Apoptosis-like programmed cell death occurs in procambium and ground meristem of pea (Pisum sativum) root tips exposed to sudden flooding

BACKGROUND AND AIMS: Pea (Pisum sativum) primary roots form long vascular cavities when grown under wet or flooded conditions at 25 degrees C. It is thought that the cavities are a form of aerenchyma. At 25 degrees C short roots continue to grow after flooding. After roots reach 10 cm long flooding causes rapid cessation of growth, and root tips often become curled. In longer roots the cavities do not extend into the base of the roots, perhaps rendering them ineffective as aerenchyma. It was hypothesized that the resulting growth arrest was due to programmed cell death (PCD) rather than necrosis. METHODS AND KEY RESULTS: Histological examination by light microscope showed that some cells in the primary meristem (elongation) zone of the primary root tips had morphological abnormalities, including misshapen and fragmented nuclei, and cytoplasmic shrinking and fragmentation. Transmission electron microscopy revealed lobing, invagination and chromatin aggregation in nuclei. The affected cells were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Extracted DNA formed a "DNA ladder" during electrophoresis. Cell death usually began in procambium at one or two protoxylem poles and seemed to spread out to nearby tissues, which asymmetrically inhibited growth and resulted in tip curling. CONCLUSIONS: The above are symptoms of apoptosis-like PCD. Programmed root tip death may rapidly reduce oxygen demand and sink strength, allowing more rapid diversion of resources to lateral roots growing in more permissive conditions.     

Bio-markers of lipid peroxidation in vivo: hydroxyoctadecadienoic acid and hydroxycholesterol

The biological role of lipid peroxidation products has continued to receive a great deal of attention not only for the elucidation of pathological mechanisms but also for their practical application to clinical use as bio-markers. In the last fifty years, lipid peroxidation has been the subject of extensive studies from the viewpoints of mechanisms, dynamics, product analysis, involvement in diseases, inhibition, and biological signaling. Lipid hydroperoxides are formed as the major primary products, however they are substrates for various enzymes and they also undergo various secondary reactions. In this decade, F2-isoprostanes from arachidonates and neuroprostanes from docosahexanoates have been proposed as bio-markers. Although these markers are formed by a free radical-mediated oxidation, the yields from the parent lipids are minimal. Compared to these markers, hydroperoxy octadecadienoates (HPODE) from linoleates and oxysterols from cholesterols are yielded by much simpler mechanisms from more abundant parent lipids in vivo. Recently, the method in which both free and ester forms of hydroperoxides and ketones as well as hydroxides of linoleic acid and cholesterol are measured as total hydroxyoctadecadienoic acid (tHODE) and 7-hydroxycholesterol (t7-OHCh), respectively, was proposed. The concentrations of tHODE and t7-OHCh determined by GC-MS analysis from physiological samples were much higher than that of 8-iso-prostagrandin F(2alpha). In addition to this advantage, hydrogen-donor activity of antioxidants in vivo could be determined by the isomeric-ratio of HODE (9- and 13-(Z,E)-HODE/9- and 13-(E,E)-HODE).     

The relation between the specific absorption rate and electromagnetic field intensity for heterogeneous exposure conditions at mobile communications frequencies

The relation between the incident electromagnetic field strength and both the whole‐body and the local specific absorption rate (SAR) was investigated for typical heterogeneous exposure scenarios for frequencies relevant for mobile communication. The results were compared to results from plane wave exposure. Heterogeneous exposure arises from multiple path propagation of the electromagnetic waves to the location of interest. It is shown that plane wave exposure does not represent worst‐case exposure conditions. When the electric field strength arising at plane wave exposure is compared to the electric field strength averaged over the volume of the human body occurring during multipath exposure, 12% of all heterogeneous cases examined represent worse exposure conditions than plane wave exposure for whole‐body exposure at 946 MHz, 15% at 1840 MHz, and 22% at 2140 MHz. The deviation between plane wave and heterogeneous whole‐body SAR ranges from ?54% to 54%. For partial‐body SAR averaged over 10 g of tissue, a range from ?93% to 209% was found when comparing multiple wave exposure to single incoming plane waves. The investigations performed using the Visible Human as phantom showed that the basic restrictions are met as long as the reference levels are not exceeded. However, this must not be necessarily the case when different phantoms are used to perform similar investigations because recent studies demonstrated that reference levels might not be conservative when phantoms of children are used. Therefore, the results of this work indicate the need to extend the investigations to numerical simulations with additional human phantoms representing parts of the human population having different anatomy and morphology compared to the phantom used within the frame of this project. This also applies to phantoms of children. Bioelectromagnetics 30:651–662, 2009. © 2009 Wiley‐Liss, Inc.     

A Genome-Scale Proteomic Screen Identifies a Role for DnaK in Chaperoning of Polar Autotransporters in Shigella

Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.The Shigella outer membrane protein IcsA is unusual in that it is secreted at the bacterial old pole (9, 13, 24). The secreted protein forms a cap at the old pole (Fig. ​(Fig.1A),1A), where during the infection of host cells, it interacts with cellular actin cytoskeletal proteins to induce the formation of propulsive actin tails (6, 43, 70). Actin tail formation is essential to the spread of Shigella spp. through cell monolayers and mammalian tissues (6, 43, 47) and is critical for Shigella virulence (15, 60). IcsA is a member of the autotransporter family of secreted proteins in gram-negative bacteria. Approximately 700 autotransporter proteins are predicted to be encoded within bacterial genomes that had been annotated as of 2003 (54). All autotransporter proteins for which the site of secretion has been determined are, like IcsA, secreted at the bacterial old pole (35).Open in a separate windowFIG. 1.Design of screen for proteins that, like IcsA, localize to potential division sites independent of FtsZ. (A) Localization of IcsA on the surface of S. flexneri. Immunofluorescence using antibody to IcsA. (B) Localization of IcsA_507-620-GFP (expressed from pBAD24-icsA_507-620::gfp) to poles of single cells of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the permissive temperature (30°C). (C) Localization of IcsA_507-620-GFP to potential cell division sites of E. coli MC4100 leu::Tn10 ftsZ84(Ts) grown at the nonpermissive temperature (42°C). (D) Diagram of the strategy used to identify proteins of E. coli that localize to potential cell division sites independent of FtsZ, displaying a localization pattern similar to that shown for IcsA in panel C. Image from DnaK-GFP localization (expressed from leaky promoter on pCA24N-dnaK, without induction) in screen well; incomplete overlay of GFP with phase-contrast microscopy is due to the movement of cells between capturing the two images, as cells were imaged live. Size bars = 2 μm (A and B) and 5 μm (C and D). Images are representative. O/N, overnight.Several other secreted bacterial proteins are also localized to one or both cell poles; these include the Listeria monocytogenes actin assembly protein ActA (39), components of the chemotaxis apparatus in Escherichia coli and Caulobacter crescentus (1, 46, 66), the Legionella pneumophila and Agrobacterium tumifaciens type IV secretion systems (14, 40), Pseudomonas aeruginosa type IV pili (8), protein components of the cell cycle regulatory pathways in C. crescentus (reviewed in reference 72), the DNA transfer apparatus in Bacillus subtilis and Streptomyces spp. (26, 28), and polar flagella in Vibrio cholerae, Campylobacter spp., Helicobacter spp., C. crescentus, and other gram-negative bacteria. In L. monocytogenes, the polarity of ActA is established after ActA secretion and likely depends on differential growth rates of the cell wall along the length of the bacterium (56). In C. crescentus, TipN serves as a polar developmental landmark (31, 42), and RcdA provides temporal and spatial specificity in the regulated proteolysis of key factors involved in polar asymmetry (50). Beyond these studies, relatively little is known about the molecular mechanisms that mediate the proper localization of polar bacterial proteins.Chemical or genetic blockade of cell division leads to the formation of filamentous cells without septa. In cells that have been filamented by either blocking FtsI or depleting functional FtsZ, a cytoplasmic derivative of IcsA localizes at or near potential cell division sites (36), which represent the sites of future cell poles. IcsA also localizes to potential division sites independent of nucleoid occlusion (36), together indicating that the positional information directing IcsA polarity is independent of these cell division proteins and chromosome positioning. The molecules that are required for the localization of IcsA to the cell pole have not been identified.One model of IcsA localization to the pole is that freely diffusing cytoplasmic IcsA recognizes and binds a protein receptor that is present at poles and future poles. Although icsA is present only in Shigella spp., upon heterologous expression, IcsA localizes to the poles of other Enterobacteriaceae (13, 58, 59), indicating that if targeting occurs via binding to a polar receptor, the receptor is likely conserved among members of this family. In addition, since IcsA localizes independently of FtsZ and FtsI, the localization of a putative polar receptor to the pole must also be independent of these cell division proteins. To find proteins that might serve as a polar receptor for IcsA, we first conducted a genome-wide screen designed to identify the subset of E. coli proteins that localize to poles and to potential cell division sites independently of functional FtsZ. For each conserved protein that displayed this localization pattern, we then tested whether it played a role in the polar localization of IcsA. We found that, under the conditions of our screen, a green fluorescent protein (GFP) fusion to the cytoplasmic chaperone DnaK localizes to cell poles and potential cell division sites. Although DnaK is not a polar receptor for IcsA, we demonstrated that it was required for the localization of IcsA to the pole in the bacterial cytoplasm in E. coli and for the secretion of both IcsA and a second Shigella autotransporter, SepA, in native Shigella flexneri, consistent with a critical role of DnaK in the chaperoning of IcsA and SepA, and perhaps autotransporter proteins more generally, in the bacterial cytoplasm.     

Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells

Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested.     

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.     

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in ΔdatA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.Initiation of DNA replication is highly regulated to coordinate with cell proliferation. It begins with a series of events in which the replication machinery is assembled at the replication origin of the chromosomal DNA (15, 26, 28, 38). Central to this process are the initiator proteins that bind to the origin of replication and eventually lead to the unwinding of the origin and to helicase loading on the unwound region. Previous biochemical studies and recent structural studies of the bacterial initiator protein DnaA have proposed the molecular mechanism of the action of ATP-DnaA in forming a large oligomeric complex to remodel the unique origin, oriC, and trigger duplex melting (12, 26). However, it is still not clear how the timing of initiation is controlled so that it takes place at a fixed time in the cell cycle. It has been reported that a basal level of DnaA molecules is bound by high-affinity DnaA binding sites (DnaA boxes R1, R2, and R4) at oriC throughout the cell cycle (9, 37). It is also suggested that noncanonical ATP-DnaA binding sites within oriC are occupied at elevated levels of the initiator molecules prior to the initiation event (18, 25). Thus, regulation of the activity and availability of DnaA is an important factor for the initiation control.At least three schemes are known to prevent untimely initiations in Escherichia coli. First, oriC is subject to sequestration, a process that prevents reinitiation, possibly by blocking ATP-DnaA from binding to newly replicated oriC (8, 24). E. coli oriC contains 11 GATC sites that are normally methylated on both strands by Dam methyltransferase. Immediately after passage of the replication fork, GATC sites are in a hemimethylated state, with the newly synthesized strands remaining unmethylated. SeqA binds specifically to such sites and, at oriC, protects these regions from reinitiation for about one-third of the cell cycle (6, 39). Second, in a process termed regulatory inactivation of DNA (RIDA), ATP-DnaA molecules are converted to an inactive ADP-bound form after initiation by the combined action of a β subunit of DNA polymerase III holoenzyme and Hda (16, 17). Newly synthesized DnaA molecules are able to bind ATP for the next initiation event, since its cellular concentration is much higher than that of ADP. ATP-DnaA is also regenerated from the inactive ADP-DnaA later in the cell cycle (21). Finally, the chromosomal segment datA serves to reduce the level of free DnaA protein by titrating a large number of DnaA molecules after replication of the site close to oriC (20).Cytological studies would be very useful for developing our understanding of the regulation mechanisms associated with the initiation step. In the present study, we tagged E. coli DnaA with a fluorescent protein in order to monitor its behavior in live cells. Microscopic observation revealed that DnaA is distributed all over the nucleoid. Remarkably, the majority of cells bore distinct foci that emitted brighter fluorescence against a weak fluorescent background on the nucleoid. We analyzed the behavior of these foci during the cell cycle with respect to oriC and datA.     

BMP and Hh signaling affects primordial germ cell division in Drosophila

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.     

The DNA Damage Checkpoint Regulates a Transition between Yeast and Hyphal Growth in Schizosaccharomyces japonicus

Dimorphic yeasts change between unicellular growth and filamentous growth. Many dimorphic yeasts species are pathogenic for humans and plants, being infectious as invasive hypha. We have studied the determinants of the dimorphic switch in the nonpathogenic fission yeast Schizosaccharomyces japonicus, which is evolutionarily close to the well-characterized fission yeast S. pombe. We report that camptothecin, an inhibitor of topoisomerase I, reversibly induced the unicellular to hyphal transition in S. japonicus at low concentrations of camptothecin that did not induce checkpoint arrest and the transition required the DNA checkpoint kinase Chk1. Furthermore, a mutation of chk1 induced hyphal transition without camptothecin. Thus, we identify a second function for Chk1 distinct from its role in checkpoint arrest. Activation of the switch from single cell bipolar growth to monopolar filamentous growth may assist cells to evade the source of DNA damage.Yeasts and molds are major members of the kingdom Fungi. Molds grow as multicellular filamentous hyphae. On the other hand, yeasts propagate in a unicellular fashion by budding or by binary fission. However, many types of yeast can switch their growth modes, changing from unicellular growth to filamentous branching multicellular hyphae. This hyphal transition can be induced by a wide variety of environmental changes ranging from pH to the nature of the carbon source, and many species of dimorphic yeasts that are pathogenic for humans and plants are infectious in the hyphal form (15, 20).Hyphal transition is a simple mode of cellular differentiation program that is turned on upon environmental changes. The fungi may differentiate to adapt to the environmental challenges. Especially in the case of Candida albicans strains that infect humans, the hyphal transition may function as an action to resist against attack from macrophages or neutrophils. Hyphae are more difficult to phagocytose (16). It can also eventually kill macrophages if hyphal transition is triggered after ingestion by macrophage (14). Indeed, C. albicans cells that cannot form hyphae are avirulent. However, inducing hyphal growth in pathogenic yeasts is not always readily achievable in the laboratory, and genetic analysis of the hyphal growth phase and transition to this phase is often limited by the lack of appropriate tools. Thus, genetically tractable nonpathogenic dimorphic yeasts are attractive models for investigating invasive hypha.The nonpathogenic fission yeast Schizosaccharomyces japonicus is evolutionarily close to the well-characterized fission yeast Schizosaccharomyces pombe (5, 24). S. japonicus is dimorphic, transiting between unicellular and hyphal growth, and thus offers itself as an appropriate model to study this differentiation mechanism and the requirements of hyphal growth (25). In S. japonicus, hyphal growth occurs naturally on most solid medium and can occur over a range of nutrient conditions (26). It has been proposed that a gradient of nitrogen in the substrate is necessary to both initiate and direct hyphal growth in S. japonicus (26). In this report we establish conditions to induce hyphal growth in a microchamber in liquid media. In addition, we show that a low dose of the topoisomerase inhibitor camptothecin (CPT) induces hyphal differentiation under rich nutrient conditions and identify a role for the DNA damage checkpoint response in promoting the CPT-dependent transition from unicellular to hyphal growth. Genetic analysis demonstrates that this role of the checkpoint is distinct from checkpoint arrest, and we suggest it may provide an opportunity for S. japonicus to grow away from sources of genotoxic stress.