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Suspension Cells (suspension + cell)
Selected AbstractsPhosphate Induces Rapid H2O2 Generation in Soybean Suspension CellsPLANT BIOLOGY, Issue 2 2000T. Shigaki Abstract: Involvement of reactive oxygen species has been implicated in plant defence against pathogens. We report here a novel pathway of H2O2 generation induced by the addition of phosphate in soybean (Glycine max L.) cell suspension cultures. This H2O2 generation was initiated shortly after the addition of phosphate, and lasted only approximately one hour, as opposed to several hours observed during an attack by an avirulent strain of the bacterial pathogen Pseudomonas syringae pv. glycinea (Psg). In addition, when cell cultures were treated with both phosphate and the avirulent pathogen, two distinct oxidative burst events were observed. In contrast to DPI-sensitive Psg -induced H2O2 generation, phosphate-induced H2O2 generation was insensitive to this NADPH oxidase inhibitor. This suggests that an NADPH oxidase-independent pathway may be involved in the phosphate-induced H2O2 accumulation, which could be involved in sensing of phosphate availability in the environment. [source] Copper treatment activates mitogen-activated protein kinase signalling in ricePHYSIOLOGIA PLANTARUM, Issue 3 2003Chuan-Ming Yeh It is well known that mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been shown that MAPKs play a role in the signalling of biotic and abiotic stresses. To characterize signalling pathways involved in heavy metal-induced stress responses, we examine whether plant MAPKs are also involved in this process. The analyses of mRNA levels of OsMAPK genes have shown that only OsMAPK2 mRNA transcripts increased within 12 h upon CuCl2 treatment in suspension cells and roots. An in-gel kinase assay revealed that three protein kinases, approximate 42, 50, and 64-kDa, were activated by CuCl2 treatments. The approximate 42-kDa protein kinase displayed MAPK properties. Antioxidant, GSH, prevented copper-induced kinase activity. Furthermore, we found that rice roots underwent a rapid cell death upon this copper treatment. The copper-induced cell death of rice roots was partially blocked by MAPK kinase inhibitor, PD98059. These results suggest that the MAPK cascades may function in the plant heavy metal induced-signalling pathway. [source] An ABA-responsive bZIP protein, OsBZ8, mediates sugar repression of , -amylase gene expressionPHYSIOLOGIA PLANTARUM, Issue 1 2003Yi-Ching Lee Expression of some , -amylase genes in cereals is suppressed by sugars and activated by sugar starvation. A 100-bp sugar response sequence (SRS) identified in the promoter of a rice , -amylase gene, ,Amy3, contains three essential motifs: the GC box, the G box, and the TATCCA element. To study the mechanism of sugar regulation of ,Amy3 transcription, an ABA-responsive bZIP protein, OsBZ8, which binds specifically to the G box in ,Amy3 SRS was characterized and function analysed. In sucrose-starved rice suspension cells and embryos, decline in OsBZ8 mRNA levels coincided with the induction of ,Amy3 mRNA accumulation. In vivo gain- and loss-of-function studies by transient expression assays in rice embryos revealed that OsBZ8 suppresses SRS activity through the G box and overrides the activity of an activator, OsMYBS1, which binds to the TATCCA element. Gel mobility shift assays revealed that OsBZ8 binds specifically to the G box in vitro. These studies suggest that OsBZ8 is a suppressor responsible for sugar repression of ,Amy3 expression, and OsMYBS1 is responsible for sugar starvation induced expression of ,Amy3. [source] Growth and movement of secondary plasmodia of Plasmodiophora brassicae in turnip suspension-culture cellsPLANT PATHOLOGY, Issue 1 2006T. Asano Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae -infected turnip cells was achieved by using P. brassicae -infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L,1 and 0·02 mg kinetin L,1. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells. [source] Proteomic analysis of rice plasma membrane reveals proteins involved in early defense response to bacterial blightPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2007Fang Chen Abstract Plant plasma membrane (PM) proteins play important roles in signal transduction during defense response to an attacking pathogen. By using an improved method of PM protein preparation and PM-bound green fluorescent protein fusion protein as a visible marker, we conducted PM proteomic analysis of the rice suspension cells expressing the disease resistance gene Xa21, to identify PM components involved in the early defense response to bacterial blight (Xanthomonas oryzae pv. oryzae). A total of 20 regulated protein spots were observed on 2-D gels of PM fractions at 12 and 24,h after pathogen inoculation, of which some were differentially regulated between the incompatible and compatible interactions mediated by Xa21, with good correlation between biological repeats. Eleven protein spots with predicted functions in plant defense were identified by MS/MS, including nine putative PM-associated proteins H+ -ATPase, protein phosphatase, hypersensitive-induced response protein (OsHIR1), prohibitin (OsPHB2), zinc finger and C2 domain protein, universal stress protein (USP), and heat shock protein. OsHIR1 was modified by the microbal challenge, leading to two differentially accumulated protein spots. Transcript analysis showed that most of the genes were also regulated at transcriptional levels. Our study would provide a starting point for functionality of PM proteins in the rice defense. [source] Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cellsTHE PLANT JOURNAL, Issue 6 2009Sally L. Hanton Summary Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES. [source] Identification of a novel family of 70 kDa microtubule-associated proteins in Arabidopsis cellsTHE PLANT JOURNAL, Issue 4 2005Andrey V. Korolev Summary Most plant microtubule-associated proteins (MAPs) have homologues across the phylogenetic spectrum. To find potential plant-specific MAPs that will have evaded bioinformatic searches we devised a low stringency method for isolating proteins from an Arabidopsis cell suspension on endogenous taxol-microtubules. By tryptic peptide mass fingerprinting we identified 55 proteins that were enriched on taxol-microtubules. Amongst a range of known MAPs, such as kinesins, MAP65 isoforms and MOR1, we detected ,unknown' 70 kDa proteins that belong to a family of five closely related Arabidopsis proteins having no known homologues amongst non-plant organisms. To verify that AtMAP70-1 associates with microtubules in vivo, it was expressed as a GFP fusion. This confirmed that the protein decorates all four microtubule arrays in both transiently infected Arabidopsis and stably transformed tobacco BY-2 suspension cells. Microtubule-directed drugs perturbed the localization of AtMAP70-1 but cytochalasin D did not. AtMAP70-1 contains four predicted coiled-coil domains and truncation studies identified a central domain that targets the fusion protein to microtubules in vivo. This study therefore introduces a novel family of plant-specific proteins that interact with microtubules. [source] Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cellsTHE PLANT JOURNAL, Issue 2 2005Christine Zalejski Summary In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling. [source] Characterization and applications of serum-free induced adhesion in jurkat suspension cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2010Julianne F. Audiffred Abstract In this study, we demonstrate that the presence of serum in different media plays an important role in inducing transient and reversible adhesion in Jurkat suspension cells. Attachment of Jurkat cells in two distinct media formulations (serum-fortified and serum-free) to untreated polystyrene (PS), plasma-treated PS, and fibronectin-coated PS was compared. Additional analysis characterized the occurrence of this transient cell adhesion, including attachment rate, reversibility of attachment, and viability and preservation of phenotype in cells during and after attachment. As a demonstration of the utility of this technique, a few applications of transiently adhering Jurkat cells are shown which would be otherwise difficult with freely suspended cells, such as increased gene delivery, confocal-based apoptosis detection, and real-time electric-field effect monitoring in Jurkat cells. Biotechnol. Bioeng. 2010;106: 784,793. © 2010 Wiley Periodicals, Inc. [source] Effect of nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cellsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2005Maojun Xu Abstract Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells. The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells, but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C. roseus cells, but had no effect on catharanthine synthesis. The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control. NO-induced catharanthine production of C. roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent. © 2004 Wiley Periodicals, Inc. [source] Gene Therapy in HIV-Infected Cells to Decrease Viral Impact by Using an Alternative Delivery MethodCHEMMEDCHEM, Issue 6 2010Teresa Gonzalo Dr. Abstract The ability of dendrimer 2G-[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I,)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole-genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer-delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase-2 gene expression in HIV-infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non-viral vector for gene therapy against HIV infection. [source] | |||||||||||||