cells. Cell viability and antioxidant enzymes were significantly increased in the co-culture compared to the monoculture under stress condition. The apoptotic, inflammatory, adipogenic, and collagen-synthesized markers were significantly altered in H 2 O 2 -induced co-culture of fibroblast and 3T3-L1 cells when compared with the monoculture of H 2 O 2 -induced fibroblast and 3T3-L1 cells. In addition, the confocal microscopical investigation indicated that the co-culture of H 2 O 2 -induced 3T3-L1 and fibroblast cells increases collagen type I and type III expression. From our results, we suggested that co-culture of fat cell (3T3-L1) and fibroblast cells may influence/regulate each other and made the cells able to withstand against oxidative stress and aging. It is conceivable that the same mechanism might have been occurring from cell to cell while animals are stressed by various environmental conditions.

HepG2 human hepatocarcinomas cells sensitization by endogenous porphyrins

NASA Astrophysics Data System (ADS)

Vonarx-Coinsmann, Veronique; Foultier, Marie-Therese; de Brito, Leonor X.; Morlet, Laurent; Patrice, Thierry

1995-03-01

We assessed the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA and analyzed ALA-induced toxicity and phototoxicity on this cell line. ALA induced a slight dose-dependent dark toxicity, with 79 and 66% cell survival respectively for ALA 50 and 100 mg/ml after 3-h incubation. Whereas the same treatment followed by laser irradiation (l equals 632 nm, 25 J/sq cm) induced dose-dependent phototoxicity, with 54 and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3-h delay before light exposure was found optimal to reach a maximal phototoxicity. Photoproducts induced by porphyrin light irradiation absorbed light in the red spectral region at longer wavelengths than did the original porphyrins. The possible enhancement of PDT effects after ALA HepG2 cell incubation was investigated by irradiating cells successively with red light (l equals 632 nm) and light (l equals 650 nm). Total fluence was kept constant at 25 J/sq cm. Phototoxicity was lower when cells were irradiated for increased periods of l equals 650 nm light than with l equals 632 nm light alone. Any photoproducts involved had either a short life or were poorly photoreactive. HepG2 cells, synthesizing enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX-PDT.

Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

PubMed Central

Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

2017-01-01

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

Overexpression of Lhx2 suppresses proliferation of human T cell acute lymphoblastic leukemia-derived cells, partly by reducing LMO2 protein levels.

PubMed

Miyashita, Kazuya; Kitajima, Kenji; Goyama, Susumu; Kitamura, Toshio; Hara, Takahiko

2018-01-15

T cell acute lymphoblastic leukemia (T-ALL) is a malignant cancer with poor prognosis. The transcriptional co-factor LIM domain only 2 (LMO2) and its target gene HHEX are essential for self-renewal of T cell precursors and T-ALL etiology. LMO2 directly associates with LDB1 in a large DNA-containing nuclear complex and controls the transcription of T-ALL-related genes. Recently, we reported that overexpression of the LIM-homeodomain transcription factor, Lhx2, results in liberation of the Lmo2 protein from the Lmo2-Ldb1 complex, followed by ubiquitin proteasome mediated degradation. Here, we found that proliferation of five human T-ALL-derived cell lines, including CCRF-CEM, was significantly suppressed by retroviral overexpression of Lhx2. The majority of Lhx2-transduced CCRF-CEM cells arrested in G 0 phase and subsequently underwent apoptosis. Expression of LMO2 protein as well as HHEX, ERG, HES1 and MYC genes was repressed in CCRF-CEM cells by transduction with Lhx2. Lhx2-mediated growth inhibition was partially rescued by simultaneous overexpression of Lmo2; however, both the C-terminal LIM domain and the homeodomain of Lhx2 were required for its growth-suppressive activity. These data indicate that Lhx2 is capable of blocking proliferation of T-ALL-derived cells by both LMO2-dependent and -independent means. We propose Lhx2 as a new molecular tool for anti-T-ALL drug development. Copyright © 2017 Elsevier Inc. All rights reserved.

Intracellular Ca2+ regulation by the leech giant glial cell.

PubMed

Nett, W; Deitmer, J W

1998-02-15

1. We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura-2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis. 2. The basal [Ca2+]i was 79 +/- 35 nM (n = 27) in cells voltage clamped at -70 to -80 mV, and 75 +/- 29 nM (mean +/- S.D., n = 82) in unclamped cells at a mean membrane potential of -67 +/- 6 mV. 3. Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 +/- 21 nM (n = 27) in cells voltage clamped at -70 to -80 mV, and of 35 +/- 18 nM (n = 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re-addition, did not change significantly with the holding potential between -110 and -60 mV. 4. The basal [Na+]i was 5.6 +/- 1.3 mM (n = 18). Increasing [Na+]i by inhibiting the Na+-K+ pump with 100 microM ouabain had no effect on the [Ca2+]i rise upon removal of external Na+. 5. The time course of recovery from a [Ca2+]i load mediated by voltage-dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+. 6. Cyclopiazonic acid (10 muM), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a transient increase in [Ca2+]i of 28 +/- 11 nM (n = 5), and significantly slowed the recovery from imposed [Ca2+]i loads. 7. Iontophoretic injection of orthovanadate, an inhibitor of P-type ATPases including the plasma membrane Ca2+-ATPase, caused a persistent increase in the basal [Ca2+]i of 163 +/- 101 nM (n = 5) in standard saline, and of 427 +/- 338 nM in Na+-free saline (n = 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 +/- 64 nM (n = 3). 8. The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+-ATPase in

SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

PubMed

Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

2011-02-04

SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

Induction of cyclo-oxygenase-2 mRNA by prostaglandin E2 in human prostatic carcinoma cells

NASA Technical Reports Server (NTRS)

Tjandrawinata, R. R.; Dahiya, R.; Hughes-Fulford, M.

1997-01-01

Prostaglandins are synthesized from arachidonic acid by the enzyme cyclo-oxygenase. There are two isoforms of cyclooxygenases: COX-1 (a constitutive form) and COX-2 (an inducible form). COX-2 has recently been categorized as an immediate-early gene and is associated with cellular growth and differentiation. The purpose of this study was to investigate the effects of exogenous dimethylprostaglandin E2 (dmPGE2) on prostate cancer cell growth. Results of these experiments demonstrate that administration of dmPGE2 to growing PC-3 cells significantly increased cellular proliferation (as measured by the cell number), total DNA content and endogenous PGE2 concentration. DmPGE2 also increased the steady-state mRNA levels of its own inducible synthesizing enzyme, COX-2, as well as cellular growth to levels similar to those seen with fetal calf serum and phorbol ester. The same results were observed in other human cancer cell types, such as the androgen-dependent LNCaP cells, breast cancer MDA-MB-134 cells and human colorectal carcinoma DiFi cells. In PC-3 cells, the dmPGE2 regulation of the COX-2 mRNA levels was both time dependent, with maximum stimulation seen 2 h after addition, and dose dependent on dmPGE2 concentration, with maximum stimulation seen at 5 microg ml(-1). The non-steroidal anti-inflammatory drug flurbiprofen (5 microM), in the presence of exogenous dmPGE2, inhibited the up-regulation of COX-2 mRNA and PC-3 cell growth. Taken together, these data suggest that PGE2 has a specific role in the maintenance of human cancer cell growth and that the activation of COX-2 expression depends primarily upon newly synthesized PGE2, perhaps resulting from changes in local cellular PGE2 concentrations.

Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

PubMed

Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

2003-10-01

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

Cell growth and water relations of the halophyte, Atriplex nummularia L., in response to NaCl.

PubMed

Casas, A M; Bressan, R A; Hasegawa, P M

1991-06-01

Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic "over adjustment" as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118-125).

5-demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis.

PubMed

Charoensinphon, Noppawat; Qiu, Peiju; Dong, Ping; Zheng, Jinkai; Ngauv, Pearline; Cao, Yong; Li, Shiming; Ho, Chi-Tang; Xiao, Hang

2013-12-01

Tangeretin (TAN) and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human nonsmall cell lung cancer (NSCLC) cells. Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50 s of 5DT were 79-fold, 57-fold, and 56-fold lower than those of TAN in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while TAN at tenfold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21(Cip1/Waf1), and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7-4.9 fold higher than those of TAN after the cells were treated with 5DT or TAN at the same concentration. Our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by TAN, which is partially due to the higher intracellular uptake of 5DT than TAN. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

PubMed

Xin, Jia-Xuan; Yue, Zhen; Zhang, Shuai; Jiang, Zhong-Hua; Wang, Ping-Yu; Li, You-Jie; Pang, Min; Xie, Shu-Yang

2013-10-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2

PubMed Central

XIN, JIA-XUAN; YUE, ZHEN; ZHANG, SHUAI; JIANG, ZHONG-HUA; WANG, PING-YU; LI, YOU-JIE; PANG, MIN; XIE, SHU-YANG

2013-01-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3′-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics. PMID:24137458

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells.

PubMed

Higaki, Takumi; Kadota, Yasuhiro; Goh, Tatsuaki; Hayashi, Teruyuki; Kutsuna, Natsumaro; Sano, Toshio; Hasezawa, Seiichiro; Kuchitsu, Kazuyuki

2008-09-01

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.

Exogenous proline enhances the sensitivity of Tobacco BY-2 cells to arsenate.

PubMed

Nahar, Mst Nur-E-Nazmun; Islam, Mohammad Muzahidul; Hoque, Md Anamul; Yonezawa, Anna; Prodhan, Md Yeasin; Nakamura, Toshiyuki; Nakamura, Yoshimasa; Munemasa, Shintaro; Murata, Yoshiyuki

2017-09-01

Arsenic causes physiological and structural disorders in plants. Proline is accumulated as a compatible solute in plants under various stress conditions and mitigates stresses. Here, we investigated the effects of exogenous proline on tobacco Bright Yellow-2 (BY-2) cultured cells under [Formula: see text] stress. Arsenate did not inhibit BY-2 cell growth at 40 and 50 μM but did it at 60 μM. Proline at 0.5 to 10 mM did not affect the cell growth but delayed it at 20 mM. At 40 μM [Formula: see text], neither 0.5 mM nor 1 mM proline affected the cell growth but 10 mM proline inhibited it. In the presence of [Formula: see text], 10 mM proline increased the number of Evans Blue-stained (dead) cells and decreased the number of total cells. Together, our results suggest that exogenous proline does not alleviate arsenate toxicity but enhances the sensitivity of BY-2 cells to arsenate.

Type 2 innate lymphoid cell suppression by regulatory T cells attenuates airway hyperreactivity and requires inducible T-cell costimulator-inducible T-cell costimulator ligand interaction.

PubMed

Rigas, Diamanda; Lewis, Gavin; Aron, Jennifer L; Wang, Bowen; Banie, Homayon; Sankaranarayanan, Ishwarya; Galle-Treger, Lauriane; Maazi, Hadi; Lo, Richard; Freeman, Gordon J; Sharpe, Arlene H; Soroosh, Pejman; Akbari, Omid

2017-05-01

Atopic diseases, including asthma, exacerbate type 2 immune responses and involve a number of immune cell types, including regulatory T (Treg) cells and the emerging type 2 innate lymphoid cells (ILC2s). Although ILC2s are potent producers of type 2 cytokines, the regulation of ILC2 activation and function is not well understood. In the present study, for the first time, we evaluate how Treg cells interact with pulmonary ILC2s and control their function. ILC2s and Treg cells were evaluated by using in vitro suppression assays, cell-contact assays, and gene expression panels. Also, human ILC2s and Treg cells were adoptively transferred into NOD SCID γC-deficient mice, which were given isotype or anti-inducible T-cell costimulator ligand (ICOSL) antibodies and then challenged with IL-33 and assessed for airway hyperreactivity. We show that induced Treg cells, but not natural Treg cells, effectively suppress the production of the ILC2-driven proinflammatory cytokines IL-5 and IL-13 both in vitro and in vivo. Mechanistically, our data reveal the necessity of inducible T-cell costimulator (ICOS)-ICOS ligand cell contact for Treg cell-mediated ILC2 suppression alongside the suppressive cytokines TGF-β and IL-10. Using a translational approach, we then demonstrate that human induced Treg cells suppress syngeneic human ILC2s through ICOSL to control airway inflammation in a humanized ILC2 mouse model. These findings suggest that peripheral expansion of induced Treg cells can serve as a promising therapeutic target against ILC2-dependent asthma. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2.

PubMed

Shankar Babu, Mani; Mahanta, Sailendra; Lakhter, Alexander J; Hato, Takashi; Paul, Subhankar; Naidu, Samisubbu R

2018-01-01

Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells.

Lapachol inhibits glycolysis in cancer cells by targeting pyruvate kinase M2

PubMed Central

Shankar Babu, Mani; Mahanta, Sailendra; Lakhter, Alexander J.; Hato, Takashi; Paul, Subhankar

2018-01-01

Reliance on aerobic glycolysis is one of the hallmarks of cancer. Although pyruvate kinase M2 (PKM2) is a key mediator of glycolysis in cancer cells, lack of selective agents that target PKM2 remains a challenge in exploiting metabolic pathways for cancer therapy. We report that unlike its structural analog shikonin, a known inhibitor of PKM2, lapachol failed to induce non-apoptotic cell death ferroxitosis in hypoxia. However, melanoma cells treated with lapachol showed a dose-dependent inhibition of glycolysis and a corresponding increase in oxygen consumption. Accordingly, in silico studies revealed a high affinity-binding pocket for lapachol on PKM2 structure. Lapachol inhibited PKM2 activity of purified enzyme as well as in melanoma cell extracts. Blockade of glycolysis by lapachol in melanoma cells led to decreased ATP levels and inhibition of cell proliferation. Furthermore, perturbation of glycolysis in melanoma cells with lapachol sensitized cells to mitochondrial protonophore and promoted apoptosis. These results present lapachol as an inhibitor of PKM2 to interrogate metabolic plasticity in tumor cells. PMID:29394289

Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.

PubMed

Sigeno, Asako; Hayashi, Sugane; Terachi, Toru; Yamagishi, Hiroshi

2009-11-01

Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.

SH2 domain-containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell-mediated Th2 immunity.

PubMed

Ahmed, Md Selim; Kang, Myeong-Ho; Lee, Ezra; Park, Yujin; Jeong, Yideul; Bae, Yong-Soo

2017-01-01

The Src homology 2 domain-containing adaptor protein B (SHB) is widely expressed in immune cells and acts as an important regulator for hematopoietic cell function. SHB silencing induces Th2 immunity in mice. SHB is also involved in T-cell homeostasis in vivo . However, SHB has not yet been studied and addressed in association with dendritic cells (DCs). The effects of SHB expression on the immunogenicity of DCs were assessed by Shb gene silencing in mouse bone marrow-derived DCs (BMDCs). After silencing, surface phenotype, cytokine expression profile, and T-cell stimulation capacity of BMDCs were examined. We investigated the signaling pathways involved in SHB expression during BMDC development. We also examined the immunogenicity of SHB-knockdown (SHB KD ) BMDCs in a mouse atopic dermatitis model. SHB was steadily expressed in mouse splenic DCs and in in vitro -generated BMDCs in both immature and mature stages. SHB expression was contingent on activation of the mitogen- activated protein kinase/Foxa2 signaling pathway during DC development. SHB KD increased the expression of MHC class II and costimulatory molecules without affecting the cytokine expression of BMDCs. When co-cultured with T cells, SHB KD in BMDCs significantly induced CD4 + T-cell proliferation and the expression of Th2 cytokines, while the regulatory T cell (Treg) population was downregulated. In mouse atopic dermatitis model, mice inoculated with SHB KD DCs developed more severe symptoms of atopic dermatitis compared with mice injected with control DCs. SHB expression in DCs plays an important role in T-cell homeostasis in vivo by regulating DC-mediated Th2 polarization.

Sensitive Detection of Single-Cell Secreted H2O2 by Integrating a Microfluidic Droplet Sensor and Au Nanoclusters.

PubMed

Shen, Rui; Liu, Peipei; Zhang, Yiqiu; Yu, Zhao; Chen, Xuyue; Zhou, Lu; Nie, Baoqing; Żaczek, Anna; Chen, Jian; Liu, Jian

2018-04-03

As an important signaling molecule, hydrogen peroxide (H 2 O 2 ) secreted externally by the cells influences cell migration, immunity generation, and cellular communications. Herein, we have developed a microfluidic approach with droplets in combination with Au nanoclusters for the sensitive detection of H 2 O 2 secreted by a single cell. Isolated in the ultrasmall volume (4.2 nL) of a microdroplet, single-cell secreted H 2 O 2 can initiate dramatic fluorescence changes of horseradish peroxidase-Au nanoclusters. We have demonstrated an ultrahigh sensitivity (200-400 attomole H 2 O 2 directly measured from a single cell) with good specificity. It offers a useful research tool to study the cell-to-cell differences of H 2 O 2 secretion at the single-cell level.

Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

PubMed

Feriotto, G; Calza, R; Bergamini, C M; Griffin, M; Wang, Z; Beninati, S; Ferretti, V; Marzola, E; Guerrini, R; Pagnoni, A; Cavazzini, A; Casciano, F; Mischiati, C

2017-03-01

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Binding, uptake, and transport of hypericin by Caco-2 cell monolayers.

PubMed

Sattler, S; Schaefer, U; Schneider, W; Hoelzl, J; Lehr, C M

1997-10-01

The biological evaluation of hypericin in various test models is hampered by its very poor water solubility. In the present study cyclodextrin formulations and liposomal preparations were investigated for improved delivery and solubility of hypericin in aqueous buffer systems. Caco-2 cells, grown to tight monolayers on 96-well tissue culture plates as well as on Transwell polycarbonate filters, were used to study the membrane binding and the epithelial transport of hypericin. Cumulative transport of hypericin, which could not be measured without the use of cyclodextrins, in apical-to-basolateral direction from cyclodextrin-hypericin buffer solutions was 3-5% at 37 degrees C and approximately 0.12% at 4 degrees C after 5 h. After an incubation time of 1 h at 37 and 4 degrees C, 12.7% +/- 2.6% and 6.5% +/- 0.8%, respectively, of hypericin were found to be bound to or taken up by Caco-2 cells. Liposomal formulations markedly increased the solubility of hypericin in Krebs-Ringer buffer, but there was no effect observed on the binding and transport of hypericin delivered by liposomes in the Caco-2 cell model. Due to the fluorescence properties of hypericin, its interaction with the cells could be visualized by confocal laser scanning microscopy. The results indicate that a significant accumulation of the drug in the cell membrane and the cell nucleus membrane takes place. We conclude that hypericin is absorbed through the intestinal epithelium by passive transcellular diffusion and that increasing its solubility by cyclodextrin appears as a promising approach to increase its oral bioavailability for pharmaceutical formulations.

Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells

PubMed Central

Cavet, M E; West, M; Simmons, N L

1997-01-01

Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 μM) or bromosulphophthalein (100 μM). Similarly tetraethylammonium and N-′methylnicotinamide (10 mM) were without effect. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4′-diisothiocyanostilbene-2-2′-disulphonic acid (DIDS, 400 μM). Net secretion of ciprofloxacin was partially inhibited by 100 μM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretion in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl− or K+ in the bathing media. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia. PMID:9283689

Live-cell imaging of phosphatidic acid dynamics in pollen tubes visualized by Spo20p-derived biosensor.

PubMed

Potocký, Martin; Pleskot, Roman; Pejchar, Přemysl; Vitale, Nicolas; Kost, Benedikt; Zárský, Viktor

2014-07-01

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors

PubMed Central

Scharfstein, Julio; Schmitz, Veronica; Morandi, Veronica; Capella, Marcia M. A.; Lima, Ana Paula C. A.; Morrot, Alexandre; Juliano, Luiz; Müller-Esterl, Werner

2000-01-01

The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease. PMID:11067878

Targeted modification of homogalacturonan by transgenic expression of a fungal polygalacturonase alters plant growth.

PubMed

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J Paul; De Lorenzo, Giulia; Cervone, Felice

2004-07-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis.

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals.

PubMed

Lucena, Rafael; Alcaide-Gavilán, Maria; Schubert, Katherine; He, Maybo; Domnauer, Matthew G; Marquer, Catherine; Klose, Christian; Surma, Michal A; Kellogg, Douglas R

2018-01-22

The size of all cells, from bacteria to vertebrates, is proportional to the growth rate set by nutrient availability, but the underlying mechanisms are unknown. Here, we show that nutrients modulate cell size and growth rate via the TORC2 signaling network in budding yeast. An important function of the TORC2 network is to modulate synthesis of ceramide lipids, which play roles in signaling. TORC2-dependent control of ceramide signaling strongly influences both cell size and growth rate. Thus, cells that cannot make ceramides fail to modulate their growth rate or size in response to changes in nutrients. PP2A associated with the Rts1 regulatory subunit (PP2A Rts1 ) is embedded in a feedback loop that controls TORC2 signaling and helps set the level of TORC2 signaling to match nutrient availability. Together, the data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals arising from the TORC2 network. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

PubMed

Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

2011-12-01

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

An Interleukin-33-Mast Cell-Interleukin-2 Axis Suppresses Papain-Induced Allergic Inflammation by Promoting Regulatory T Cell Numbers.

PubMed

Morita, Hideaki; Arae, Ken; Unno, Hirotoshi; Miyauchi, Kousuke; Toyama, Sumika; Nambu, Aya; Oboki, Keisuke; Ohno, Tatsukuni; Motomura, Kenichiro; Matsuda, Akira; Yamaguchi, Sachiko; Narushima, Seiko; Kajiwara, Naoki; Iikura, Motoyasu; Suto, Hajime; McKenzie, Andrew N J; Takahashi, Takao; Karasuyama, Hajime; Okumura, Ko; Azuma, Miyuki; Moro, Kazuyo; Akdis, Cezmi A; Galli, Stephen J; Koyasu, Shigeo; Kubo, Masato; Sudo, Katsuko; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

2015-07-21

House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Iodine-131 treatment of thyroid cancer cells leads to suppression of cell proliferation followed by induction of cell apoptosis and cell cycle arrest by regulation of B-cell translocation gene 2-mediated JNK/NF-κB pathways.

PubMed

Zhao, L M; Pang, A X

2017-01-16

Iodine-131 (131I) is widely used for the treatment of thyroid-related diseases. This study aimed to investigate the expression of p53 and BTG2 genes following 131I therapy in thyroid cancer cell line SW579 and the possible underlying mechanism. SW579 human thyroid squamous carcinoma cells were cultured and treated with 131I. They were then assessed for 131I uptake, cell viability, apoptosis, cell cycle arrest, p53 expression, and BTG2 gene expression. SW579 cells were transfected with BTG2 siRNA, p53 siRNA and siNC and were then examined for the same aforementioned parameters. When treated with a JNK inhibitor of SP600125 and 131I or with a NF-κB inhibitor of BMS-345541 and 131I, non-transfected SW579 cells were assessed in JNK/NFκB pathways. It was observed that 131I significantly inhibited cell proliferation, promoted cell apoptosis and cell cycle arrest. Both BTG2 and p53 expression were enhanced in a dose-dependent manner. An increase in cell viability by up-regulation in Bcl2 gene, a decrease in apoptosis by enhanced CDK2 gene expression and a decrease in cell cycle arrest at G0/G1 phase were also observed in SW579 cell lines transfected with silenced BTG2 gene. When treated with SP600125 and 131I, the non-transfected SW579 cell lines significantly inhibited JNK pathway, NF-κB pathway and the expression of BTG2. However, when treated with BMS-345541 and 131I, only the NF-κB pathway was suppressed. 131I suppressed cell proliferation, induced cell apoptosis, and promoted cell cycle arrest of thyroid cancer cells by up-regulating B-cell translocation gene 2-mediated activation of JNK/NF-κB pathways.

Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by sulphur starvation.

PubMed

Wawrzyńska, Anna; Lewandowska, Małgorzata; Sirko, Agnieszka

2010-03-01

Sulphur deficiency severely affects plant growth and their agricultural productivity leading to diverse changes in development and metabolisms. Molecular mechanisms regulating gene expression under low sulphur conditions remain largely unknown. AtSLIM1, a member of the EIN3-like (EIL) family was reported to be a central transcriptional regulator of the plant sulphur response, however, no direct interaction of this protein with any sulphur-responsive promoters was demonstrated. The focus of this study was on the analysis of a promoter region of UP9C, a tobacco gene strongly induced by sulphur limitation. Cloning and subsequent examination of this promoter resulted in the identification of a 20-nt sequence (UPE-box), also present in the promoters of several Arabidopsis genes, including three out of four homologues of UP9C. The UPE-box, consisting of two parallel tebs sequences (TEIL binding site), proved to be necessary to bind the transcription factors belonging to the EIL family and of a 5-nt conserved sequence at the 3'-end. The yeast one-hybrid analysis resulted in the identification of one transcription factor (NtEIL2) capable of binding to the UPE-box. The interactions of NtEIL2, and its homologue from Arabidopsis, AtSLIM1, with DNA were affected by mutations within the UPE-box. Transient expression assays in Nicotiana benthamiana have further shown that both factors, NtEIL2 and AtSLIM1, activate the UP9C promoter. Interestingly, activation by NtEIL2, but not by AtSLIM1, was dependent on the sulphur-deficiency of the plants.

HSP90 inhibitor 17-DMAG enhances EphA2+ tumor cell recognition by specific CD8+ T cells

PubMed Central

Kawabe, Mayumi; Mandic, Maja; Taylor, Jennifer L.; Vasquez, Cecilia A.; Wesa, Amy K.; Neckers, Leonard M.; Storkus, Walter J.

2009-01-01

EphA2, a member of the receptor tyrosine kinase (RTK) family, is commonly expressed by a broad range of cancer types, where its level of (over)expression correlates with poor clinical outcome. Since tumor cell expressed EphA2 is a non-mutated “self” protein, specific CD8+ T cells are subject to self-tolerance mechanisms and typically exhibit only moderate-to-low functional avidity, rendering them marginally competent to recognize EphA2+ tumor cells in vitro or in vivo. We have recently reported that the ability of specific CD8+ T cells to recognize EphA2+ tumor cells can be augmented after the cancer cells are pretreated with EphA2 agonists that promote proteasomal degradation and upregulated expression of EphA2/class I complexes on the tumor cell membrane (Wesa et al., J. Immunol. 2008;181:7721-7). In the current study we show that treatment of EphA2+ tumor cells with the irreversible HSP90 inhibitor, 17-DMAG, similarly enhances their recognition by EphA2-specific CD8+ T cell lines and clones in vitro via a mechanism that is dependent on proteasome and TAP function, as well as, the retrotranslocation of EphA2 into the tumor cytoplasm. When 17-DMAG and agonist anti-EphA2 mAb are co-applied, T cell recognition of tumor cells is further increased over that observed for either agent alone. These studies suggest that EphA2 represents a novel HSP90 client protein and that the treatment of cancer patients with 17-DMAG-based “pulse” therapy may improve the anti-tumor efficacy of CD8+ T effector cells reactive against EphA2-derived epitopes. PMID:19690146

MicroRNA-196b Inhibits Cell Growth and Metastasis of Lung Cancer Cells by Targeting Runx2.

PubMed

Bai, Xiaoxue; Meng, Lin; Sun, Huijie; Li, Zhuo; Zhang, Xiufang; Hua, Shucheng

2017-01-01

Lung cancer is one of the most common causes of cancer related deaths worldwide. The role of several microRNAs (miRNAs) including miR-196b in different cancers has already been established. The study was aimed to explore the role of miR-196b in lung cancer and its possible underlying mechanism. Human lung cancer cell line A549 was transfected with miR-196b mimic, miR-196b inhibitor and corresponding controls. Then cell viability, migration, invasion, and apoptosis of A549 lung cancer cells either with overexpression or with suppression of miR-196b were estimated sequentially. Next, dual luciferase activity assay was performed to clarify whether Runx2 was a direct target of miR-196b. Finally, the expressions of main factors associated with epithelial mesenchymal transition (EMT), PI3K/AKT/GSK3β, Smad, and JNK pathways were detected by western blot. MiR-196b expression was significantly decreased in A549, H1650 and H1299 cell lines compared with in WI-38 and HEL-1 cell lines. Overexpression of miR-196b suppressed cell viability, migration, invasion, and induced apoptosis as well as inhibited TGF-β induced EMT process in A549 cells. In addition, Runx2 was a putative target of miR-196b, and Runx2 silence remarkably increased cell apoptosis and abolished the promotive effects of miR-196b suppression on cell viability, migration and invasion. Finally, miR-196b also mediated its action by inactivation of PI3K/AKT/GSK3β, Smad, and JNK pathways by down-regulation of Runx2. MiR-196b functions as a tumor suppressor that inhibited cell growth and metastasis of lung cancer cells by targeting Runx2. These findings provided further evidences for treatment of lung cancer. The Author(s). Published by S. Karger AG, Basel.

Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

PubMed Central

Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

2012-01-01

Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis—total protein content, thiols—reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

The role of accessory cells in polyclonal T cell activation. I. Both induction of interleukin 2 production and of interleukin 2 responsiveness by concanavalin A are accessory cell dependent.

PubMed

Hünig, T; Loos, M; Schimpl, A

1983-01-01

Recent studies from other laboratories have shown that concanavalin A (Con A) acts at two separate steps in polyclonal T cell activation: interleukin 2 (IL2) production, and induction of responsiveness to IL2. Using a combination of techniques for the depletion of accessory cells from lymph node T cells, we have investigated which of these steps, if not both, is responsible for the known requirement for accessory cells in the Con A response. It was found that with increasing T cell purification, first the ability is lost to produce sufficient levels of endogenous IL2, whereas induction of IL2 responsiveness can still take place. Further removal of accessory cells however yields a population of resting T cells that cannot be induced by Con A to become IL2-reactive. It was concluded that both IL2 production and induction of reactivity to IL2 are accessory cell-dependent events.

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth1

PubMed Central

Capodicasa, Cristina; Vairo, Donatella; Zabotina, Olga; McCartney, Lesley; Caprari, Claudio; Mattei, Benedetta; Manfredini, Cinzia; Aracri, Benedetto; Benen, Jacques; Knox, J. Paul; De Lorenzo, Giulia; Cervone, Felice

2004-01-01

Pectins are a highly complex family of cell wall polysaccharides comprised of homogalacturonan (HGA), rhamnogalacturonan I and rhamnogalacturonan II. We have specifically modified HGA in both tobacco (Nicotiana tabacum) and Arabidopsis by expressing the endopolygalacturonase II of Aspergillus niger (AnPGII). Cell walls of transgenic tobacco plants showed a 25% reduction in GalUA content as compared with the wild type and a reduced content of deesterified HGA as detected by antibody labeling. Neutral sugars remained unchanged apart from a slight increase of Rha, Ara, and Gal. Both transgenic tobacco and Arabidopsis were dwarfed, indicating that unesterified HGA is a critical factor for plant cell growth. The dwarf phenotypes were associated with AnPGII activity as demonstrated by the observation that the mutant phenotype of tobacco was completely reverted by crossing the dwarfed plants with plants expressing PGIP2, a strong inhibitor of AnPGII. The mutant phenotype in Arabidopsis did not appear when transformation was performed with a gene encoding AnPGII inactivated by site directed mutagenesis. PMID:15247378

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells.

PubMed

Monetti, Emanuela; Kadono, Takashi; Tran, Daniel; Azzarello, Elisa; Arbelet-Bonnin, Delphine; Biligui, Bernadette; Briand, Joël; Kawano, Tomonori; Mancuso, Stefano; Bouteau, François

2014-03-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·(-)) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·(-) generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca(2+)]cyt increase and singlet oxygen production, do not seem to be involved in PCD.

A Cyclin D2-derived peptide acts on specific cell cycle phases by activating ERK1/2 to cause the death of breast cancer cells.

PubMed

Russo, Lilian C; Araujo, Christiane B; Iwai, Leo K; Ferro, Emer S; Forti, Fabio L

2017-01-16

Protein degradation by the proteasome generates functional intracellular peptides. Pep5, a peptide derived from Cyclin D2, induces cell death in tumor cell lines and reduces the volume of rat C6 glioblastoma tumors in vivo. Here, we chose the human MDA-MB-231 breast cancer cells to evaluate the mechanism of cell death induced by pep5 in different phases of the cell cycle. Fluorescently labeled pep5, monitored by real time confocal microscopy, entered the MDA-MB-231 cells 3min after application and localized to the nucleus and cytoplasm. Pep5-induced cell death was increased when the MDA-MB-231 cell population was arrested at the G1/S transition or in S phase compared to asynchronous cells. Pep5 induced permanent extracellular signal-regulated kinase (ERK1/2) phosphorylation in MDA-MB-231 cells synchronized in G1/S or S phase. Affinity chromatography followed by mass spectrometry identified CLIC1 and Plectin as the only two proteins that interacted with pep5 in both asynchronous and synchronized MDA-MB-231 cells. These interactions could explain the long-lasting ERK1/2 phosphorylation and the cytoskeleton perturbations in the MDA-MB-231 cells, in which the stress fibers' integrity is affected by pep5 treatments. These data suggest that pep5 has potential therapeutic properties for treating specific types of cancers, such as breast cancer cells. Pep5, a natural intracellular peptide formed by the degradation of Cyclin D2 through the ubiquitin-proteasome system, induces cell death when reintroduced into MDA-MB-231 breast cancer cells, which express low levels of Cyclin D2, specifically in G1/S arrested cells or in cells that are passing through S phase. Under these conditions, pep5 is able to interact with different intracellular proteins, primarily cytoskeleton and proteasome components, which can lead to cellular apoptosis. Together, our data suggest that pep5 is an intracellular peptide with therapeutic potential for treating specific types of tumors with low

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Effect of 2,4-Dichlorophenoxyacetic Acid on Glucosylation of Scopoletin to Scopolin in Tobacco Tissue Culture

PubMed Central

Hino, Fumitsugu; Okazaki, Mitsuo; Miura, Yoshiharu

1982-01-01

2,4-Dichlorophenoxyacetic acid (2,4-D) stimulated the formation of scopoletin and scopolin in tobacco (Nicotiana tabacum L. `Bright Yellow') cell culture. It especially stimulated the uptake of scopoletin from culture medium into the cells and the glucosylation of scopoletin to its monoglucoside, scopolin. This phenomenon is peculiar to 2,4-D, in contrast to other plant hormones. 2,4-D (1 μg/ml) stimulated the glucosylation of scopoletin to scopolin by enhancing UDP-glucose:scopoletin glucosyltransferase (SGTase) activity. The enhancement of SGTase activity caused by treatment with 2,4-D was observed when the syntheses of RNA and protein were inhibited by either actinomycin-D and/or cycloheximide. However, the stimulatory effect of 2,4-D was inhibited by treatment with dinitrophenol. Furthermore, SGTase with or without treatment by 2,4-D in vivo for 24 hours, was isolated from cultured tobacco cells. The enzymes were purified about 200-fold by precipitation with (NH4)2SO4 and chromatography with Sephadex G-100, DEAE-cellulose, and hydroxyapatite. The specific activity of 2,4-D-treated SGTase was 10 times higher than that of untreated SGTase even in the purified fraction, which showed one protein band under electrophoresis. These results suggest that the enhancement of SGTase activity by 2,4-D is due to the energy-dependent activation of the enzyme already present, but not due to the de novo synthesis of the enzyme. Images PMID:16662301

Crystallization of the photosystem II core complex and its chlorophyll binding subunit CP43 from transplastomic plants of Nicotiana tabacum.

PubMed

Piano, Dario; El Alaoui, Sabah; Korza, Henryk J; Filipek, Renata; Sabala, Izabela; Haniewicz, Patrycja; Buechel, Claudia; De Sanctis, Daniele; Bochtler, Matthias

2010-12-01

Photosystem II from transplastomic plants of Nicotiana tabacum with a hexahistidine tag at the N-terminal end of the PsbE subunit (α-chain of the cytochrome b(559)) was purified according to the protocol of Fey et al. (BBA 12:1501-1509, 2008). The protein sample was then subjected to two additional gel filtration runs in order to increase its homogeneity and to standardize the amount of detergent. Large three dimensional crystals of the core complex were obtained. Crystals of one of its chlorophyll binding subunits (CP43) in isolation grew in very similar conditions that differed only in the concentration of the detergent. Diffraction of Photosystem II and CP43 crystals at various synchrotron beamlines was limited to a resolution of 7 and 14 Å, respectively. In both cases the diffraction quality was insufficient for an unambiguous assignment of the crystallographic lattice or space group.

Involvement of P2X7 receptors in retinal ganglion cell apoptosis induced by activated Müller cells.

PubMed

Xue, Bo; Xie, Yuting; Xue, Ying; Hu, Nan; Zhang, Guowei; Guan, Huaijin; Ji, Min

2016-12-01

Müller cell reactivation (gliosis) is an early response in glaucomatous retina. Previous studies have demonstrated that activation of P2X 7 receptors results in retinal ganglion cell (RGC) apoptosis. Here, the issues of whether and how activated Müller cells may contribute to RGC apoptosis through P2X 7 receptors were investigated. Either intravitreal injection of (S)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR I) agonist, in normal rat retinas, or DHPG treatment of purified cultured rat retinal Müller cells induced an increase in glial fibrillary acidic protein (GFAP) expression, indicative of Müller cell gliosis. In addition, an increase in adenosine triphosphate (ATP) release from purified cultured Müller cells was detected during DHPG treatment (for 10 min to 48 h), which was mediated by the intracellular mGluR5/Gq/PI-PLC/PKC signaling pathway. Intravitreal injection of DHPG mimicked the reduction in the number of fluorogold retrogradely labeled RGCs in chronic ocular hypertension (COH) rats. Treatment with the conditioned culture medium (CM) obtained from the DHPG-activated Müller cell medium induced an increase in the number of TUNEL-positive cells in cultured RGCs, which was mimicked by benzoylbenzoyl adenosine triphosphate (BzATP), a P2X 7 receptor agonist, but was partially blocked by brilliant blue G (BBG), a P2X 7 receptor antagonist. Moreover, the CM treatment of cultured RGCs significantly increased Bax protein level and decreased Bcl-2 protein level, which was also mimicked by BzATP and partially blocked by BBG, respectively. These results suggest that reactivated Müller cells may release excessive ATP, in turn leading to RGC apoptosis through activating P2X 7 receptors in these cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dendritic cells' death induced by contact sensitizers is controlled by Nrf2 and depends on glutathione levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

El Ali, Zeina

Dendritic cells (DC) are known to play a major role during contact allergy induced by contact sensitizers (CS). Our previous studies showed that Nrf2 was induced in DC and controlled allergic skin inflammation in mice in response to chemicals. In this work, we raised the question of the role of Nrf2 in response to a stress provoked by chemical sensitizers in DC. We used two well-described chemical sensitizers, dinitrochlorobenzene (DNCB) and cinnamaldehyde (CinA), known to have different chemical reactivity and mechanism of action. First, we performed a RT-qPCR array showing that CinA was a higher inducer of immune and detoxificationmore » genes compared to DNCB. Interestingly, in the absence of Nrf2, gene expression was dramatically affected in response to DNCB but was slightly affected in response to CinA. These observations prompted us to study DC's cell death in response to both chemicals. DNCB and CinA increased apoptotic cells and decreased living cells in the absence of Nrf2. The characterization of DC apoptosis induced by both CS involved the mitochondrial-dependent caspase pathway and was regulated via Nrf2 in response to both chemicals. Oxidative stress induced by DNCB, and leading to cell death, was regulated by Nrf2. Unlike CinA, DNCB treatment provoked a significant reduction of intracellular GSH levels and up-regulated bcl-2 gene expression, under the control of Nrf2. This work underlies that chemical reactivity may control Nrf2-dependent gene expression leading to different cytoprotective mechanisms in DC. - Highlights: • Nrf2 controls cell death induced by contact sensitizers in dendritic cells. • DNCB reduced GSH levels and up-regulated bcl-2 gene expression unlike CinA. • Chemical reactivity controls Nrf2-dependent genes having protective effect in DC.« less

Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells

PubMed Central

Ogasawara, Takashi; Kohashi, Yuko; Ikari, Jun; Taniguchi, Toshibumi; Tsuruoka, Nobuhide; Watanabe-Takano, Haruko; Fujimura, Lisa; Sakamoto, Akemi; Hatano, Masahiko; Hirata, Hirokuni; Fukushima, Yasutsugu; Fukuda, Takeshi; Kurasawa, Kazuhiro; Tatsumi, Koichiro; Tokuhisa, Takeshi; Arima, Masafumi

2018-01-01

Transcriptional repressor B-cell lymphoma 6 (Bcl6) appears to regulate TH2 immune responses in allergies, but its precise role is unclear. We previously reported that Bcl6 suppressed IL-4 production in naïve CD4+ T cell-derived memory TH2 cells. To investigate Bcl6 function in allergic responses in naturally occurring memory phenotype CD4+ T (MPT) cells and their derived TH2 (MPTH2) cells, Bcl6-manipulated mice, highly conserved intron enhancer (hcIE)-deficient mice, and reporter mice for conserved noncoding sequence 2 (CNS2) 3′ distal enhancer region were used to elucidate Bcl6 function in MPT cells. The molecular mechanisms of Bcl6-mediated TH2 cytokine gene regulation were elucidated using cellular and molecular approaches. Bcl6 function in MPT cells was determined using adoptive transfer to naïve mice, which were assessed for allergic airway inflammation. Bcl6 suppressed IL-4 production in MPT and MPTH2 cells by suppressing CNS2 enhancer activity. Bcl6 downregulated Il4 expression in MPTH2 cells, but not MPT cells, by suppressing hcIE activity. The inhibitory functions of Bcl6 in MPT and MPTH2 cells attenuated allergic responses. Bcl6 is a critical regulator of IL-4 production by MPT and MPTH2 cells in TH2 immune responses related to the pathogenesis of allergies. PMID:29696026

CCNG2 Overexpression Mediated by AKT Inhibits Tumor Cell Proliferation in Human Astrocytoma Cells.

PubMed

Zhang, Danfeng; Wang, Chunhui; Li, Zhenxing; Li, Yiming; Dai, Dawei; Han, Kaiwei; Lv, Liquan; Lu, Yicheng; Hou, Lijun; Wang, Junyu

2018-01-01

The cyclin family protein CCNG2 has an important inhibitory role in cancer initiation and progression, but the exact mechanism is still unknown. In this study, we examined the relationship between CCNG2 and the malignancy of astrocytomas and whether the AKT pathway, which is upregulated in astrocytomas, may inhibit CCNG2 expression. CCNG2 expression was found to be negatively associated with the pathological grade and proliferative activity of astrocytomas, as the highest expression was found in control brain tissue ( N  = 31), whereas the lowest expression was in high-grade glioma tissue ( N  = 31). Additionally, CCNG2 overexpression in glioma cell lines, T98G and U251 inhibited proliferation and arrested cells in the G0/G1 phase. Moreover, CCNG2 overexpression could increase glioma cells apoptosis. In contrast, AKT activity increased in glioma cells that had low CCNG2 expression. Expression of CCNG2 was higher in cells treated with the AKT kinase inhibitor MK-2206 indicating that the presence of phosphorylated AKT may inhibit the expression of CCNG2. Inhibition of AKT also led to decreased colony formation in T98G and U251 cells and knocked down of CCNG2 reversed the result. Finally, overexpression of CCNG2 in glioma cells reduced tumor volume in a murine model. To conclude, low expression of CCNG2 correlated with the severity astrocytoma and CCNG2 overexpression could induce apoptosis and inhibit proliferation. Inhibition of AKT activity increased the expression of CCNG2. The present study highlights the regulatory consequences of CCNG2 expression and AKT activity in astrocytoma tumorigenesis and the potential use of CCNG2 in anticancer treatment.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response

PubMed Central

Feinerman, Ofer; Jentsch, Garrit; Tkach, Karen E; Coward, Jesse W; Hathorn, Matthew M; Sneddon, Michael W; Emonet, Thierry; Smith, Kendall A; Altan-Bonnet, Grégoire

2010-01-01

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL-2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression. PMID:21119631

Bcl-2 protects tubular epithelial cells from ischemia/reperfusion injury by dual mechanisms.

PubMed

Isaka, Y; Suzuki, C; Abe, T; Okumi, M; Ichimaru, N; Imamura, R; Kakuta, Y; Matsui, I; Takabatake, Y; Rakugi, H; Shimizu, S; Takahara, S

2009-01-01

Ischemia/reperfusion (I/R) injury, which induces extensive loss of tubular epithelial cells, is associated with delayed graft function following kidney transplantation. Recent reports have suggested that cell death by I/R injury occurs by autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, as well as by apoptosis. Recently, we demonstrated that overexpression of the anti-apoptotic factor, Bcl-2, inhibited tubular apoptosis and subsequent tubulointerstitial damage after I/R injury. Autophagy is also observed in cells undergoing cell death in several diseases. Therefore, we hypothesized that increased Bcl-2 protein may protect tubular epithelial cells by suppressing autophagy and inhibiting apoptosis. In the present study, a transgenic mouse model (LC3-GFP TG) in which autophagosomes are labeled with LC3-GFP and Bcl-2/LC3-GFP double transgenic mice (Bcl-2/LC3-GFP TG) were used to examine the effect of Bcl-2 on I/R-induced autophagy. I/R injury, which is associated with marked disruption of normal tubular morphology, promoted the formation of LC3-GFP dots, representing extensively induced autophagosomes. On electron microscopy, the autophagosomes contained mitochondria in I/R-injured tubular epithelial cells. In contrast, Bcl-2 augmentation suppressed the formation of autophagosomes and there was less tubular damage. In conclusion, Bcl-2 augmentation protected renal tubular epithelial cells from I/R injury by suppressing autophagosomal degradation and inhibiting tubular apoptosis.

Hyperoside protects human kidney‑2 cells against oxidative damage induced by oxalic acid.

PubMed

Chen, Yongliang; Ye, Lihong; Li, Wangjian; Li, Dongzhang; Li, Feng

2018-05-02

The majority of renal calculi (kidney stones) are calcium stones. Oxidative damage to renal tubular epithelial cells induced by reactive oxygen species (ROS) is the predominant cause of calcium oxalate stone formation. Hyperoside (Hyp) is a flavonol glycoside extracted from medicinal plants and appears to exhibit potent antioxidant activity in various cells. The aim of the present study was to investigate the protective effect of Hyp on renal cells exposed to oxidative stress simulated by oxalic acid (OA), and to determine whether the underlying mechanism involves the nuclear factor E2‑related factor2 (Nrf2)‑antioxidative response element signaling pathway. The study determined the indicators of high oxidative stress, including ROS and hydrogen peroxide (H2O2) in human kidney‑2 cells and the results demonstrated that the levels of ROS, as evaluated by flow cytometry, and H2O2 were significantly increased following treatment with OA (5 mmol/l) for 24 h (OA group), compared with those in the untreated control group. The increased activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in these cells explained this observation, as it is a major source of ROS. The results demonstrated that, in the OA group, the adhesion of calcium oxalate crystals and lactate dehydrogenase (LDH) were significantly increased, and MTT assay demonstrated that cell viability was inhibited, compared with the control, which indicated that severe injury of cells was induced by OA. However, when the cells were pre‑treated with Hyp prior to treatment with OA (drug group), the levels of ROS and H2O2, and the activities of NADPH oxidase and LD were increased, and the adhesion of calcium oxalate crystals to cells was reduced, compared with the OA group. Western blot analysis and reverse transcription‑quantitative polymerase chain reaction demonstrated that the protein and mRNA expression levels of Nrf2, heme oxygenase‑1 (HO‑1) and NAD(P)H: quinineoxidoreductase 1

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

PubMed

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-12-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Regulation of Kv2.1 K+ conductance by cell surface channel density

PubMed Central

Fox, Philip D.; Loftus, Rob J.; Tamkun, Michael M.

2013-01-01

The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues. PMID:23325261

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

PubMed Central

Danson, Christopher M.; Pocha, Shirin M.; Bloomberg, Graham B.; Cory, Giles O.

2009-01-01

Summary The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration. PMID:18032787

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity.

PubMed

Danson, Christopher M; Pocha, Shirin M; Bloomberg, Graham B; Cory, Giles O

2007-12-01

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Bone regeneration by implantation of adipose-derived stromal cells expressing BMP-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li Huiwu; Health and Science Center, SIBS CAS and SSMU, 225 South Chongqing Road, Shanghai 200025; Dai Kerong

2007-05-18

In this study, we reported that the adipose-derived stromal cells (ADSCs) genetically modified by bone morphogenetic protein 2 (BMP-2) healed critical-sized canine ulnar bone defects. First, the osteogenic and adipogenic differentiation potential of the ADSCs derived from canine adipose tissue were demonstrated. And then the cells were modified by the BMP-2 gene and the expression and bone-induction ability of BMP-2 were identified. Finally, the cells modified by BMP-2 gene were applied to a {beta}-tricalcium phosphate (TCP) carrier and implanted into ulnar bone defects in the canine model. After 16 weeks, radiographic, histological, and histomorphometry analysis showed that ADSCs modified bymore » BMP-2 gene produced a significant increase of newly formed bone area and healed or partly healed all of the bone defects. We conclude that ADSCs modified by the BMP-2 gene can enhance the repair of critical-sized bone defects in large animals.« less

Elucidating the physiological and biochemical responses of different tobacco (Nicotiana tabacum) genotypes to lead toxicity.

PubMed

Maodzeka, Antony; Hussain, Nazim; Wei, Liquan; Zvobgo, Gerald; Mapodzeke, James Mutemachani; Adil, Muhammad Faheem; Jabeen, Salma; Wang, Feng; Jiang, Lixi; Shamsi, Imran Haider

2017-01-01

In the present study, the effects of lead (Pb) uptake and toxicity were investigated in a hydroponic culture using 7 tobacco (Nicotiana tabacum L.) genotypes (Bina 1 [B1], Kutsaga Mammoth 10 [KM10], Nanjing 3 [N3], Kutsaga 35 [K35], Kutsaga E1 [KE1], Cocker 176 [C176], and Kutsaga RK6 [KRK6]) that differed in Pb tolerance. Lead was applied as a solution of Pb nitrate at concentrations of 0 μM, 10 μM, 250 μM, and 500 μM. After 4 wk of Pb treatment, tissue biomass and photosynthetic parameters were measured and elemental analysis was performed. The results showed decreases in growth and photosynthetic parameters with increases in Pb concentration compared with the control. The least reduction in the recorded physiological parameters was noted in K35, whereas the greatest reduction was observed in N3, which is an obvious indication of genotypic differences. Activities of peroxidase, catalase, and malondialdehyde increased significantly with increases in Pb concentration, with genotypes K35 and N3 showing the least and the greatest reduction, respectively. The results demonstrate the phototoxic nature of Pb on plants, and it can be concluded that in Pb-prone areas genotypes K35 and B1 can be used for cultivation because they can grow efficiently in the presence of high Pb concentrations while restricting Pb uptake in the aboveground parts, as seen by the higher Pb tolerance index. Environ Toxicol Chem 2017;36:175-181. © 2016 SETAC. © 2016 SETAC.

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells

PubMed Central

Monetti, Emanuela; Kadono, Takashi; Bouteau, François

2014-01-01

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca2+ concentration ([Ca2+]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·–) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·– generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca2+]cyt increase and singlet oxygen production, do not seem to be involved in PCD. PMID:24420571

Cytokinin-induced cell death is associated with elevated expression of alternative oxidase in tobacco BY-2 cells.

PubMed

Mlejnek, Petr

2013-10-01

N(6)-benzyladenine (BA) and N(6)-benzyladenosine ([9R]BA) induce massive production of reactive oxygen species (ROS) that is eventually followed by a loss of cell viability in tobacco BY-2 cells (Mlejnek et al. Plant Cell Environ 26:1723-1735, 2003, Plant Sci 168:389-395, 2005). Results presented in this work suggest that the main sources of ROS are likely mitochondria and that the maintenance of the mitochondrial transmembrane potential is crucial for ROS production in cytokinin-treaded BY-2 cells. Therefore, the possible involvement of alternative oxidase (AOX) in cell death process induced by BA and [9R]BA was studied. About three- to fourfold increase in mRNA levels of AOX1 was observed a few hours after the BA and [9R]BA addition into the growth medium. The elevated expression of AOX1 mRNA could be prevented by adding adenine and adenosine which simultaneously reduced the cytotoxic effects of BA and [9R]BA, respectively. N(6)-benzyladenine 7-β-D-glucoside ([7G]BA) which is a common non-toxic metabolite of BA and [9R]BA did not affect the AOX1 mRNA expression. Although AOX1 seemed to be involved in protection of BY-2 cells against the abiotic stress induced by BA and [9R]BA, the results do not support the idea that it protects cells from death exclusively by scavenging of reactive oxygen species. Indeed, N-propyl gallate, an inhibitor of AOX, decreased cell survival despite it concomitantly decreased the ROS production. This finding is in contrast to the effect of salicylhydroxamic acid, another well-known inhibitor of AOX, which also increased the number of dying cells while it increased the ROS production.

Up-regulation of K{sub ir}2.1 by ER stress facilitates cell death of brain capillary endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kito, Hiroaki; Yamazaki, Daiju; Department of Biological Chemistry, Kyoto University, Graduate School of Pharmaceutical Sciences, Kyoto

Highlights: {yields} We found that application of endoplasmic reticulum (ER) stress with tunicamycin to brain capillary endothelial cells (BCECs) induced cell death. {yields} The ER stress facilitated the expression of inward rectifier K{sup +} channel (K{sub ir}2.1) and induced sustained membrane hyperpolarization. {yields} The membrane hyperpolarization induced sustained Ca{sup 2+} entry through voltage-independent nonspecific cation channels and consequently facilitated cell death. {yields} The K{sub ir}2.1 up-regulation by ER stress is, at least in part, responsible for cell death of BCECs under pathological conditions. -- Abstract: Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cellmore » turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K{sup +} channel (K{sub ir}2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K{sub ir} channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca{sup 2+} concentration due to Ca{sup 2+} influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K{sub ir}2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.« less

Melatonin partially protects 661W cells from H2O2-induced death by inhibiting Fas/FasL-caspase-3.

PubMed

Sánchez-Bretaño, Aída; Baba, Kenkichi; Janjua, Uzair; Piano, Ilaria; Gargini, Claudia; Tosini, Gianluca

2017-01-01

Previous studies have shown that melatonin (MEL) signaling is involved in the modulation of photoreceptor viability during aging. Recent work by our laboratory suggested that MEL may protect cones by modulating the Fas/FasL-caspase-3 pathway. In this study, we first investigated the presence of MEL receptors (MT 1 and MT 2 ) in 661W cells, then whether MEL can prevent H 2 O 2 -induced cell death, and last, through which pathway MEL confers protection. The mRNA and proteins of the MEL receptors were detected with quantitative PCR (q-PCR) and immunocytochemistry, respectively. To test the protective effect of MEL, 661W cells were treated with H 2 O 2 for 2 h in the presence or absence of MEL, a MEL agonist, and an antagonist. To study the pathways involved in H 2 O 2 -mediated cell death, a Fas/FasL antagonist was used before the exposure to H 2 O 2 . Finally, Fas/FasL and caspase-3 mRNA was analyzed with q-PCR and immunocytochemistry in cells treated with H 2 O 2 and/or MEL. Cell viability was analyzed by using Trypan Blue. Both MEL receptors (MT 1 and MT 2 ) were detected at the mRNA and protein levels in 661W cells. MEL partially prevented H 2 O 2 -mediated cell death (20-25%). This effect was replicated with IIK7 (a melatonin receptor agonist) when used at a concentration of 1 µM. Preincubation with luzindole (a melatonin receptor antagonist) blocked MEL protection. Kp7-6, an antagonist of Fas/FasL, blocked cell death caused by H 2 O 2 similarly to what was observed for MEL. Fas, FasL, and caspase-3 expression was increased in cells treated with H 2 O 2 , and this effect was prevented by MEL. Finally, MEL treatment partially prevented the activation of caspase-3 caused by H 2 O 2 . The results demonstrate that MEL receptors are present and functional in 661W cells. MEL can prevent photoreceptor cell death induced by H 2 O 2 via the inhibition of the proapoptotic pathway Fas/FasL-caspase-3.

Two aspartate residues at the putative p10 subunit of a type II metacaspase from Nicotiana tabacum L. may contribute to the substrate-binding pocket.

PubMed

Acosta-Maspons, Alexis; Sepúlveda-García, Edgar; Sánchez-Baldoquín, Laura; Marrero-Gutiérrez, Junier; Pons, Tirso; Rocha-Sosa, Mario; González, Lien

2014-01-01

Metacaspases are cysteine proteases present in plants, fungi, prokaryotes, and early branching eukaryotes, although a detailed description of their cellular function remains unclear. Currently, three-dimensional (3D) structures are only available for two metacaspases: Trypanosoma brucei (MCA2) and Saccharomyces cerevisiae (Yca1). Furthermore, metacaspases diverged from animal caspases of known structure, which limits straightforward homology-based interpretation of functional data. We report for the first time the identification and initial characterization of a metacaspase of Nicotiana tabacum L., NtMC1. By combining domain search, multiple sequence alignment (MSA), and protein fold-recognition studies, we provide compelling evidences that NtMC1 is a plant metacaspase type II, and predict its 3D structure using the crystal structure of two type I metacaspases (MCA2 and Yca1) and Gsu0716 protein from Geobacter sulfurreducens as template. Analysis of the predicted 3D structure allows us to propose Asp353, at the putative p10 subunit, as a new member of the aspartic acid triad that coordinates the P1 arginine/lysine residue of the substrate. Nevertheless, site-directed mutagenesis and expression analysis in bacteria and Nicotiana benthamiana indicate the functionality of both Asp348 and Asp353. Through the co-expression of mutant and wild-type proteins by transient expression in N. benthamiana leaves we found that polypeptide processing seems to be intramolecular. Our results provide the first evidence in plant metacaspases concerning the functionality of the putative p10 subunit.

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

PubMed

Kim, Hyung-Sik; Shin, Tae-Hoon; Lee, Byung-Chul; Yu, Kyung-Rok; Seo, Yoojin; Lee, Seunghee; Seo, Min-Soo; Hong, In-Sun; Choi, Soon Won; Seo, Kwang-Won; Núñez, Gabriel; Park, Jong-Hwan; Kang, Kyung-Sun

2013-12-01

Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2. Copyright © 2013 AGA

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana).

PubMed

Jones, A Maxwell P; Chattopadhyay, Abhishek; Shukla, Mukund; Zoń, Jerzy; Saxena, Praveen K

2012-05-30

Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

PubMed Central

2012-01-01

Background Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. Results This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. Conclusions This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable

Protection against Chlamydia psittaci in mice conferred by Lyt-2+ T cells.

PubMed Central

Buzoni-Gatel, D; Guilloteau, L; Bernard, F; Bernard, S; Chardès, T; Rocca, A

1992-01-01

A murine model was used to study the respective roles of L3T4+ and Lyt-2+ T cells in protection against Chlamydia psittaci. Donor mice were intravenously (i.v.) infected with 1 x 10(5) plaque-forming units (PFU) per mice of live C. psittaci. One month after inoculation, splenic cells from donors were transferred into syngenic recipients (5 x 10(7) cells/mouse). As measured by splenic colonization on Day 6 after i.v. challenge (1 x 10(5) PFU/mouse), transfer with primed (untreated) cells conferred a 3 log protection in this model. In vitro treatment, before transfer, of splenic cells with anti-Lyt-2 monoclonal antibody (mAb) and complement, markedly impaired the protection in comparison with control mice transferred with primed untreated cells, whereas treatment with anti-L3T4 mAb did not reduce the transferred protection. Resistance to a reinfection with C. psittaci was also studied after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. One month after primary infection, mice were treated with anti-L3T4 or anti-Lyt-2 mAb and challenged thereafter (i.v., 1 x 10(5) PFU). The splenic colonization on Day 6 after challenge demonstrated that treatment with anti-Lyt-2 mAb impaired resistance against a subsequent infection with C. psittaci. Treatment with anti-L3T4 mAb in vivo had no effect on protection, as previously described in vitro. The mechanisms by which Lyt-2+ T cells could participate in the elimination of bacteria were discussed. PMID:1427980

Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

PubMed

Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F; Moran, Nava

2009-02-01

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oue, Erika; Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Global Center of Excellence

Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cellmore » lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is

Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE.

PubMed

Lai, Shan-Shan; Xue, Bin; Yang, Yang; Zhao, Li; Chu, Chao-Shun; Hao, Jia-Yin; Wen, Chuan-Jun

2012-11-01

The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE. Copyright © 2012. Published by Elsevier Inc.

Inhibition of T Helper Cell Type 2 Cell Differentiation and Immunoglobulin E Response by Ligand-Activated Vα14 Natural Killer T Cells

PubMed Central

Cui, Junqing; Watanabe, Naohiro; Kawano, Tetsu; Yamashita, Masakatsu; Kamata, Tohru; Shimizu, Chiori; Kimura, Motoko; Shimizu, Eiko; Koike, Jyunzo; Koseki, Haruhiko; Tanaka, Yujiro; Taniguchi, Masaru; Nakayama, Toshinori

1999-01-01

Murine Vα14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Vα14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-γ upon in vivo stimulation with a specific ligand, α-galactosylceramide (α-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Vα14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Vα14 NKT-deficient mice that Vα14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with α-GalCer, Vα14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Vα14 NKT– or IFN-γ–deficient mice. Consistent with these results, we also found that ligand-activated Vα14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Vα14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-γ. In marked contrast, our studies have revealed that IL-4 produced by Vα14 NKT cells has only a minor effect on Th2 cell differentiation. PMID:10499917

Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

PubMed Central

Greenberg, Milton L; Jairaman, Amit; Akunwafo, Chijioke; Leverrier, Sabrina; Yu, Ying; Parker, Ian; Dynes, Joseph L

2017-01-01

Ca2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here, we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration ‘sparkles’ and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance. PMID:29239723

Isoprene emission protects photosynthesis but reduces plant productivity during drought in transgenic tobacco (Nicotiana tabacum) plants.

PubMed

Ryan, Annette C; Hewitt, C Nicholas; Possell, Malcolm; Vickers, Claudia E; Purnell, Anna; Mullineaux, Philip M; Davies, William J; Dodd, Ian C

2014-01-01

Isoprene protects the photosynthetic apparatus of isoprene-emitting plants from oxidative stress. The role of isoprene in the response of plants to drought is less clear. Water was withheld from transgenic isoprene-emitting and non-emitting tobacco (Nicotiana tabacum) plants, to examine: the response of isoprene emission to plant water deficit; a possible relationship between concentrations of the drought-induced phytohormone abscisic acid (ABA) and isoprene; and whether isoprene affected foliar reactive oxygen species (ROS) and lipid peroxidation levels. Isoprene emission did not affect whole-plant water use, foliar ABA concentration or leaf water potential under water deficit. Compared with well-watered controls, droughted non-emitting plants significantly increased ROS content (31-46%) and lipid peroxidation (30-47%), concomitant with decreased operating and maximum efficiencies of photosystem II photochemistry and lower leaf and whole-plant water use efficiency (WUE). Droughted isoprene-emitting plants showed no increase in ROS content or lipid peroxidation relative to well-watered controls, despite isoprene emission decreasing before leaf wilting. Although isoprene emission protected the photosynthetic apparatus and enhanced leaf and whole-plant WUE, non-emitting plants had 8-24% more biomass under drought, implying that isoprene emission incurred a yield penalty. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nedelkoska, T.V.; Doran, P.M.

Hairy roots were used to investigate cadmium uptake by Thlaspi caerulescens, a metal hyperaccumulator plant with potential applications in phytoremediation and phytomining. Experiments were carried out in nutrient media under conditions supporting root growth. Accumulation of Cd in short-term (9-h) experiments varied with initial medium pH and increased after treating the roots with H{sup +}-ATPase inhibitor. The highest equilibrium Cd content measured in T. caerulescens roots was 62,800 {micro}g g{sup {minus}1} dry weight, or 6.3% dry weight, at a liquid Cd concentration of 3,710 ppm. Cd levels in live T. caerulescens roots were 1.5- to 1.7-fold those in hairy rootsmore » of nonhyperaccumulator species exposed to the same Cd concentration, but similar to the Cd content of auto-claved T. caerulescens roots. The ability to grow at Cd concentrations of up to 100 ppm clearly distinguished T. caerulescens hairy roots from the nonhyperaccumulators. The specific growth rate of T. caerulescens roots was essentially unaffected by 20 to 50 ppm Cd in the culture medium; in contrast, N. tabacum roots turned dark brown at 20 ppm and growth was negligible. Up to 10,600 {micro}g g{sup {minus}1} dry weight Cd was accumulated by growing T. caerulescens hairy roots. Measurement of Cd levels in while roots and in the cell wall fraction revealed significant differences in the responses of T. caerulescens and N. tabacum roots to 20 ppm Cd. Most metal was transported directly into the symplasm of N. tabacum roots within 3 days of exposure; in contrast, T. caerulescens roots stored virtually all of their Cd in the wall fraction for the first 7 to 10 days. This delay in transmembrane uptake may represent an important defensive strategy against Cd poisoning in T. caerulescens, allowing time for activation of intracellular mechanisms for heavy metal detoxification.« less

Pest and disease resistance enhanced by heterologous suppression of a Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2.

PubMed

Smigocki, Ann C; Wilson, Dennis

2004-12-01

The functional role of the Nicotiana plumbaginifolia cytochrome P450 gene CYP72A2 was investigated in transgenic plants. N. tabacum plants transformed with a sense or antisense CYP72A2 construct exhibited diminished heights, branched stems, smaller leaves and deformed flowers. Western blot analysis revealed reduced levels of a 58 kDa protein corresponding to CYP72A2, suggesting that the CYP72A2 homolog was suppressed in the sense and antisense plants. Transgenic plants had increased resistance to Manduca sexta larvae that consumed about 35 to 90 less of transgenic versus control leaves. A virulent strain of Pseudomonas syringae pv. tabaci induced a disease-limiting response followed by a delayed and decreased development of disease symptoms in the transgenics. CYP72A2 gene mediated resistance suggests that the plant-pest or -pathogen interactions may have been modified by changes in bioactive metabolite pools.

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

PubMed Central

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

Tissue-autonomous promotion of palisade cell development by phototropin 2 in Arabidopsis.

PubMed

Kozuka, Toshiaki; Kong, Sam-Geun; Doi, Michio; Shimazaki, Ken-ichiro; Nagatani, Akira

2011-10-01

Light is an important environmental information source that plants use to modify their growth and development. Palisade parenchyma cells in leaves develop cylindrical shapes in response to blue light; however, the photosensory mechanism for this response has not been elucidated. In this study, we analyzed the palisade cell response in phototropin-deficient mutants. First, we found that two different light-sensing mechanisms contributed to the response in different proportions depending on the light intensity. One response observed under lower intensities of blue light was mediated exclusively by a blue light photoreceptor, phototropin 2 (PHOT2). Another response was elicited under higher intensities of light in a phototropin-independent manner. To determine the tissue in which PHOT2 perceives the light stimulus to regulate the response, green fluorescent protein (GFP)-tagged PHOT2 (P2G) was expressed under the control of tissue-specific promoters in the phot1 phot2 mutant background. The results revealed that the expression of P2G in the mesophyll, but not in the epidermis, promoted palisade cell development. Furthermore, a constitutively active C-terminal kinase fragment of PHOT2 fused to GFP (P2CG) promoted the development of cylindrical palisade cells in the proper direction without the directional cue provided by light. Hence, in response to blue light, PHOT2 promotes the development of cylindrical palisade cells along a predetermined axis in a tissue-autonomous manner.

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

PubMed

Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

2012-04-01

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the

Modulation of Human Leukocyte Antigen-C by Human Cytomegalovirus Stimulates KIR2DS1 Recognition by Natural Killer Cells

PubMed Central

van der Ploeg, Kattria; Chang, Chiwen; Ivarsson, Martin A.; Moffett, Ashley; Wills, Mark R.; Trowsdale, John

2017-01-01

The interaction of inhibitory killer cell Ig-like receptors (KIRs) with human leukocyte antigen (HLA) class I molecules has been characterized in detail. By contrast, activating members of the KIR family, although closely related to inhibitory KIRs, appear to interact weakly, if at all, with HLA class I. KIR2DS1 is the best studied activating KIR and it interacts with C2 group HLA-C (C2-HLA-C) in some assays, but not as strongly as KIR2DL1. We used a mouse 2B4 cell reporter system, which carries NFAT-green fluorescent protein with KIR2DS1 and a modified DAP12 adaptor protein. KIR2DS1 reporter cells were not activated upon coculture with 721.221 cells transfected with different HLA-C molecules, or with interferon-γ stimulated primary dermal fibroblasts. However, KIR2DS1 reporter cells and KIR2DS1+ primary natural killer (NK) cells were activated by C2-HLA-C homozygous human fetal foreskin fibroblasts (HFFFs) but only after infection with specific clones of a clinical strain of human cytomegalovirus (HCMV). Active viral gene expression was required for activation of both cell types. Primary NKG2A−KIR2DS1+ NK cell subsets degranulated after coculture with HCMV-infected HFFFs. The W6/32 antibody to HLA class I blocked the KIR2DS1 reporter cell interaction with its ligand on HCMV-infected HFFFs but did not block interaction with KIR2DL1. This implies a differential recognition of HLA-C by KIR2DL1 and KIR2DS1. The data suggest that modulation of HLA-C by HCMV is required for a potent KIR2DS1-mediated NK cell activation. PMID:28424684

Enhanced degradation of 2-nitrotoluene by immobilized cells of Micrococcus sp. strain SMN-1.

PubMed

Mulla, Sikandar I; Talwar, Manjunatha P; Bagewadi, Zabin K; Hoskeri, Robertcyril S; Ninnekar, Harichandra Z

2013-02-01

Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate-polyvinyl alcohol (SA-PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-nitrotoluene than freely suspended cells and the cells immobilized in SA-PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Transient Expression of Lumbrokinase (PI239) in Tobacco (Nicotiana tabacum) Using a Geminivirus-Based Single Replicon System Dissolves Fibrin and Blood Clots.

PubMed

Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju

2017-01-01

Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.

COX-2 Promotes Migration and Invasion by the Side Population of Cancer Stem Cell-Like Hepatocellular Carcinoma Cells

PubMed Central

Guo, Zhe; Jiang, Jing-Hang; Zhang, Jun; Yang, Hao-Jie; Yang, Fu-Quan; Qi, Ya-Peng; Zhong, Yan-Ping; Su, Jie; Yang, Ri-Rong; Li, Le-Qun; Xiang, Bang-De

2015-01-01

Abstract Cancer stem cells (CSCs) are thought to be responsible for tumor relapse and metastasis due to their abilities to self-renew, differentiate, and give rise to new tumors. Cyclooxygenase-2 (COX-2) is highly expressed in several kinds of CSCs, and it helps promote stem cell renewal, proliferation, and radioresistance. Whether and how COX-2 contributes to CSC migration and invasion is unclear. In this study, COX-2 was overexpressed in the CSC-like side population (SP) of the human hepatocellular carcinoma (HCC) cell line HCCLM3. COX-2 overexpression significantly enhanced migration and invasion of SP cells, while reducing expression of metastasis-related proteins PDCD4 and PTEN. Treating SP cells with the selective COX-2 inhibitor celecoxib down-regulated COX-2 and caused a dose-dependent reduction in cell migration and invasion, which was associated with up-regulation of PDCD4 and PTEN. These results suggest that COX-2 exerts pro-metastatic effects on SP cells, and that these effects are mediated at least partly through regulation of PDCD4 and PTEN expression. These results further suggest that celecoxib may be a promising anti-metastatic agent to reduce migration and invasion by hepatic CSCs. PMID:26554780

miR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2

PubMed Central

Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

2016-01-01

Objective(s): Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). Materials and Methods: The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Results: Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. Conclusion: These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein. PMID:27746866

miR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2.

PubMed

Jin, Qiao; Li, Xiang Jun; Cao, Pei Guo

2016-08-01

Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein.

ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP.

PubMed

Lee, Wing-Kee; Chakraborty, Prabir K; Roussa, Eleni; Wolff, Natascha A; Thévenod, Frank

2012-10-01

Upon endoplasmic reticulum (ER) stress induction, cells endeavor to survive by engaging the adaptive stress response known as the unfolded protein response or by removing aggregated proteins via autophagy. Chronic ER stress culminates in apoptotic cell death, which involves induction of pro-apoptotic CHOP. Here, we show that bestrophin-3 (Best-3), a protein previously associated with Ca(2+)-activated Cl(-) channel activity, is upregulated by the ER stressors, thapsigargin (TG), tunicamycin (TUN) and the toxic metal Cd(2+). In cultured rat kidney proximal tubule cells, ER stress, CHOP and cell death were induced after 6h by Cd(2+) (25μM), TG (3μM) and TUN (6μM), were associated with increased cytosolic Ca(2+) and downstream formation of reactive oxygen species and attenuated by the Ca(2+) chelator BAPTA-AM (10μM), the antioxidant α-tocopherol (100μM), or overexpression of catalase (CAT). Immunofluorescence staining showed Best-3 expression in the plasma membrane, nuclei and intracellular compartments, though not in the ER, in cultured cells and rat kidney cortex sections. Best-3 mRNA was augmented by ER stress and signaled through increased Ca(2+), oxidative stress and ERK1/2 phosphorylation, because it was attenuated by α-tocopherol, CAT expression, BAPTA-AM, calmodulin kinase inhibitor calmidazolium (40μM), ERK1/2 inhibitor U0126 (10μM), and ERK1/2 RNAi. Knockdown of Best-3 resulted in decreased cell number consequentially of cell death, as determined by nuclear staining and PARP-1 cleavage. Furthermore, reduced ER stress-cell death by Best-3 overexpression is attributed to diminished CHOP. Since Best-3 overexpression did not affect upstream signaling pathways, we hypothesize that Best-3 possibly interferes with CHOP transcription. From our novel observations, we conclude that ERK1/2-dependent Best-3 activation regulates cell fate decisions during ER stress by suppressing CHOP induction and death. Copyright © 2012 Elsevier B.V. All rights reserved.

Ca(2+) signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca(2+) channel blockade.

PubMed

Gryshchenko, Oleksiy; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

2016-01-15

Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions. In this study, we have investigated bradykinin-elicited Ca(2+) signal generation in normal mouse pancreatic lobules. We found complete separation of Ca(2+) signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca(2+) signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca(2+) signals in PACs, never elicited Ca(2+) signals in PSCs. The bradykinin-elicited Ca(2+) signals were due to initial Ca(2+) release from inositol trisphosphate-sensitive stores followed by Ca(2+) entry through Ca(2+) release-activated channels (CRACs). The Ca(2+) entry phase was effectively inhibited by a CRAC blocker. B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis-promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca(2+) signals. We have compared Ca(2+) signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca(2+) signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto-digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca(2+) signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca(2+) signals in PSCs. The BK-induced Ca(2+) signals were mediated by B2 receptors and B2

Adrenaline Stimulates Glucagon Secretion by Tpc2-Dependent Ca2+ Mobilization From Acidic Stores in Pancreatic α-Cells.

PubMed

Hamilton, Alexander; Zhang, Quan; Salehi, Albert; Willems, Mara; Knudsen, Jakob G; Ringgaard, Anna K; Chapman, Caroline E; Gonzalez-Alvarez, Alejandro; Surdo, Nicoletta C; Zaccolo, Manuela; Basco, Davide; Johnson, Paul R V; Ramracheya, Reshma; Rutter, Guy A; Galione, Antony; Rorsman, Patrik; Tarasov, Andrei I

2018-06-01

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca 2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca 2+ ] i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca 2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca 2+ ] i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca 2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca 2+ ] i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca 2+ ] i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca 2+ release from the acidic stores and further amplified by Ca 2+ -induced Ca 2+ release from the sarco/endoplasmic reticulum. © 2018 by the American Diabetes Association.

Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

PubMed Central

1996-01-01

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point. PMID:8642331

Induction of apoptosis and cell cycle arrest in L-1210 murine lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one.

PubMed

Pedrini, Fernanda Spezia; Chiaradia, Louise Domeneghini; Licínio, Marley Aparecida; de Moraes, Ana Carolina Rabello; Curta, Juliana Costa; Costa, Aline; Mascarello, Alessandra; Creczinsky-Pasa, Tânia Beatriz; Nunes, Ricardo José; Yunes, Rosendo Augusto; Santos-Silva, Maria Cláudia

2010-09-01

New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic leukemia cells L-1210. A series of ten chalcones derived from 2-naphtaldehyde and corresponding acetophenones were prepared by aldolic condensation, using methanol as solvent under basic conditions, at room temperature for 24 h. The cell viability was determined by MTT colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after propidium iodide staining. The apoptosis induction was assessed by exposure to phosphatidylserine (ANNEXIN V-FITC). Cytometric analysis was performed to evaluate the expression of p53, Bcl-2 and Bax protein. The caspase-3 expression was studied by immunoblotting analysis. A preliminary screening of a series of ten chalcones derived from 2-naphtaldehyde showed that chalcone 8, (2E)-3-(2-naphtyl)-1-(3'-methoxy-4'-hydroxy-phenyl)-2-propen-1-one, had the highest cytotoxic effect (IC50 of 54 microM), but not in normal human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8, its effect on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in Bax:Bcl-2 ratio and increased p53 expression and caspase-3 activation. The studied chalcone 8 has cytotoxic effect against L-1210 lymphoblastic leukaemic cells, and this effect is associated with increase of p-53 and Bax expression.

Effect of prostaglandin E2 on cytotoxic activity and granzyme A protease release by murine adherent IL-2 activated killer cells.

PubMed

Vaillier, D; Daculsi, R; Gualde, N

1994-04-01

The effects of prostaglandin E2 (PGE2) have been studied on a highly purified population of murine IL-2 activated killer cells obtained by selecting plastic-adherent splenocytes (AK cells) after incubation with high doses of recombinant IL-2. AK cells were highly cytotoxic for YAC-1 target cells. The cytotoxic activity was detectable at one hour after initiation of the cytotoxic assay and then increased with time. Cytotoxic activity of AK cells was inhibited by the addition of PGE2 or forskolin during the cytotoxic assay. When AK cells were generated in the presence of PGE2, the yielding cytotoxic activity was lower than the one expressed by "regular" AK cells but were insensitive to the inhibitory effect of PGE2 even if their lytic capability was still suppressed by forskolin. The presence of PGE2 during the AK cell culture had no effect on the cellular proliferation. Moreover, using tetrazolium-based colorimetric assay which reflects the cellular activation, it was observed that AK cells cultured in presence of PGE2 had an increased capacity to cleave the tetrazolium salt to formazan. Since the cytotoxic activity of killer cells is related to expression of serine esterase enzymes we evaluated the effects of PGE2 on serine esterase (Granzyme A) release after one hour of incubation of AK cells either alone or in presence of PGE2, YAC-1 cells or both. We observed that (i) AK cells spontaneously release granzyme A, (ii) the level of granzyme A was significantly increased when AK cells were incubated either with YAC-1 cells or PGE2 but did not change when YAC-1 cells and PGE2 were both associated with AK cells.

Analyzing Cell Wall Elasticity After Hormone Treatment: An Example Using Tobacco BY-2 Cells and Auxin.

PubMed

Braybrook, Siobhan A

2017-01-01

Atomic force microscopy, and related nano-indentation techniques, is a valuable tool for analyzing the elastic properties of plant cell walls as they relate to changes in cell wall chemistry, changes in development, and response to hormones. Within this chapter I will describe a method for analyzing the effect of the phytohormone auxin on the cell wall elasticity of tobacco BY-2 cells. This general method may be easily altered for different experimental systems and hormones of interest.

Sites and Regulation of Polyamine Catabolism in the Tobacco Plant. Correlations with Cell Division/Expansion, Cell Cycle Progression, and Vascular Development1

PubMed Central

Paschalidis, Konstantinos A.; Roubelakis-Angelakis, Kalliopi A.

2005-01-01

We previously gave a picture of the homeostatic characteristics of polyamine (PA) biosynthesis and conjugation in tobacco (Nicotiana tabacum) plant organs during development. In this work, we present the sites and regulation of PA catabolism related to cell division/expansion, cell cycle progression, and vascular development in the tobacco plant. Diamine oxidase (DAO), PA oxidase (PAO), peroxidases (POXs), and putrescine N-methyltransferase expressions follow temporally and spatially discrete patterns in shoot apical cells, leaves (apical, peripheral, and central regions), acropetal and basipetal petiole regions, internodes, and young and old roots in developing plants. DAO and PAO produce hydrogen peroxide, a plant signal molecule and substrate for POXs. Gene expression and immunohistochemistry analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with nascent nuclear DNA and also with POXs and lignification. In mature and old tissues, flow cytometry indicates that amine oxidase and POX activities, as well as pao gene and PAO protein levels, coincide with G2 nuclear phase and endoreduplication. In young versus the older roots, amine oxidases and POX expression decrease with parallel inhibition of G2 advance and endoreduplication, whereas putrescine N-methyltransferase dramatically increases. In both hypergeous and hypogeous tissues, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization. DNA synthesis early in development and the advance in cell cycle/endocycle are temporally and spatially related to PA catabolism and vascular development. PMID:16040649

Cell proliferation by silk gut incorporating FGF-2 protein microcrystals.

PubMed

Kotani, Eiji; Yamamoto, Naoto; Kobayashi, Isao; Uchino, Keiro; Muto, Sayaka; Ijiri, Hiroshi; Shimabukuro, Junji; Tamura, Toshiki; Sezutsu, Hideki; Mori, Hajime

2015-06-08

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential.

BIM mediates synergistic killing of B-cell acute lymphoblastic leukemia cells by BCL-2 and MEK inhibitors.

PubMed

Korfi, K; Smith, M; Swan, J; Somervaille, T C P; Dhomen, N; Marais, R

2016-04-07

B-cell acute lymphoblastic leukemia (B-ALL) is an aggressive hematological disease that kills ~50% of adult patients. With the exception of some BCR-ABL1(+) patients who benefit from tyrosine kinase inhibitors, there are no effective targeted therapies for adult B-ALL patients and chemotherapy remains first-line therapy despite adverse side effects and poor efficacy. We show that, although the MEK/ERK pathway is activated in B-ALL cells driven by different oncogenes, MEK inhibition does not suppress B-ALL cell growth. However, MEK inhibition synergized with BCL-2/BCL-XL family inhibitors to suppress proliferation and induce apoptosis in B-ALL cells. We show that this synergism is mediated by the pro-apoptotic factor BIM, which is dephosphorylated as a result of MEK inhibition, allowing it to bind to and neutralize MCL-1, thereby enhancing BCL-2/BCL-XL inhibitor-induced cell death. This cooperative effect is observed in B-ALL cells driven by a range of genetic abnormalities and therefore has significant therapeutic potential.

Nitric Oxide- and Hydrogen Peroxide-Responsive Gene Regulation during Cell Death Induction in Tobacco1[W

PubMed Central

Zago, Elisa; Morsa, Stijn; Dat, James F.; Alard, Philippe; Ferrarini, Alberto; Inzé, Dirk; Delledonne, Massimo; Van Breusegem, Frank

2006-01-01

Nitric oxide (NO) and hydrogen peroxide (H2O2) are regulatory molecules in various developmental processes and stress responses. Tobacco (Nicotiana tabacum) leaves exposed to moderate high light dramatically potentiated NO-mediated cell death in catalase-deficient (CAT1AS) but not in wild-type plants, providing genetic evidence for a partnership between NO and H2O2 during the induction of programmed cell death. With this experimental model system, the specific impact on gene expression was characterized by either NO or H2O2 alone or both molecules combined. By means of genome-wide cDNA-amplified fragment length polymorphism analysis, transcriptional changes were compared in high light-treated CAT1AS and wild-type leaves treated with or without the NO donor sodium nitroprusside. Differential gene expression was detected for 214 of the approximately 8,000 transcript fragments examined. For 108 fragments, sequence analysis revealed homology to genes with a role in signal transduction, defense response, hormone interplay, proteolysis, transport, and metabolism. Surprisingly, only 16 genes were specifically induced by the combined action of NO and H2O2, whereas the majority were regulated by either of them alone. At least seven transcription factors were mutually up-regulated, indicating significant overlap between NO and H2O2 signaling pathways. These results consolidate significant cross-talk between NO and H2O2, provide new insight into the early transcriptional response of plants to increased NO and H2O2 levels, and identify target genes of the combined action of NO and H2O2 during the induction of plant cell death. PMID:16603664

MicroRNA-138 Regulates DNA Damage Response in Small Cell Lung Cancer Cells by Directly Targeting H2AX.

PubMed

Yang, Huan; Luo, Jinwen; Liu, Zhiguang; Zhou, Rui; Luo, Hong

2015-04-01

Lung cancer is the leading cause of cancer death worldwide and small cell lung cancer (SCLC) accounts for a significant proportion of all lung cancer cases. Even so, the underlying mechanism governing SCLC development remains poorly understood and SCLC related cancer death stands high despite decades of intensive investigation. We noted that both miR-138 and H2AX have been implicated in development of various malignancies. Also, there is a recent report showing the role of miR-138 in mediating DNA damage response by targeting H2AX. In light of these data, we sought to characterize the role of miR-138 for SCLC cell growth and cell-cycle progression by regulating H2AX expression. Results showed that miR-138 is significantly down-regulated in SCLC tumor tissues as well as in three SCLC cell lines. After successfully engineering miR-138 overexpression in one of the SCLC cell lines, NCI-H2081, we observed a remarkable reduction of cell growth and a significant inhibition on cell-cycle progression. Moreover, we were able to show that miR-138 potently inhibits H2AX expression, which suggests that H2AX may serve as a downstream executor for miR-138. Consistent with this hypothesis, we found that engineered H2AX knockdown achieves a similar effect as observed for miR-138 overexpression in terms of SCLC growth and cell cycle regulation. We also showed that H2AX overexpression largely abolished miR-138-mediated SCLC cancer cell growth and cell-cycle progression inhibition, which strongly suggests, at least in vitro, that miR-138 potently regulates SCLC development by targeting H2AX. In addition, we found lower miR-138 expression confers SCLC cells with greater DNA damage repair capacity. Finally, we were able to show miR-138 overexpression inhibits DNA damage repair in SCLC cells while miR-138 knockdown further facilitates DNA damage repair in these cells after IR. To date, there has been no study showing the role of miR-138/H2AX machinery in SCLC development. Our results may

Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells