113 research outputs found
Caspase inhibitors affect the kinetics and dimensions of tracheary elements in xylogenic Zinnia (<it>Zinnia elegans</it>) cell cultures
No full textAbstract Background The xylem vascular system is composed of fused dead, hollow cells called tracheary elements (TEs) that originate through trans-differentiation of root and shoot cambium cells. TEs undergo autolysis as they differentiate and mature. The final stage of the formation of TEs in plants is the death of the involved cells, a process showing some similarities to programmed cell death (PCD) in animal systems. Plant proteases with functional similarity to proteases involved in mammalian apoptotic cell death (caspases) are suggested as an integral part of the core mechanism of most PCD responses in plants, but participation of plant caspase-like proteases in TE PCD has not yet been documented. Results Confocal microscopic images revealed the consecutive stages of TE formation in Zinnia cells during trans-differentiation. Application of the caspase inhibitors Z-Asp-CH2-DCB, Ac-YVAD-CMK and Ac-DEVD-CHO affected the kinetics of formation and the dimensions of the TEs resulting in a significant delay of TE formation, production of larger TEs and in elimination of the 'two-wave' pattern of TE production. DNA breakdown and appearance of TUNEL-positive nuclei was observed in xylogenic cultures and this was suppressed in the presence of caspase inhibitors. Conclusions To the best of our knowledge this is the first report showing that caspase inhibitors can modulate the process of trans-differentiation in Zinnia xylogenic cell cultures. As caspase inhibitors are closely associated with cell death inhibition in a variety of plant systems, this suggests that the altered TE formation results from suppression of PCD. The findings presented here are a first step towards the use of appropriate PCD signalling modulators or related molecular genetic strategies to improve the hydraulic properties of xylem vessels in favour of the quality and shelf life of plants or plant parts.</p
Cell proliferation, cell shape, and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space
No full textThe microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleto
Efficacy and safety of AEZS-108 (INN: Zoptarelin Doxorubicin Acetate) an LHRH agonist linked to doxorubicin in women with platinum refractory or resistant ovarian cancer expressing LHRH receptors: A multicenter Phase II trial of the ago-study group (AGO GYN 5)
No full textObjectives. To evaluate the activity and toxicity of AEZS-108 (Zoptarelin Doxorubicin Acetate) an LHRH agonist linked to doxorubicin in women with platinum refractory or resistant ovarian cancer expressing LHRH receptors. Methods. Women with epithelial ovarian, fallopian tube or primary peritoneal cancer, expressing LHRH receptors were eligible for this trial, when they had progression during treatment with a platinum based regimen or within 6 months after receiving a platinum based regimen and a previous taxane treatment At least one measurable target lesion (RECIST) or CA-125 levels higher than twice the upper limit of normal range (GCIG-criteria) were required. Patients received AEZS-108 (267 mg/m(2) equimolar to 76.8 mg/m(2) of free doxorubicin) every 3 weeks as a two hour i.v. infusion. Results. Fifty-five of 59 (93%) of ovarian cancer samples screened expressed LHRH receptors. 42 patients were enrolled in this study and received at least 1 infusion of AEZS-108 (ITT population). Of these 42 patients 6 (143%) had a partial response, 16 (38%) stable disease, 16 (38%) progressive disease and 4 patients were not evaluable. Median time to progression was 12 weeks (95% CI: 8-20 weeks), and median overall survival was 53 weeks (95% CI: 39-73 weeks). Toxicity profile was favorable. Conclusion. AEZS-108 has a clinical activity in platinum refractory/resistant ovarian cancer which, seems to be comparable to that of pegylated liposomal doxorubicin or to topotecan. Toxicity was comparably low. These data support the concept of a targeted chemotherapy for tumors expressing LHRH receptors. (C) 2014 Elsevier Inc. All rights reserved
Efficacy and safety of AEZS-108 (LHRH agonist linked to doxorubicin) in women with advanced or recurrent endometrial cancer expressing LHRH receptors : a multicenter phase 2 trial (AGO-GYN5)
No full textObjective: Advanced or recurrent endometrial cancer (EC) no longer amenable to surgery or radiotherapy is a life-threatening disease with limited therapeutic options left. Eighty percent of ECs express receptors for luteinizing hormone-releasing hormone (LHRH), which can be targeted by AEZS-108 (zoptarelin doxorubicin acetate). This phase 2 trial was performed to assess the efficacy and safety of AEZS-108 in this group of patients.
Methods: Patients had FIGO (Fédération Internationale de Gynécologie et d'Obstétrique) III or IV or recurrent EC, LHRH receptor-positive tumor status, and at least had 1 measurable lesion (Response Evaluation Criteria in Solid Tumors). Prior anthracycline therapy was not allowed. Patients received AEZS-108 as a 2-hour infusion on day 1 of a 21-day cycle. The treatment was continued for a maximum of 6 to 8 cycles. The primary end point was the response rate determined by the Response Evaluation Criteria in Solid Tumors.
Results: From April 2008 to November 2009, 44 patients were included in the study at 8 centers in Germany (AGO) and 3 centers in Bulgaria. Forty-three of these patients were eligible. Two (5%) patients had a complete remission, and 8 (18%) achieved a partial remission. Stable disease for at least 6 weeks was observed in 44%. The median time to progression was 7 months, and the median overall survival was 15 months. The most frequently reported grade 3 or 4 adverse effects were neutropenia (12%) and leucopenia (9%).
Conclusions: AEZS-108, an LHRH-agonist coupled to doxorubicin, has significant activity and low toxicity in women with advanced or recurrent LHRH receptor-positive EC, supporting the principle of receptor-mediated targeted chemotherapy
Accuracy Versus Falsification Costs: The Optimal Amount of Evidence under Different Procedures
No full textAn arbiter can decide a case on the basis of his priors or he can ask for further evidence from the two parties to the conflict. The parties may misrepresent evidence in their favor at a cost. The arbiter is concerned about accuracy and low procedural costs. When both parties testify, each of them distorts the evidence less than when they testify alone. When the fixed cost of testifying is low, the arbiter hears both, for intermediate values one, and for high values no party at all. The arbiter's ability to remain uninformed as well as sequential testifying makes it more likely that the arbiter requires evidence. (JEL D82, K41, K42) The Author 2007. Published by Oxford University Press on behalf of Yale University. All rights reserved. For permissions, please email: [email protected], Oxford University Press.
Microtubules in legume root hairs: cell polarity and response to rhizobial signal molecules
No full textMicrotubules, which occur as hollow protein tubes with a diameter of 25 nanometers, are an important compound of the cytoskeleton and occur in plant cells as a highly organized and dynamic array, which actual arrangement will depend on its tasks during the cell cycle. Microtubules play a key-role in the spatial distribution of the cytoplasm, cell growth, and cell division, and they respond to external or internal stimuli. In this thesis the microtubule cytoskeleton in developing Medicago truncatula root hairs is described. Cortical microtubules are present in all developmental stages, but the sub-apical cytoplasmic dense region of tip-growing root hairs contains a specific microtubule configuration that was not described for interphase cells in detail before. When tip growth begins, the hairs acquire an extensive endoplasmic microtubule array in their sub-apex, which they retain until growth stops. These endoplasmic microtubules may be specific for legume root hairs. They are essential in maintaining the highly polarized distribution of cytoplasm in growing root hairs, and in keeping the growth of these hairs at a high rate. Further, the endoplasmic microtubule cytoskeleton appears to be a target of early Nod factor signaling. Nod factors are rhizobacterial signal molecules, which cause several cellular responses in legume root hairs. The endoplasmic microtubule array in elongating hairs responds to Nod factors with a subtle and short termed shortening, whereas cortical microtubules are not obviously affected. The presented results demonstrate that the microtubule cytoskeleton contributes to sustaining tip-growth and to determining the growth direction of hairs after Nod factor application. The latter two features are crucial for root hair curling around rhizobia, which is one of the first steps in establishing symbiosis between legumes and rhizobia. A hypothesis how microtubules may determine the site of tip-growth and thereby the directionality of root hair growth is discussed
From signal to form: Nod factor as a morhogenetic signal molecule to induce symbiotic responses in legume root hairs
No full textIn this thesis, research is presented which contributes to a better understanding of nod factor (NF) induced signalling in Iegume root hairs, leading to a successful symbiosis. We mainly use root hairs of the model Iegume Medicago truncatula ('barrel medic') as an experimental system. In the different chapters, different aspects of the NF induced changes in root hair morphology that are required for establishing a successful symbiosis between rhizobia and legumes are covered.Chapter 1 is a review article that describes the different roles of the actin cytoskeleton in Iegume root hairs: its different configurations in relation to root hair growth, its function as backbone of cytoplasmic strands and highway for cellular transport, and its target for NF-induced signalling.Chapter 2 describes a new experimental assay to test the effects of NF on legume root hairs. The advantage of this assay, in comparison with the classical global application assays, is that it better mimics the natural situation in which rhizobia are locally present on the hosts' root hairs. It tests a theoretical computer model explaining root hair curling around bacteria. With a microinjection needie, a small droplet of purified NF was applied on the side o1 the tip of growing root hairs. The result of this is that the root hair under study reoriented its growth axis - it curls, toward the site of NF application, and i1 expresses the early nodulin geneMutagenesis screens are nowadays widely performed to genetically dissec1 signal transduction pathways. In chapter 3, we studied the root hair phenotype in the non nodulating M. truncatula dmi2/N0RK mutant which was found in such a screen. This mutant, and its two orthologues in alfalfa and Lotus japonicus, appeared to exhibit an enhanced touch response to experimenta handling. When care was taken to not induce this touch response, the mutan1 root hairs responded morphologically like wild-type root hairs to NF application. A global application resulted in root hair deformation and NF spot application induced root hair reorientation or - branching, depending on the position of application on the root hair. In addition, dmi2/N0RK root hairs make 180° curls in the presence of rhizobia, but as soon as the root hair tip touches its own shank, the root hair stops growing/curling, and as such is unable to entrap bacteria in a three-dimensional pocket. Because dmi2 root hairs do not express the ProMtENOon-GUS reporter gene after NF application we propose a split in NF-induced signalling, with one branch to root hair curling, the other to ProutENOon-GUS expression.Pea plants can be successfully nodulated by certain strains of rhizobia that oroduce hardly detectable amounts of NF. In addition, very low concentrations of purified NF elicit changes in root hair morphology and gene expression in other legume species. Therefore, we tested what is the lowest NF concentration at which root hair reorientation and ProMtENooii-GUS expression.still occur. In chapter 4, we show the exciting result that one single NF molecule is sufficient to induce root hair reorientation and ProMtENoon-GUS expression,In chapter 5 we describe the results that we obtained after spot application of nod factor mixed with pharmacological agonists or antagonists of signal transduction pathways. As such, we show that NF-induced root hair reorientation can be blocked with gadolinium ions, ions that specifically block calcium influxes in plant cells. Moreover, we show that we can induce root hair reorientation in a number of hairs after spot application of a mixture of calcium ions and the ionophore A23187. Pertussis toxin specifically inhibits heterotrimeric G-proteins. Upon spot application of a mixture of NF and pertussis toxin, root hairs do reorient their growth axis, but do not express the ProMtENODii-GUS reporter gene. Spot application of mastoparan, a small peptide from wasp venom that activates heterotrimeric G-proteins, does not result in root hair reorientation, but does induce Pro^tENOon-GUS expression. Heterotrimeric G-proteins activate phospholipase C. Upon spot application of a mixture of NF and neomycin or U~73122, two known antagonists of phospholipase Ct ProMtENOon-GUS expression was inhibited, but root hair reorientation not Phospholipase C is an enzyme that cleaves phosphoinositolbisphosphate (PIP2) into diacylglycerolphosphate (DAG) and inositoltriphosphate (IP3). With microinjection of caged IP3 into growing M. truncatula root hairs and subsequent uncaging with an UV laser, we got expression of ProMtENooirGUS in a number of root hairs. This all shows that heterotrimeric G-protein coupled phosphoinositide signalling is involved in NF-induced Pr0MtEN0Di1-GUS expression in M. truncatula root hairs.Chapter 6 is a review which covers the current state of the art in the research of the Rhizobium-\egume symbiosis, with a special focus on signal transduction. It not only compares genetic dissection with pharmacological approaches, but also covers the cell biological aspects that are necessary to fully understand NF induced signal transduction.Chapter 7 is a chapter which describes that SHAGGY-kinase signalling is involved in root hair deformation in thale cress (Arabidopsis). This is a small weed that is used as a non~legume model plant. We show that application of lithium ions induces root hair deformation, and that in roots which lack a SHAGGY-kinase, a significant higher percentage of root hairs deform upon lithium application. In plant cells, lithium ions influence ethylene and phosphoinositide signalling, but with the use of specific agonists and antagonists of these pathways, we show that lithium induced root hair deformation in Arabidopsis is not caused by disrupted ethylene or phosphoinositide signalling
Distribution of callose synthase, cellulose synthase and sucrose synthase in tobacco pollen tube is controlled in dissmilar ways by actin filaments and microtubules
No full textCallose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthetized by distinct enzymes, callose synthase and cellulose synthase respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (Latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin and Brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially co-aligned with cortical microtubules and unrelated to callose plugs. Callose synthase also co-migrates with tubulin by Blue Native PAGE. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments dependently on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubule
Genetics of Root Hair Development
No full textThere has been a great deal of recent progress in our understanding of the genetic control of root hair development, particularly in Arabidopsis thaliana . This chapter summarizes the genes and gene products that have been identified using forward and reverse genetic approaches. The involvement of these genes at various stages of root hair development is described, including the specification of the root hair cell type, the initiation of the root hair outgrowth, and the elongation (tip growth) of the root hair.There has been a great deal of recent progress in our understanding of the genetic control of root hair development, particularly in Arabidopsis thaliana . This chapter summarizes the genes and gene products that have been identified using forward and reverse genetic approaches. The involvement of these genes at various stages of root hair development is described, including the specification of the root hair cell type, the initiation of the root hair outgrowth, and the elongation (tip growth) of the root hair
From Golgi body movement to cellulose microfibril alignment
No full text The shape and strength of plant cells is determined by a combination of turgor pressure and constraining cell wall. The main load bearing structures in the cell wall, cellulose microfibrils (CMFs), are deposited in highly organized textures. For more than 50 years scientists have tried to elucidate how the organized CMF textures are being generated and what role cortical microtubules (CMTs) play in CMF deposition. In 2006 Paredez et al. caused a breakthrough by live imaging of cellulose synthase (CESA) complexes that move along CMTs. However, the mechanism by which CMTs guide CESA complexes is still unknown at the moment of writing this thesis and many questions related to the CMF organization still are unanswered. This thesis illuminates the mechanism behind the highly organized CMF textures. To analyze the positioning and patterning of CESA complexes in the cell we studied the following three aspects: (1) the distribution and delivery (close) to the plasma membrane of CESA complexes via Golgi bodies, (2) the distribution and movement of CESA complexes inside the plasma membrane while producing CMFs and finally (3) their product, the CMF texture of the cell wall. We chose epidermal root cells of Arabidopsis thaliana and compared cells of different growth stages. Chapter 1 is an introduction into cellulose deposition and an outline of this thesis. In chapter 2 the movement and distribution of Golgi bodies is studied in the cortex of cells of different growth stages, early elongation zone compared to late elongation zone, in relation to the configuration of the actin cytoskeleton. Golgi bodies in the cortex of cells in the early elongation zone, where growth accelerates to rapid growth, show slow random oriented movement, called wiggling. In the cortex of cells in the late elongation zone, where cell elongation ceases, they also show a second kind of motility, fast directed movement with velocities of up to 7 µm.s-1, like in cytoplasmic strands in the same cells. The cortical areas where Golgi body movement is slow and random co-localize with fine F-actin, a configuration of single or thin bundles of filaments. On the other hand, areas where Golgi body movement is fast and directed co-localize with thick actin filament bundles. When Golgi bodies enter an area with a different actin cytoskeleton configuration they change their type of motility concomitantly. We conclude that Golgi body dynamics correlate with the actin cytoskeleton organization. CESA complexes are known to run in rows along CMTs in Arabidopsis hypocotyl cells. In chapter 3 we studied the orientation, density, alignment and movement of CMTs and CESA complexes using immunocytochemistry and live cell imaging. Furthermore we studied the orientation and density of the product of the CESA complexes, the CMFs, in the innermost wall layer with Field Emission Scanning Electron Microscopy (FESEM). The CMTs, the tracks of CESA complexes and the innermost CMFs lay in the same orientation, approximately transverse to the elongation axis in both the inner and outer periclinal cell face in the elongation zone and root hair zone, where cell elongation ceases. CESA complexes predominantly move in rows along CMTs in both directions. While the CMFs form a uniform cell wall layer, CESA complexes run one after the other along CMTs that are wider spread from each other than the CMFs and only few CESA complexes move in between the CMTs. To understand how CESA complexes can produce a uniform layer of CMFs, instead of local CMF thickenings, we studied whether the CMTs change position during CMF production. Time lapse movies of CMTs show that CMTs reposition over time, so that CESA complexes produce an even CMF layer. In this way we can understand how the density of CMFs in the nascent cell wall can be higher than that of the CMTs and the moving rows of CMFs in the plasma membrane. CMFs are deposited consecutively next to earlier deposited ones in the same orientation. In chapter 4 we used several different electron microscopy techniques to visualize CMF texture: transmission Electron Microscopy (TEM) of ultrathin sections after mild or complete matrix extraction, TEM of surface preparations and FESEM of surface preparations. We used root hairs of three different species; Arabidopsis thaliana, Medicago truncatula and Vicia sativa. We compare and discuss the results of the techniques for the capacity to measure orientation, density, length and width of the CMFs. In ultrathin sections and surface preparations we observed that the three species studied have root hairs with an axial/helical wall texture. Surface preparations are best suitable for density and orientation measurements of CMFs within the most inner cell wall layer. Ultrathin sections showed that the thickness of CMFs in Arabidopsis is approximately 3 nm. which indicates that these CMFs are produced by single CESA complexes. Chapter 5 is a general discussion of our work in relation to the field. It describes the role of the actin cytoskeleton , Golgi body motility and CMTs in the deposition of an organized texture of CMFs. </p
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